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Methods in
Molecular Biology 2352

Henrik Ahlenius Editor

Neural
Reprogramming
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Neural Reprogramming

Methods and Protocols

Edited by

Henrik Ahlenius
Stem Cells, Aging and Neurodegeneration Group, Faculty of Medicine, Department of Clinical
Sciences, Neurology, Lund Stem Cell Center, Lund University, Lund, Sweden
Editor
Henrik Ahlenius
Stem Cells, Aging and
Neurodegeneration Group
Faculty of Medicine
Department of Clinical
Sciences, Neurology
Lund Stem Cell Center
Lund University
Lund, Sweden

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1600-0 ISBN 978-1-0716-1601-7 (eBook)
https://doi.org/10.1007/978-1-0716-1601-7

© Springer Science+Business Media, LLC, part of Springer Nature 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
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This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

The seminal discovery, by Takahashi and Yamanaka, that somatic cells can be reprogrammed
to become pluripotent has led to a whole new field of research. We now know that somatic
cells can be reprogrammed not only to induced pluripotent stem cells but also to other
lineage-restricted progenitors or somatic cells within a specific lineage or to cells of distant
lineages.
The relative inaccessibility of human neural cells has previously impeded neuroscience
research. Scientists were restricted to immortalized or cancer cell lines, fetal tissue, surgical
resection, or post-mortem material which have been very useful and informative but all
come with different limitations.
Reprogramming technologies have become a real game changer making normal and
patient-derived neural cells readily available for research and potential therapeutic purposes.
It is now in principle possible to generate neural cells in vitro from any living human being or
any bio-banked cells.
For instance, mouse or human fibroblasts of the mesoderm lineage can be repro-
grammed to become neurons of the ectoderm lineage by overexpression of neuronal
transcription factors. The concept of reprogramming has also been applied to pluripotent
or neural stem cells to fast forward differentiation to neurons, astrocytes, and oligodendrog-
lia by the use of lineage-specific transcription factors.
These technologies enable the development of potential cell replacement therapies,
studies of physiological cellular processes, disease modeling, drug screening, and much
more. For instance, it is now possible to generate neurons and astrocytes from somatic
cells and culture them together to study synapse formation as well as neuron–glia interac-
tions. Similarly, cocultures of reprogrammed neurons and oligodendrocytes could be used
to study myelination. Neural cells can be generated directly from healthy and patient-
derived cells, or by first reprogramming to pluripotency. Alone or in combination with
genome engineering, this offers the opportunity to study in dynamic ways neurological
disorders in the dish.
Currently neural cells derived from pluripotent stem cells are explored and tested in
clinical trials for cell replacement therapies and extensively used for modeling of mainly
monogenetic but also sporadic neurological disorders. Neural cells reprogrammed from
somatic cells without going through a pluripotent stem cell stage are highly relevant for
disease modeling of sporadic and late onset diseases as they, in contrast to cells derived from
pluripotent stem cells, have been shown to retain aging phenotypes. Another exciting
avenue is in vivo neuronal reprogramming of glial cells in the brain, which has shown in
animal models promising therapeutic potential.
The field of reprogramming is constantly developing, and new methods are rapidly
emerging. Here we bring together a number of state-of-the-art and recent protocols to
generate different types of neural cells. In addition, the book covers different aspects of
functional evaluation and applications of reprogrammed neural cells as well as in silico
methods to aid reprogramming.
The book covers reprogramming of somatic mouse (Chapters 1–5) and human cells
(Chapters 5–7) to neurons and astrocytes driven by transcription factors as well as by the use
of small molecules. It also details the use of transcription factors for forward programming

v
vi Preface

of human pluripotent stem cells to neurons, astrocytes, and oligodendrocytes (Chapters

8–11). Furthermore, we provide protocols for transcriptional profiling during neuronal
reprogramming (Chapter 12) and functional assessment of reprogrammed neural cells
using electrophysiology and electrochemistry (Chapters 13 and 14). In addition, the book
covers protocols for combining reprogramming with protein engineering to understand
transcription factor biology (Chapter 15), Crispr/Cas9 genome engineering for neural
disease modeling (Chapter 16), and the establishment of human brain organotypical slice
cultures which should prove useful for preclinical testing of reprogrammed neural cells
(Chapter 17).
The field of neural reprogramming has in the last decade opened up previously unimag-
inable possibilities to study the central nervous system in health and disease. The field is still
in its infancy, and many findings need further validation and development. Nonetheless, we
can expect to see great discoveries being made in neuroscience through the use of repro-
gramming technologies in the coming years. The protocols presented here provide ample
experimental experience and guidance for anyone, be it experienced or beginner, to gener-
ate, validate, and apply reprogrammed neural cells in their research.
Finally, I would like to extend my sincere gratitude to all authors as well as the
production team during the lengthy process of making this book a reality.

Lund, Sweden Henrik Ahlenius

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Isolation and Neuronal Reprogramming of Mouse Embryonic Fibroblasts . . . . . 1

Juan M. Adrian-Segarra, Bettina Weigel, and Moritz Mall
2 Direct In Vitro Reprogramming of Astrocytes into Induced Neurons . . . . . . . . . 13
Nesrin Sharif, Filippo Calzolari, and Benedikt Berninger
3 Direct Cell Reprogramming of Mouse Fibroblasts into Functional
Astrocytes Using Lentiviral Overexpression of the Transcription
Factors NFIA, NFIB, and SOX9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Boning Qiu, Ruben J. de Vries, and Massimiliano Caiazzo
4 Direct Reprogramming of Fibroblasts to Astrocytes Using Small
Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
E Tian, Mingzi Zhang, and Yanhong Shi
5 Bcl-2-Assisted Reprogramming of Mouse Astrocytes and Human
Fibroblasts into Induced Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Amel Falco, Rocı́o Bartolomé-Cabrero, and Sergio Gascon
6 Direct Conversion of Human Fibroblasts to Induced Neurons . . . . . . . . . . . . . . . 73
Lucia Zhou-Yang, Sophie Eichhorner, Lukas Karbacher,
Lena Böhnke, Larissa Traxler, and Jerome Mertens
7 Generation of Induced Dopaminergic Neurons from Human
Fetal Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Emilie M. Legault and Janelle Drouin-Ouellet
8 Direct Differentiation of Functional Neurons from Human Pluripotent
Stem Cells (hPSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Ruiqi Hu, Xiaoting Zhu, and Nan Yang
9 Generation of Motor Neurons from Human ESCs/iPSCs Using Sendai
Virus Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Keiko Imamura, Jitsutaro Kawaguchi, Tsugumine Shu, and Haruhisa
Inoue
10 Transcription Factor Programming of Human Pluripotent Stem Cells
to Functionally Mature Astrocytes for Monocultures and Cocultures
with Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Ella Quist, Henrik Ahlenius, and Isaac Canals
11 Single Transcription Factor-Based Differentiation Allowing Fast
and Efficient Oligodendrocyte Generation via SOX10 Overexpression. . . . . . . . . 149
Katrien Neyrinck and Juan A. Garcı́a-Leon
12 Transcriptional Profiling During Neural Conversion . . . . . . . . . . . . . . . . . . . . . . . . 171
Yohannes Afeworki, Hannah Wollenzien, and Michael S. Kareta
13 Functional Assessment of Direct Reprogrammed Neurons In Vitro
and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Srisaiyini Kidnapillai and Daniella Rylander Ottosson
vii
viii Contents

14 Neurotransmitter Release of Reprogrammed Cells Using Electrochemical

Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Andreas Heuer
15 Combining Cell Fate Reprogramming and Protein Engineering to Study
Transcription Factor Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Juan M. Adrian-Segarra, Bettina Weigel, and Moritz Mall
16 CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem
Cells for Modeling of Neurological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Isaac Canals and Henrik Ahlenius
17 Derivation of Adult Human Cortical Organotypic Slice Cultures
for Coculture with Reprogrammed Neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . 253
Giedre Kvist

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Contributors

JUAN M. ADRIAN-SEGARRA • Cell Fate Engineering and Disease Modeling Group, German
Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany;
HITBR Hector Institute for Translational Brain Research gGmbH, Heidelberg, Germany;
Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University,
Mannheim, Germany
YOHANNES AFEWORKI • The Functional Genomics and Bioinformatics Core, University of
South Dakota, Vermillion, SD, USA
HENRIK AHLENIUS • Stem Cells, Aging and Neurodegeneration Group, Faculty of Medicine,
Department of Clinical Sciences, Neurology, Lund Stem Cell Center, Lund University,
Lund, Sweden; Division of Neurology, Department of Clinical Sciences, University of Lund,
Lund, Sweden
ROCÍO BARTOLOMÉ-CABRERO • Department of Pharmacology and Toxicology, Faculties of
Veterinary and Medicine, Complutense University of Madrid, Madrid, Spain; Department
of Molecular, Cellular and Developmental Neurobiology, Instituto Cajal – CSIC, Madrid,
Spain; Department of Biochemistry, Universidad Autonoma de Madrid (UAM), Instituto
de Investigaciones Biomedicas Alberto Sols (CSIC-UAM), Madrid, Spain
BENEDIKT BERNINGER • Institute of Physiological Chemistry, University Medical Center
Johannes Gutenberg University Mainz, Mainz, Germany; Institute of Psychiatry,
Psychology, and Neuroscience, Centre for Developmental Neurobiology, King’s College
London, London, UK; MRC Centre for Neurodevelopmental Disorders, King’s College
London, London, UK
LENA BÖHNKE • Institute of Molecular Biology and CMBI, Leopold-Franzens-University,
Innsbruck, Austria; Laboratory of Genetics, The Salk Institute for Biological Studies, La
Jolla, CA, USA
MASSIMILIANO CAIAZZO • Department of Pharmaceutics, Utrecht Institute for
Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands;
Department of Molecular Medicine and Medical Biotechnology, University of Naples
‘Federico II’, Naples, Italy
FILIPPO CALZOLARI • Institute of Physiological Chemistry, University Medical Center
Johannes Gutenberg University Mainz, Mainz, Germany
ISAAC CANALS • Stem Cells, Aging and Neurodegeneration Group, Faculty of Medicine,
Department of Clinical Sciences, Neurology, Lund Stem Cell Center, Lund University,
Lund, Sweden
RUBEN J. DE VRIES • Department of Pharmaceutics, Utrecht Institute for Pharmaceutical
Sciences (UIPS), Utrecht University, Utrecht, The Netherlands
JANELLE DROUIN-OUELLET • Faculté de Pharmacie, Université de Montréal, Montreal, QC,
Canada
SOPHIE EICHHORNER • Institute of Molecular Biology and CMBI, Leopold-Franzens-
University, Innsbruck, Austria
AMEL FALCO • Department of Pharmacology and Toxicology, Faculties of Veterinary and
Medicine, Complutense University of Madrid, Madrid, Spain; Department of Molecular,
Cellular and Developmental Neurobiology, Instituto Cajal – CSIC, Madrid, Spain

ix
x Contributors

JUAN A. GARCÍA-LEÓN • Departamento Biologia Celular, Genetica y Fisiologia, Facultad de

Ciencias, Instituto de Investigacion Biomedica de Malaga-IBIMA, Universidad de
Malaga, Malaga, Spain; Centro de Investigacion Biomedica en Red Sobre Enfermedades
Neurodegenerativas (CIBERNED), Madrid, Spain
SERGIO GASCÓN • Department of Pharmacology and Toxicology, Faculties of Veterinary and
Medicine, Complutense University of Madrid, Madrid, Spain; Institute of Physiology,
Department of Physiological Genomics, Biomedical Center (BMC), Ludwig-Maximilians
University Munich, Planegg-Martinsried, Germany
ANDREAS HEUER • Behavioural Neuroscience Laboratory, Department of Experimental
Medical Sciences, Lund University, Lund, Sweden
RUIQI HU • Department of Neuroscience, Icahn School of Medicine at Mount Sinai, Black
Family Stem Cell Institute, Friedman Brain Institute, New York, NY, USA
KEIKO IMAMURA • Center for iPS Cell Research and Application (CiRA), Kyoto University,
Kyoto, Japan; iPSC-Based Drug Discovery and Development Team, RIKEN BioResource
Research Center (BRC), Kyoto, Japan; Medical-Risk Avoidance Based on iPS Cells Team,
RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
HARUHISA INOUE • Center for iPS Cell Research and Application (CiRA), Kyoto University,
Kyoto, Japan; iPSC-Based Drug Discovery and Development Team, RIKEN BioResource
Research Center (BRC), Kyoto, Japan; Medical-Risk Avoidance Based on iPS Cells Team,
RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan; Institute for
Advancement of Clinical and Translational Science, Kyoto University, Kyoto, Japan
LUKAS KARBACHER • Institute of Molecular Biology and CMBI, Leopold-Franzens-University,
Innsbruck, Austria
MICHAEL S. KARETA • The Functional Genomics and Bioinformatics Core, University of
South Dakota, Vermillion, SD, USA; Division of Basic Biomedical Sciences, Sanford School
of Medicine, University of South Dakota, Vermillion, SD, USA; Genetics and Genomics
Group, Sanford Research, Sioux Falls, SD, USA; Cellular Therapies and Stem Cell Biology
Group, Sanford Research, Sioux Falls, SD, USA; Department of Pediatrics, Sanford School
of Medicine, Sioux Falls, SD, USA; Department of Chemistry and Biochemistry, South
Dakota State University, Brookings, SD, USA
JITSUTARO KAWAGUCHI • R&D Center, ID Pharma Co. Ltd., Tsukuba, Japan
SRISAIYINI KIDNAPILLAI • Department of Experimental Medical Science and Lund Stem Cell
Centre, BMC, Lund University, Lund, Sweden
GIEDRE KVIST • Lund Stem Cell Center, Lund University, Lund, Sweden
EMILIE M. LEGAULT • Faculté de Pharmacie, Université de Montréal, Montreal, QC, Canada
MORITZ MALL • Cell Fate Engineering and Disease Modeling Group, German Cancer
Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany; HITBR
Hector Institute for Translational Brain Research gGmbH, Heidelberg, Germany; Central
Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University,
Mannheim, Germany
JEROME MERTENS • Institute of Molecular Biology and CMBI, Leopold-Franzens-University,
Innsbruck, Austria; Laboratory of Genetics, The Salk Institute for Biological Studies, La
Jolla, CA, USA
KATRIEN NEYRINCK • Department of Development and Regeneration, Stem Cell Institute,
KU Leuven, Leuven, Belgium
BONING QIU • Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences
(UIPS), Utrecht University, Utrecht, The Netherlands
 Contributors xi

ELLA QUIST • Stem Cells, Aging and Neurodegeneration Group, Faculty of Medicine,
Department of Clinical Sciences, Neurology, Lund Stem Cell Center, Lund University,
Lund, Sweden
DANIELLA RYLANDER OTTOSSON • Department of Experimental Medical Science and Lund
Stem Cell Centre, BMC, Lund University, Lund, Sweden
NESRIN SHARIF • Institute of Physiological Chemistry, University Medical Center Johannes
Gutenberg University Mainz, Mainz, Germany; International PhD Programme on Gene
Regulation, Epigenetics and Genome Stability, Mainz, Germany
YANHONG SHI • Division of Stem Cell Biology Research, Department of Developmental and
Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
TSUGUMINE SHU • R&D Center, ID Pharma Co. Ltd., Tsukuba, Japan
E. TIAN • Division of Stem Cell Biology Research, Department of Developmental and Stem
Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
LARISSA TRAXLER • Institute of Molecular Biology and CMBI, Leopold-Franzens-University,
Innsbruck, Austria; Laboratory of Genetics, The Salk Institute for Biological Studies, La
Jolla, CA, USA
BETTINA WEIGEL • Cell Fate Engineering and Disease Modeling Group, German Cancer
Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany; HITBR
Hector Institute for Translational Brain Research gGmbH, Heidelberg, Germany; Central
Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University,
Mannheim, Germany
HANNAH WOLLENZIEN • Division of Basic Biomedical Sciences, Sanford School of Medicine,
University of South Dakota, Vermillion, SD, USA; Genetics and Genomics Group, Sanford
Research, Sioux Falls, SD, USA
NAN YANG • Department of Neuroscience, Icahn School of Medicine at Mount Sinai, Black
Family Stem Cell Institute, Friedman Brain Institute, New York, NY, USA
MINGZI ZHANG • Division of Stem Cell Biology Research, Department of Developmental and
Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
LUCIA ZHOU-YANG • Institute of Molecular Biology and CMBI, Leopold-Franzens-
University, Innsbruck, Austria
XIAOTING ZHU • Department of Neuroscience, Icahn School of Medicine at Mount Sinai,
Black Family Stem Cell Institute, Friedman Brain Institute, New York, NY, USA
 Chapter 1

Isolation and Neuronal Reprogramming of Mouse

Embryonic Fibroblasts
Juan M. Adrian-Segarra, Bettina Weigel, and Moritz Mall

Abstract
Forced expression of specific neuronal transcription factors in mouse embryonic fibroblasts (MEFs) can lead
to their direct conversion into functional neurons. Direct neuronal reprogramming has become a powerful
tool to characterize individual factors and molecular mechanisms involved in forced and normal neurogen-
esis and to generate neuronal cell types for in vitro studies. Here we provide a detailed protocol for the
isolation of MEFs devoid of neural tissue and their direct reprogramming into functional neurons by
overexpression of neuronal reprogramming factors (Ascl1, Brn2, and Myt1l) using lentiviral vectors. This
method enables quick and efficient generation of mouse neurons in vitro for versatile functional and
mechanistic characterization.

Key words MEF, Cell fate conversion, Neuronal reprogramming, Ascl1, Brn2, Myt1l, Induced
neurogenesis

1 Introduction

Direct neuronal reprogramming is based on the same factors that

operate during normal neuronal development and constitutes a
powerful new tool to investigate the molecular mechanisms gov-
erning neurogenesis [1, 2]. As an example, neuronal reprogram-
ming made it possible to identify and uncover specific functions of
previously poorly characterized neuronal transcription factors
[3–8].
In addition, neuronal reprogramming offers an interesting
application to model neurological diseases in the culture dish
[9]. Recent progress has been made to develop protocols to gener-
ate human neurons from fibroblasts or stem cells for in vitro disease
modeling [10–12]. However, neuronal reprogramming of murine
cells such as MEFs is still of great relevance due to the availability of
many transgenic lines, which greatly facilitate studying the contri-
butions of particular genes/risk factors to neurogenesis and neuro-
nal function (Fig. 1).

Henrik Ahlenius (ed.), Neural Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 2352,
https://doi.org/10.1007/978-1-0716-1601-7_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

1
2 Juan M. Adrian-Segarra et al.

Fig. 1 (a) Live cell time-lapse microscopy of MEFs derived from TauEGFP reporter mice transduced with Ascl1,
Brn2, and Myt1l at indicated time points after transgene induction using doxycycline. After 3–5 days, clear
morphological changes such as rounding of the cell body and neurite extension can be observed along with
induction of neuronal genes such as the TauEGFP reporter. The same cell is highlighted by an arrowhead.
Scale bar 20μm. (b) Immunofluorescence microscopy images of cells reprogrammed as in (a) after 14 days
and stained with antibodies against the neuronal markers TauEGFP (green) and Tuj1 (red), as well as the DNA
stain DAPI (blue). Scale bar 50μm

Here we provide a detailed protocol covering all steps involved

in direct neuronal reprogramming of mouse embryonic fibroblasts
(MEFs), which include MEF isolation and culture, lentiviral pro-
duction, MEF transduction, and neuronal reprogramming
(Fig. 2a). By following this protocol, MEFs and lentiviral solutions
can be prepared in less than 1 week, with fully functional repro-
grammed neurons being reliably obtained after 2–3 weeks
(Fig. 2b).
Before undertaking this protocol, researchers should ensure
compliance with their institution’s guidelines and requirements
regarding both animal and biosafety level 2 lentiviral work.

2 Materials

2.1 MEF Isolation 1. Class II laminar flow hood.

and Culture 2. Dissecting microscope.
2.1.1 Equipment 3. CO2 cell culture incubator (37  C, 5% CO2).
4. Surgical tools: scissors, spring scissors, forceps.
5. Freezing box providing a controlled temperature decrease rate.
6. Regular 10-cm dishes.
7. Cell culture dishes (10 and 15 cm).
8. 1.5-mL microfuge tubes.
9. Freezing vials (1 mL).
 Direct Neuronal Reprogramming 3

Fig. 2 (a) Schematic overview of the distinct steps comprising MEF isolation, lentiviral transduction, and
neuronal conversion described in this direct neuronal reprogramming protocol. (b) Expected timeline for the
different parts of the procedure described in detail in the protocol

2.1.2 Solutions 1. Ice-cold 80% ethanol.

2. Ice-cold Hank’s Balanced Salt Solution (HBSS).
3. 0.25% trypsin-EDTA solution.
4. Fetal bovine serum (FBS).
5. 2 cryopreservation medium: FBS + 20% (v/v) dimethyl sulf-
oxide (DMSO).
6. MEF medium: Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with GlutaMAX (1), penicillin-streptomycin
(1), MEM non-essential amino acids (1), sodium pyruvate
(1 mM), β-mercaptoethanol (0.1 mM) (see Note 1), and
bovine calf serum fortified with iron and growth-promoting
factors (10% v/v).

2.1.3 Other Materials 1. Pregnant mouse of the desired strain (e.g., C57BL/6 mice) at
embryonic day 13.5 (E13.5).

2.2 Lentivirus 1. Access to a biosafety level 2 cell culture room with laminar flow
Production hood and CO2 incubator.
2.2.1 Equipment 2. Cell culture dishes (10 and 15 cm).
3. 30- or 50-mL sterile syringes.
4. 45-μm filters.
Other documents randomly have
different content
the remnant of the cigarette hanging from her long white fingers, while she
stared into the fire. There was a curious sense of repose in the whole body,
and a queer sadness too. She might have been quite alone. He had the
feeling that she had forgotten his existence.
He worked nervously, with long, sure strokes, and with each one he
knew that he was succeeding. In the end he would fix her thus forever on a
fragment of paper. And then suddenly he heard some one enter the stable
below, and, fumbling with the door, open it and hurry up the steps. He went
on, pressed by the fear that if he were disturbed now the thing would never
be finished. He had to have it. It would be a kind of fetish to keep off
despair.
It was Lily Shane who moved first, stirred perhaps by a sense of being
watched. As she moved, Philip turned too, and there, half-way up the stairs
where she had halted at sight of them, stood Naomi, staring.
She was breathless, and beneath a carelessly pinned hat, from which
wisps of hair escaped, her face showed red and shining as a midsummer
day. For one dreadful moment the three remained silent, staring at each
other. Lily Shane stared with a kind of bored indifference, but there was in
Naomi’s eyes a hurt look of bewilderment. Suddenly she turned back, as if
she meant to go away again without speaking to either of them. Philip knew
the expression at once. She had looked thus on the day that Lady Millicent
appeared out of the forest with the Arab marching before her. It was the
look of one who was shut out from something she could not understand,
which frightened her by its strangeness.
It was Lily Shane who moved first. The burnt cigarette dropped from her
fingers; and she stamped on it. The action appeared to stir Naomi into life.
“Philip,” she said. “I came to tell you that your Pa has come home.”

14
It was Emma herself who saw him first. Returning flustered and upset
from the call upon Mary Conyngham, she entered the slate-colored house
closing the door stormily behind her. She would have passed the darkened
parlor (where since Naomi’s departure the shades were always kept drawn
to protect the carpet), but, as she explained it afterward, she “felt” that there
was some one in the room. Peering into the darkness, she heard a faint
sound of snoring, and, as her eyes grew accustomed to the darkness, she
discerned the figure of a man lying on her best sofa, with his feet resting on
the arm. He was sleeping with his mouth open a little way beneath a black
mustache, waxed and curled with the care of a dandy.
As she stood there in the midst of the room, the figure in the shadows
took form slowly, and suddenly she knew it ... the dapper, small body,
dressed so dudishly, the yellow waistcoat with its enormous gold watch-
chain, and cluster of seals. She knew, with a sudden pang, even the small,
well-shaped hand, uncalloused by any toil, that lay peacefully at rest on the
Brussels carpet. For a second she thought, “I’ve gone suddenly crazy from
all the trouble I’ve had. What I’m seeing can’t be true.”
It took a great deal of courage for her to move toward the sofa, for it
meant moving in an instant, not simply across the Brussels carpet, but
across the desert of twenty-six years. It meant giving up Moses Slade and
all that resplendent future which had been taking form in her mind only a
moment before. It was like waking the dead from the shadows of the
tomblike parlor.
She did not lack courage, Emma; or perhaps it was not courage, but the
headlong thrust of an immense vitality which now possessed her. She went
over to the sofa and said, “Jason! Jason Downes!” He did not stir, and
suddenly the strange thought came to her that he might be dead. The wicked
idea threw her into an immense confusion, for she did not know whether
she preferred the unstable companionship of the fascinating Jason to the
bright future that would be hers as the wife of Moses. Then, all at once, she
saw that the gaudy watch-chain was moving up and down slowly as he
breathed, and she was smitten abruptly by memories twenty-six years old of
morning after morning when she had wakened, full of energy, to find Jason
lying beside her sleeping in the same profound, conscienceless slumber.
“Jason!” she said again. “Jason Downes!” And this time there was a
curious tenderness in her voice that was almost a sob.
He did not stir, and she touched his shoulder. He moved slowly, and
then, opening his eyes, sat up and put his feet on the floor. He awakened
lazily, and for a moment he simply sat staring at her, looking as neat and
dapper as if he had just finished an elaborate toilet. Again memory smote
Emma. He had always been like this: he had always wakened in the
mornings, looking fresh and neat, with every hair in place. It was that hair-
oil he persisted in using. Now that he’d come home, she would have to get
antimacassars to protect the furniture against Jason’s oily head.
Suddenly he grinned and said, “Why! Hello! It’s you, Em.” It wasn’t a
sheepish grin, but a smile of cocky assurance, such as was frozen forever
upon the face of the enlarged portrait.
“Jason ... Jason! Oh, my God! Jason!” She collapsed suddenly and fell
into the mahogany-veneer rocker. It was a strange Emma, less strange
perhaps to Jason Downes than she would have been to the world outside,
for suddenly she had become all soft and collapsed and feminine. All those
twenty-six years had rolled away, leaving her helpless.
As if he had left the house only that morning, he sat on the arm of the
chair and kissed her. He patted her hands and said, “You mustn’t cry like
that, Em. I can’t bear to hear you. It breaks me all up.”
“If you knew how long I’d waited!” she sobbed. “Why didn’t you even
write? Why didn’t you tell me you were coming?”
He seemed a little proud of himself. “I wanted it to be a surprise.”
He led her to the sofa and sat there, patting her hand and smiling, and
comforting her while she wept and wept. “A surprise,” she echoed. “A
surprise ... after twenty-six years....” After a time she grew more calm, and
suddenly she began to laugh. She kept saying at little intervals, “If you
knew how I’ve waited!”
“I’m rich now, Emma,” he said with the shadow of a swagger. “I’ve
done well out there.”
“Out where ... Jason?”
“Out in Australia ... where I went.”
“You were in Australia?” He wasn’t in China at all, then. The story was
so old that she had come to believe it, and with a sudden shock of horror
she saw that they would now have to face the ancient lie. He hadn’t been in
China, and he hadn’t been killed by bandits. Here he was back again, and
you couldn’t keep a man like Jason shut up forever in the house. The Town
would see him. She began once more to cry.
“There, there, Em!” he said, patting her hand again, almost amorously.
“Don’t take it so hard. You’re glad I did come back, ain’t you?”
“I don’t know ... I don’t know. You don’t deserve anything ... even tears
... after treating a wife the way you’ve treated me. Don’t think I’m crying
because I’m glad you’re back. It’s not that. I ought to turn you out. I’d do it,
too, if I was an ordinary woman.”
She saw then that she still had to manage everything, including Jason.
She saw that he was as useless as he had always been. She would have to
“take hold.” The feminine softness melted away, and, sitting up, she blew
her nose and said, “It’s like this, Jason. When you went away, I said you’d
gone to China on business. And when you didn’t come back, I said I hadn’t
had any letters from you and something must be wrong. You see I pretended
I heard from you regularly because ... I wanted to protect you and because I
was ashamed. I didn’t want people to think you’d deserted me after
everybody had warned me against you. And so Elmer....”
“And how’s he?” said Jason. “Cold boiled mutton, I call ’im.”
“Wait till I finish my story, Jason. Try to keep your mind on what I’m
saying. And so Elmer set the Government to investigating....”
“They were looking for me? The United States Government itself?”
There was in his voice and manner a sudden note of gratification at his
importance.
“Yes ... they hunted all over China.”
Jason was grinning now. “It’s lucky they was looking in China, because I
was in Australia all the time.”
“And they said you must have been killed by bandits ... so I put on black
and set out to support myself and Philip.”
“Why didn’t old pious Elmer help you out? I wouldn’t have gone away,
except that I knew ’e was rich enough to look out for you.”
“Elmer’s tight, and besides I didn’t want him to be pitying me and
saying, ‘I told you so’ every time I asked him for a cent.”
“And Philip? You haven’t told me about him yet.”
“We’ll come to him. We’ve got to settle this other thing first. You see,
Jason, we’ve got to do something about that lie I told ... it wasn’t really a lie
because I told it for your sake and Philip’s—to protect you both.”
“Yes, it is kind-a awkward.” He sat for a moment, trying to bring his
volatile mind into profitable operation. At last he said, “You oughtn’t to
have told that lie, Em.”
“I told you why I told it. God will understand me if no one else will.”
 “Now, Em, don’t begin on that line.... It was always the line I couldn’t
stand.... You ain’t no bleedin’ martyr.”
She looked at him with a sudden suspicion. “Jason, where did you pick
up this queer talk ... all the queer words you’ve been using?”
“Australia, I guess ... living out among the cockneys out there.” He rose
suddenly. “Em, I can’t sit any more in this dark. I can’t think in a tomb.” He
went over and drew up the window-shades. As the fading winter light filled
the room, he looked around him. “Why, it ain’t changed at all! Just the same
... wedding parlor suite and everything.” His glance fell on the wall above
the fireplace. “And you still got my picture, Em. That was good of you.”
She showed signs of sobbing again. “It’s all I had....”
He was looking at the picture with a hypnotic fascination. “It’s funny, I
ain’t changed much. You’d never think that picture was taken twenty-six
years ago.” He took out a pocket mirror and began comparing his features
with those in the enlarged photograph. What he said was true enough. Time
had left no marks on the smooth, good-looking face, nor even on a mind
that was like a shining, darting minnow. He was as slim and dapper as ever.
The hair was much thinner, but it was still dark, and with the aid of grease
and shrewd manipulation you couldn’t tell that he was really bald. Emma,
watching him, had an awful suspicion that it was dyed as well; and the
elegant mustaches too. She would be certain to discover, now that he had
come back to share the same room and bed. She had a sudden, awful fear
that she must look much older than he.
“I’m a little bald,” he said ruefully, “but nothing very much.”
“Jason,” she said sternly. “Jason ... we’ve got to settle this thing ... now
... before we do anything else. Did any one see you?”
“No, I don’t think so.” He replaced the pocket mirror with a mild, comic
air of alarm at the old note of authority in her voice.
“You must think of something ... you’re better at such things than I am.”
He had, she remembered, the proper kind of an imagination. She knew from
experience how it had worked long ago when he had given her excuses for
his behavior.
He looked at her with an absurd air of helplessness. “What can we say? I
suppose you could say I lost my memory ... that I got hit on the head.”
Suddenly a great light burst upon the empty face. “I did get a fall on the
steamer going out. I fell down a stairway and for three days I didn’t know a
thing. A fall like that might easily make you lose your memory.... A thing
like that might happen.” As if the possibilities of such a tale had suddenly
dawned upon him, his face became illumined with that look which must
come at times into the faces of great creative artists. He said, “Yes, I might
have lost my memory, not knowing who I was, or where I came from, and
then, after twenty-six years, I got another fall ... how?... well out of the
mow on my ranch in Australia, and when I came to, I remembered
everything—that I had a wife in America. It’s true—it might happen. I’ve
read of such things.”
Listening to him, Emma felt the story seemed too preposterous, and yet
she knew that only heroic measures could save the situation. The bolder the
tale, the better. It was, as he said, a story that might be true. Such things had
happened. She could trust him, too, to make the tale a convincing one: the
only danger lay in the possibility of his doing it too well. It occurred to her
in the midst of her desperate planning that it was strange what wild,
incredible things had happened in her life ... a life devoted always to hard
work and Christian living.
Jason’s glittering mind had been working rapidly. He was saying, “You
see, there’s the scar and everything.” He bent down, exposing the bald spot
that was the only sign of his decay. “You see, there it is—the scar.”
She looked at him scornfully, for the crisis of her emotion had passed
now, and she was beginning to feel herself once more. “Now, Jason,” she
said, “I haven’t forgotten where that scar came from. You’ve always had it.
You got it in Hennessey’s saloon.”
For a second the dash went out of him. “Now, Em, you’re not going to
begin on that, the minute I get home.” And then quickly his imagination set
to work again, and with an air of brightness, as if the solution he had
thought of vindicated him completely, he said, “Besides I wasn’t bald in
those days and nobody ever saw the scar. And the funny thing is that it was
on that exact spot that I fell on the boat. It enlarged the scar.” He looked at
her in the way he had always done when he meant to turn her mind into
more amiable channels. “Now, isn’t that queer? It enlarged the scar.”
It was clear that she meant not to be diverted from the business at hand.
“I suppose that’s as good a story as any. We’ve got to have a story of some
kind. But you must stick to it, Jason, and don’t make it too good. That’s
what you always do ... make it too good.” (Hadn’t she, years ago, trapped
him time after time in a lie, because he could not resist a too elaborate
pattern of embroidery?)
She said, “But there’s one thing I’ve got to do right away, and that is
send word to Naomi to tell Philip.”
“Who’s Naomi?”
“She’s Philip’s wife.”
“He’s married?”
“He’s been married for five years.”
He made a clucking sound. “We’re getting on, Em.”
“And there’s more than that. You’re a grandfather.”
The smooth face wrinkled into a rueful expression. “It’s hard to think of
myself as a grandfather. How old is the child, or the children?”
“They’re twins.”
He chuckled. “He did a good job, Philip.”
“Now, Jason....”
“All right, but how old are they?”
“Four months ... nearly five.”
“I must say that Philip took ’is time about it. Married five years.... Well,
we didn’t waste any time, did we, Em.”
“Jason!”
She hated him when he was vulgar. She decided not to go into the
reasons why Philip and Naomi had been married four years without
children, because it was a thing which Jason wouldn’t understand—
sacrificing the chance of children to devote yourself to God. There was
nothing spiritual about Jason. It was one of his countless faults.
“But who did ’e marry, Em? You haven’t told me.”
“Her name was Naomi Potts. You wouldn’t know who she was. Her
people were missionaries, and she was a missionary too.”
“Oh, my God!”
“I won’t have you blaspheming.”
“And what’s Philip like?”
“He was a missionary too.... He was three years in Africa ... until his
health broke.”
 “Oh, my God!” He grew suddenly thoughtful, moved perhaps by the
suspicion that she had succeeded in doing to his son what she had failed to
do to him.
She was at the door now. “I won’t listen to you talking like that any
longer.” She turned in the doorway. “Don’t go out till I come back. You
mustn’t be seen till we’ve worked this thing out. I’ve got to send word to
them all.”
When she had gone, he picked up his hat, took a cigar from his vest
pocket and lighted it. In the hallway, he shouted at her, “Are we still using
the same room, Em? I’ll just move in my things and wash up a bit.”
In the sitting-room Emma sat down and wrote three notes—one to
Naomi, one to Mabelle, and the third to Moses Slade. With a trembling
hand she wrote to him, “God has sent Jason, my husband, back to me. He
came to-day. It is His will that we are not to marry. Your heartbroken
Emma.”
She summoned the slattern Essie, and, giving her instructions of a
violence calculated to impress Essie’s feeble mind, she bade her deliver the
three notes, Mr. Slade’s first of all. But once outside the sight of Emma, the
hired girl had her own ideas of the order in which she meant to deliver
them, and so the note to Moses Slade arrived last. But it made no
difference, as the Honorable Mr. Slade, bearing a copy of the Labor
Journal, was at the same moment on his way to Emma’s to break off the
engagement, for he had discovered the author of the libelous drawings. The
latest one was signed boldly with the name, “Philip Downes.” He never
arrived at Emma’s house, for on his way he heard in Smollett’s Cigar Store
that Jason Downes had returned, and so he saved himself the trouble of an
unpleasant interview. For Essie, in the moment after the returned prodigal
had made known to her his identity, had put on a cast-off hat of Emma’s and
set out at once to spread the exciting news through the Town.
When she returned at last from delivering the three notes, Emma was
“getting Jason settled” in the bedroom he had left twenty-six years before.
Essie, tempted, fell, and, listening outside the door, heard him recounting to
his wife a wonderful story of having lost his memory for a quarter of a
century. But one thing tormented the brain of the slattern Essie. She could
not understand how Emma seemed to know the whole story and to put in a
word now and then correcting him.
 At the sound of Emma’s footsteps approaching the door, Essie turned
and, fleeing, hid in the hall closet, from which she risked her whole future
by opening the door a little way to have a look at the fascinating Mr.
Downes. Her heart thumped wildly under her cotton blouse at the proximity
of so romantic a figure.

15
It seemed that something in the spirit of the irrepressible Jason Downes
took possession of the house, for Emma turned almost gay, and at times
betrayed signs of an ancient coquetry (almost buried beneath so many
hardening years) in an actual tendency to bridle. For the first time since
Jason had slipped quietly out of the back door, the sallow dining-room was
enlivened by the odors, the sounds, the air of banqueting: a dinner was held
that very night to celebrate the prodigal’s return. Elmer came, goaded by an
overpowering curiosity, and Mabelle, separated for once from Jimmy, her
round, blue eyes dilated with excitement and colored by that faintly bawdy
look which so disturbed Emma. And Philip was there, of course, and
Naomi, paler than usual, dressed in a badly fitting new foulard dress, which
she and Mabelle had “run up at home” in the hope of pleasing Philip. The
dress had been saved for an “occasion.” They had worked over it for ten
days in profound secrecy, keeping it to dazzle Philip. It was thick about the
waist, and did not hang properly in the back, and it made her look all lumpy
in the wrong places. In case Philip did not notice it, Mabelle was to say to
him, “You haven’t spoken about Naomi’s pretty new dress. She made it all
herself—with her own hands.” They had carefully rehearsed the little plot
born of Mabelle’s romantic brain.
But when Naomi arrived at the slate-colored house, she took Mabelle
quickly into a corner and said, “Don’t speak of the dress to him.” And when
Mabelle asked, “Why not?” she only answered, “You can do it later, but not
to-night. I can’t explain why just now.”
She couldn’t explain to Mabelle that she was ashamed of the dress, nor
why she was ashamed of it. She couldn’t say that as she stood on the stairs
of the stable and saw a handsome woman, in a plain black dress, with her
knees crossed, and furs thrown back over her fine shoulders, that the pride
of the poor little foulard dress had turned to ashes. She couldn’t explain
how she had become suddenly sick at the understanding that she must seem
dowdy and ridiculous, standing there, all red and hot and disheveled, staring
at them, and wanting all the time to turn and run, anywhere, on and on,
without stopping. She couldn’t explain how the sight of the other woman
had made the foulard dress seem poor and frowzy, even when she put on the
coral beads left her by her mother, and pinned on the little gold fleur-de-lys
watch her father had given her.
When she first arrived, she kept on her coat, pretending that the house
was cold, but Emma said, “It’s nonsense, Naomi. The house is warm
enough,” and the irrepressible Mabelle echoed, “That’s what I say, Emma.
She ought to take it off and show her pretty new dress.”
Naomi had looked quickly about her, but Philip hadn’t been listening. He
was standing with Uncle Elmer beside his father, who was in high spirits,
talking and talking. He wouldn’t notice the dress if only she could keep
people from speaking of it.
She hadn’t spoken of Lily Shane to Philip. All the way back to the flat by
the railroad they had talked of nothing but his father and the poor bits of
information she had been able to wring from the excited Essie; and when
they arrived it was to find Mabelle waiting breathlessly to discuss it with
them. She had been already to the slate-colored house and seen him with
her own eyes. She didn’t stay long (she said) because she felt as if she were
intruding on honeymooners. Did they know that he had lost his memory by
a fall on the boat going out to China, and that it had only come back to him
when he had a fall six months ago out of the mow on his ranch in Australia?
Yes, it was Australia he had been to all this time....
She went on and on. “Think of it,” she said. “The excitement of
welcoming home a husband you hadn’t seen in twenty-six years ... like a
return from the dead. I don’t wonder your Ma is beside herself.”
Naomi heard it all, dimly, as if all Mabelle’s chatter came to her from a
great distance. She should have been excited, but she couldn’t be, with
something that was like a dull pain in her body. She could only keep seeing
Lily Shane, who made her feel tiny and miserable and ridiculous—Lily
Shane, whom Philip said he didn’t even know, and had never spoken to. Yet
he knew her well enough to be making a picture of her. He never thought of
making a picture of his own wife.
She felt sick, for it was the first time she had ever seen herself. She
seemed to see at a great distance a pale, thin, freckled woman, with sandy
hair, dressed in funny clothes.
And then she would hear Mabelle saying through a fog, “Your Ma wants
you to come right up to supper. You can get Mrs. Stimson—the druggist’s
wife—to sit with the twins.”
Mabelle hurried off presently, and Mrs. Stimson came in duly to sit with
the twins. She gave up the evening at her euchre club because the
excitement of sitting up with the grandchildren of a man who had returned
after being thought dead for twenty-six years was not to be overlooked. She
would hear all the story at first hand when Philip and Naomi returned,
before any one else in the Town had heard it. She could say, “I sat with the
twins so that Philip and Naomi could go to supper with Mr. Downes
himself. I heard the whole thing from them.”
As they went up the hill to the slate-colored house, Naomi said nothing,
and so they walked in silence. She had begun to understand a little Philip’s
queer moods, and she knew now that he was nervous and irritable. She had
watched him so closely of late that she had become aware of a queer sense
of strain which once she had passed over unnoticed. She had learned not to
speak when Philip was like that. And as they climbed the hill, the silence,
the strain, seemed to become unbearable. It was Philip who broke it by
crying out suddenly, “I know what you’re thinking. You’re thinking I lied to
you about Lily Shane. Well, I didn’t. Before God, I never spoke to her until
to-day, and I wouldn’t have, even then, but she came to my room without
my asking her.”
For a moment, she wanted to lie down in the snow and, burying her face
in it, cry and cry. She managed to say, “I wasn’t even thinking of her.
Honestly I wasn’t, Philip. And I believe you.”
“If that’s so, why do you sulk and not say anything?”
“I wasn’t sulking. I only thought you didn’t want to talk just now.”
“I hate it when you act like a martyr.” This time she was silent, and he
added, “I suppose all women do it ... or most women ... it’s what Ma does
when she wants to get her way. I hate it.”
She thought, “He said ‘most women’ because he meant all women but
Lily Shane.” But she was silent. They did not speak again until they reached
the slate-colored house.
It wasn’t really Naomi who lay at the bottom of his irritation, but the
thought of his father. The return troubled him. Why should he have come
now after twenty-six years? It was, he thought, almost indecent and unfair,
in a way, to his mother. He tried, when he was not talking to Naomi, to
imagine what he must be like—a man who Emma said had gone out to
China to make money for his wife and child, a man who adored her and
worshipped his son. He was troubled, because the moral image created by
his mother seemed not to fit the enlarged, physical portrait in the parlor. In
these last years he had come to learn a lot about the world and about people,
and one of the things he had learned was that people are like their faces. His
mother was like her large, rather coarse and energetic face; Naomi was like
her pale, weak one; and Lily Shane and Mary and Uncle Elmer and even
Krylenko and McTavish were like theirs. It was impossible to escape your
own face. His father, he thought, couldn’t escape that face that hung in the
parlor.
When the door opened and he stepped into the parlor, he saw that his
father hadn’t escaped his face. He felt, with a sudden sensation of sickness,
that his father was even worse than his face. It was the same, only a little
older, and the outlines had grown somehow dim and vague from weakness
and self-indulgence. Why, he thought again, did he ever come back?
But his mother was happy again. Any one could see that.
And then his father turned and looked at him. For a moment he stared,
astonished by something in the face of his son, something which he himself
could not perhaps define, but something which, with all the sharp instincts
of a sensual nature he recognized as strange, which had little to do with
either himself or Emma. And then, perhaps because the astonishment had
upset him, the meeting fell flat. The exuberance flowed out of Jason
Downes. It was almost as if he were afraid of his son—this son who, unlike
either himself or Emma, was capable of tragedy and suffering. His eyes
turned aside from the burning eyes of his son.
“Well, Philip,” he said, with a wild effort at hilarity, “here’s your Pa ...
back again.”
Philip shook hands with him, and then a silence fell between them.
But it was Jason Downes who dominated the family gathering. Philip,
silent, watched his father’s spirits mounting. It seemed to him that Jason
had set himself deliberately to triumph over his dour, forbidding brother-in-
law, and to impress his own son. It was as if he felt that his son had a poor
opinion of him, and meant to prove that he was wrong in his judgment.
 He told the whole story of the voyage out, of his fall down a
companionway, and the strange darkness that followed. Once more he
bowed his head and exhibited the scar.
“But,” said the skeptical Elmer sourly, “you always had that scar, Jason.
You got it falling on the ice at the front gate.”
“Oh, no. The one before was only a small one. The funny thing was that
I struck my head in exactly the same place. Wasn’t that queer? And then
when I fell out of the mow I hit it a third time. That’s what the doctors in
Sydney said made it so serious.” For a moment, conscious that the
embroidering had begun, Emma looked troubled and uneasy.
And Mabelle, with a look of profound speculation, asked, “And what if
you hit it a fourth time? Would that make you lose your memory about
Australia?”
Jason coughed and looked at her sharply, and then said, “Well, no one
could say about that. If it happened again, it would probably kill me.”
“Well,” said Mabelle, “I must say I never heard a more interesting story
... I never read as interesting a one in any of the magazines ... not even in
the Ladies’ Home Journal.”
For a moment Philip wanted to laugh at Mabelle’s question, but it wasn’t
a natural desire to laugh: it sprang from a blend of anger and hysterics. He
loathed the whole party, with Mabelle and her half-witted questions, his
mother with all her character gone in the silly blind admiration for her
husband, Uncle Elmer and his nasty, mean questions, and Naomi, silent,
and looking as if she were going to cry. (If only she wouldn’t sulk and play
the martyr!) And Mabelle’s half-witted questions were worse than Uncle
Elmer’s cynical remarks, for they made him see suddenly that his father
was lying. He was creating a whole story that wasn’t true, and he was
enjoying himself immensely. If it was a lie, if he had deliberately deserted
his wife and child, why had he come back now?
Jason went on and on, talking, talking, talking. He told of his ranch of
eighteen hundred acres and of the thousands of sheep he owned and of the
sixty herders employed to take care of them. He described the long drouths
that sometimes afflicted them, and told a great deal about Melbourne and
Sydney.
“Your Pa,” he said, addressing Philip, “is an important man out there.”
And the implication was, “You don’t think much of him, but you ought to
see him in Australia.”
But Philip was silent, and thought, “He’s probably lying about that, too,”
and, as the conversation went on, he thought, “He’s never said anything
about women out there. He’s never spoken about that side of his life, and
he’s not the kind to leave women alone.”
“And I suppose you’ll be wanting to take Emma back to Australia,” said
Uncle Elmer, regarding Jason over his steel-rimmed spectacles.
“No ... I won’t be doing that. After all, her life is here, ain’t it? I shall
have to go back from time to time to look after my affairs, but....”
“Don’t speak of that now,” Emma interrupted, “when you’ve only just
arrived.”
“But we have to face these things,” said Jason.
Suddenly Emma turned away from the table to the doorway where Essie,
in terror of interrupting the party, yet fascinated still by the spell of Jason’s
narrative, stood waiting. She was standing, as she always stood, on the sides
of her shoes.
“What is it, Essie? What are you standing there for?”
“There’s a man come to see Mr. Downes.”
“What does he want?”
“He’s from the newspaper.”
“Tell him to come back to-morrow.”
But Jason had overheard. He rose with the napkin still tucked into the
fawn-colored vest. “No, Essie.... Tell him I’ll speak to him now.”
“But, Jason....”
“Yes, Em.... I might as well get it over.”
There was no holding him now; but Emma succeeded in thrusting
forward a word of advice.
“Remember, Jason, what the newspapers are like. Don’t tell them too
much.”
A shadow crossed her face, and Philip thought suddenly, “Ma knows
he’s lying too, and she’s afraid he’ll overdo it.” And then a more fantastic
thought occurred to him—that she knew for a good reason that he was
lying, that perhaps she had planned the lie to cover up an earlier one.
 “I must say it’s all very remarkable ... how Jason’s affairs have turned
out,” said Elmer. “I never would have thought it.”
“You never believed in him,” said Emma, with an air of triumph, “and
now you see.”
To Philip the whole room, the table, the people about it, the figure of the
slattern Essie standing in the doorway, all their petty boasting and piety and
lying, became suddenly vulgar and loathsome. And then, almost at once, he
became ashamed of himself for being ashamed, for they were his people.
He had no others. It was a subtle, sickening sort of torture.

16
Emma was herself forced to go in at last and send away the newspaper
man, for Jason would have kept him there the rest of the night, telling a
story which became more and more embroidered with each rash recounting.
And when, at last, the reporter had gone, the others came in and sat about
while Jason continued his talk. But the evening died slowly, perhaps
because of Elmer’s suspicions, or Naomi’s curious depression, or Philip’s
own disgust and low spirits. Jason found himself talking presently against a
curious, foreboding silence, of which he took no notice. Only Emma and
Mabelle were still listening.
It was Elmer who at last broke up the party, pushing the rotund and
breathless Mabelle before him. In the door Mabelle turned, and, shaking her
head a little coquettishly, said, “Well, good-night, Jason. Good-night,
Emma. I feel like I was saying ‘good-night’ to a honeymoon couple.” And
the bawdy look came into her eyes. “There’ll never be any second
honeymoon for Elmer and me. We’ve got our family now and that’s all
done.”
Still tittering, she was dragged off by her husband. When she had gone,
Jason said, “Mabelle is a cute one, ain’t she, and a funny one too, to be
married to a mausoleum like Elmer.”
“Now, Jason, it’s all patched up between you and Elmer. There’s no use
beginning all over again.”
Naomi and Philip had put on their wraps, and were standing by the door,
when Jason suddenly slapped his son on the back. “We’ve got to get better
acquainted, son. You’ll like your Pa when you know him better. Nobody
can resist him.” He winked at Emma, who turned crimson. “Ain’t it so, Em.
Least of all, the ladies.” And then to Philip again, “I’ll come and see you in
the morning.”
Philip turned quickly. “No, I’ll come and fetch you myself. You wouldn’t
find the way.”
“I want to see the twins the first thing.”
“I’ll come for you.”
He had resolved that his father was not to come to the stable. He saw
that Emma hadn’t even told his father that he wasn’t living with his wife.
The stable had suddenly become to him a kind of temple, a place dedicated
to that part of him which had escaped. There were things there which his
father wouldn’t understand, and could only defile. The stable belonged to
him alone. It was apart from all the others—his father, his mother, Naomi,
Uncle Elmer and Aunt Mabelle.
Emma was standing before Naomi, holding her coat open, so that she
might examine the dress underneath. She was saying, “You must come up
some afternoon, Naomi, and I’ll help you make the dress right. It hangs all
wrong at the back, and it’s all bunchy around the armholes. You could make
it all right, but, as it is, it’s ... it’s sort of funny-looking.”
All the way back to the Flats neither of them spoke at all: Philip, because
there was a black anger and rebellion burning in him, and Naomi, because if
she had tried to speak, she would have wept. She felt as though she were
dead, as if in a world made up of Philip and his father and Emma she no
longer had any existence. She was only a burden who annoyed them all.
And the dress ... it was only sort of “funny-looking.”
He left Naomi at the door of the flat with an abrupt “good-night.” It was
after midnight, and the moon was rising behind the hill crowned by Shane’s
Castle, throwing a blue light on the mist that hung above the Flats. In the far
distance the mist was all rosy with the light from four new furnaces that had
begun once more to work. The strike was slipping slowly into defeat, and
he understood that it meant nothing to him any longer. He had almost
forgotten Krylenko.
As he passed through the rusted gates of the park, there drifted toward
him from among the trunks of the dead trees, a faint, pungent odor that was
hauntingly familiar and, as he climbed the drive between the dead trees, it
grew stronger and stronger, until at last he recognized, in a sudden flash of
memory which brought back all the hot panorama of the lake and the forest
at Megambo, that it was the smell of gunpowder, the smell that clung to his
rifle when he had stood there by the barricade beside Lady Millicent killing
those poor niggers. It was a faint, ghostly smell that sometimes died away
altogether and sometimes came in strong waves on a warm breeze filled
with the dampness of the melting snow.
At the top of the hill, the big house lay dead and blind, without a sign of
life, and, as he turned the corner, he saw that near the stable lay the
remnants of a fire which had burnt to a heap of embers. His foot touched
something that was wet and slippery. He looked down to discover a great
stain of black on the snow. For a moment he stared at the stain, fascinated,
and suddenly he knew what it was. It was a great stain of blood.
In the distance, among the trees, he discerned a light, and after a moment
he discovered a little group of men ... three or four ... carrying a lantern,
which they held high from time to time, as if searching for something. And
then, all at once, as he moved forward again, he almost stepped upon a
woman who lay in the snow at the entrance to the rotting arbor covered
with the vines of the dying wistaria. She lay face down with one arm above
her head in a posture that filled him for a moment with a sense of having
lived through this same experience before, of having seen this same woman
lying face down ... dead ... for she was unmistakably dead. He knelt beside
her, and, turning the body on its side, he remembered suddenly. She lay like
the black virgin they had found dead across the path in the tall grass at
Megambo ... the one they had left to the leopards.
Trembling, he peered at the white face in the moonlight. The woman was
young, and across one side of the face there was a little trickle of blood that
came from a hole in the temple. She was dressed in rags, and her feet were
wrapped in rolls of sacking. She was the wife or daughter of some striker. It
occurred to him suddenly that there was something pitifully lonely in the
sight of the body left there, forgotten, by the embers in the dead park; it had
the strangest effect upon him. He rose and tried to call to the little group of
searchers, but no sound came from his throat, and he began suddenly to cry.
Leaning against one of the pillars of the arbor, he waited until his body had
ceased to tremble. It was a strange, confused feeling, as if the whole
spectacle of humanity were suddenly revealed in all its pathos, its
meanness, its grandeur, and its cruelty. It was a brilliant flash of
understanding, but it passed almost at once, leaving him weak and sick. And
then, after a moment, he found his voice again, and shouted. The little party
halted, and looked about, and he shouted a second time. Then they came
toward him, and he saw that two of them carried shotguns and that one of
them was McTavish.
The woman was dead. They picked her up and laid her carefully on one
of the blackened marble benches of the garden, and McTavish told him
what had happened. In the Town they had forbidden the strikers to hold
meetings, hoping thus to break the strike, but the Shanes, Irene and Lily (for
the old woman was dead), had sent word to Krylenko that they might meet
in the dead park. And so the remnant of those who had held out in the face
of cold and starvation had come here to listen to Krylenko harangue them
from a barrel by the light of a great fire before the stables. There had been
shouting and disorder, and then some one inside the Mill barrier—one of
the hooligans (they hadn’t yet discovered who did it) turned a machinegun
on the mob around the fire. It had only lasted an instant—the sharp, vicious,
staccato sound, but it had taken its toll.
“It’s a dirty business,” concluded McTavish in disgust. He wasn’t jolly
to-night. All the old, cynical good-humor had gone out of him, as if he, too,
had seen what Philip saw in that sudden flash as he leaned against the
decaying arbor.
They took a shutter from the windows of the stable and, placing the body
of the girl upon it, set off down the hill between the dead walls of the pine-
trees. For a long time Philip stood in the soiled, trampled snow, looking
after them, until a turn in the drive hid the lantern from view behind the
pine-trees.

17
The room above the stable was in darkness, but as he came up out of the
staircase he saw that there was a woman sitting by the window, silhouetted
against the moonlight beyond. He thought, “It must be Lily Shane, but why
is she here at this hour of the night?” And then a low, familiar voice came
out of the darkness, “It’s only me, Philip ... Mary.” She spoke as if he must
have known she was there, waiting for him.
He struck a match quickly and lighted the kerosene lamp, at which she
rose and came over to him. By the flickering, yellow light he saw that she
had been crying.
“It’s been horrible, Philip. I saw it all from the window while I was
waiting for you.”
“I know ... we just found a dead woman in the snow.”
He was possessed by a curious feeling of numbness, in which Mary
seemed to share, as if the horror of what had taken place outside wiped out
all the strangeness of their meeting thus. Death, it seemed, had brushed by
them so closely that it had swept away all but those things which lay at the
foundation of existence—the fact that they loved each other, that they were
together now, and that nothing else was of any importance. They were, too,
like people stunned by horror. They sat by the stove, Philip in silence, while
Mary told him what she had seen. For a long time it did not even appear
strange to him that she should be there in his room at two o’clock in the
morning.
He heard her saying, “Who was the woman they killed?”
“I don’t know. She looked Italian.”
There was a long silence and at last it was Mary, the practical Mary, who
spoke. “You must wonder why I came here, Philip ... after ... after not
seeing you at all for all this time.”
He looked at her slowly, as if half-asleep. “I don’t know. I hadn’t even
thought of it, Mary ... anything seems possible to-night, anything seems
possible in this queer park.” And then, stirring himself, he reached across
the table and touched her hand. She did not draw it away, and the touch
gave him the strangest sense of a fathomless intimacy which went back and
back into their childhood, into the days when they had played together in
the tree-house. She had belonged to him always, only he had been stupid
never to have understood it. He could have spoken out once long ago. If
only he, the real Philip, had been born a little sooner, they would both have
been saved.
And then, suddenly, he knew why she had come, and he was frightened.
He said, “You heard about my father?”
She started a little, and said, “No.”
“He came back to-night. It was awful, Mary. If he’d only stayed away! If
he’d never have come back....”
 So he told her the whole story, even to his suspicion that his father was a
liar, and had deserted him and his mother twenty-six years before. He told
her of the long agony of the reunion, describing his father in detail. And at
the end, he said, “You see why I wish he’d never come back. You do see,
don’t you, Mary ... if he’d stayed away, I’d never have thought of him at all,
or at least only as my mother thought of him. But he isn’t like that at all. I
don’t see how she can take him back ... how she can bear to have him
about.”
She wanted to cry out, “Don’t you see, Philip? Don’t you see the kind of
woman she is? If you don’t see, nothing can save you. She’s worse than he
is, because he’s harmless.” But she only said quietly, “Perhaps she’s in love
with him. If that’s true, it explains anything.”
“Maybe it’s that. She must be in love with him.”
Mary thought, “Oh, Philip! If you’d only forget all the things that don’t
matter and just live, you’d be so much happier!” She wanted him to be
happy more than anything in the world. She would, she knew, do anything
at all to make him happy.
Presently she said, “She came to see me this afternoon, Philip ... your
mother. That’s why I’m here now. She said horrible things ... that weren’t
true at all. She said ... she said ... that I’d been living with you all along, and
she’d just found out about it. She said that I came here to meet you in the
stable. She’s hated me always ... just because I’ve always been fond of you.
She said I’d tried to steal you from her.”
For a moment he simply sat very still, staring at her. She felt his hand
grow cold and relax its grasp. At last he whispered, “She said that? She said
such things to you?”
“Yes ... I ran away from her in the end. It was the only thing I could do.”
Then all at once he fell on his knees and laid his head in her lap. She
heard him saying, “There’s nothing I can say, Mary. I didn’t think she’d do
a thing like that ... and now I know, I know what kind of a woman she is.
Oh, I’m so tired, Mary ... you don’t know how tired I am!”
She began to stroke his dark hair, and the sudden thought came to her
with horror that in her desire for vengeance upon Emma Downes, it was not
Emma she had hurt, but Philip.
He said, “You don’t know what it is, Mary,—for months now ... for years
even, I’ve been finding out bit by bit ... to have something gone that you’ve
always believed in, to have some one you loved destroyed bit by bit, in
spite of anything you can do. I tried and tried, but it was no good. And now
... I can’t hold out any more. I can’t do it ... I hate her ... but I can never let
her know it. I can never hurt her ... because she really loves me, and it’s true
what she says ... that she did everything for me. She fed and clothed me
herself with her own hands.”
Again Mary wanted to cry out, “She doesn’t love you. She doesn’t love
any one but herself!” and again she kept silent.
“And now it’s true ... what she said ... you’ve stolen me away from her,
Mary. She’s made it so. I’m through now ... I can’t go on trying any more.”
Still stroking his head, she thought, “He’s like a little boy. He’s never
grown up at all.” And she said, “I was so angry, Philip, that I came here. I
didn’t care what happened; I only thought, ‘If she thinks that’s the truth, it
might as well be, because she’ll tell about it as the truth.’ I didn’t care any
longer for anything but myself and you.”
His head stirred, and he looked up at her, seizing her hands. “Is that true,
Mary?” He kissed her hand suddenly.
“It’s true ... or why else should I be here, at this hour?” He was hopeless,
she thought: he didn’t live for a moment in reality.
He hadn’t even thought it queer of her to be sitting there in his room
long after midnight with his head on her knees. And suddenly she thought
again, “If I’m his mistress, I can save him from her altogether. Nothing else
can break it off forever.”
He was kissing her hands, and the kisses seemed to burn her. He was
saying, “Mary, I’ve loved you always, always ... since the first time I saw
you, but I only knew it when it was too late.”
“It isn’t too late, Philip. It isn’t too late.”
He was silent for a time, but she knew what he was thinking. He wasn’t
strong enough to take life into his own hands and bend it to his own will, or
perhaps it wasn’t a lack of strength, but only a colossal confusion that kept
him caught and lost in an immense and hopeless tangle. Until to-night she
hadn’t herself been strong enough to act, but now a kind of intoxicating
recklessness had seized her—the sober, sensible Mary Conyngham. She
meant to-night to take him and comfort him, to make them both, for a little
time, happy. To-morrow didn’t matter. It would have been better if there
were no to-morrow, if they could never wake at all.
 It was Philip who spoke first. After a long silence, he said in a whisper,
“I can’t do it, Mary ... I can’t. It isn’t only myself that matters. It’s you and
Naomi too. It isn’t her fault any more than mine.”
For a moment she wished wickedly that he had been a little more like
John Conyngham, and then almost at once she saw that it was his decency,
the very agony of his struggle, that made her love him so profoundly. And
she was afraid that he would think her wicked and brazen and fleshly. It was
a thing she couldn’t explain to him.
There were no words rich enough, strong enough, to make him
understand what it was that had brought her here. She had thought it all out,
sitting for hours there by the window, in the light of the rising moon. She
had felt life rushing past her. She was growing old with the passing of each
second. She had seen a man killed, and afterwards Philip had himself come
upon the body of a dead woman lying in the snow. Nothing mattered, save
that they come together. What happened to her was of no consequence.
Some terrible force, stronger than either of them, had meant them for each
other since the beginning, and to resist it, to fight against it unnaturally as
Philip was doing, seemed to her all at once a black and wicked sin.
He freed himself suddenly and stood up. “I can’t do it, Mary. I’ll go
away.... You can spend the night here and leave in the morning. No one in
the Town will know you haven’t spent the night at Shane’s Castle.”
“Where will you go?”
“I’ll go to the tents. I’ll be all right.”
She suddenly put her hand over her eyes, and, in a low voice, asked,
“And ... what’s to come after, Philip?”
“I don’t know ... I don’t know. I don’t know what I’m doing.”
“We can’t go on ... I can’t....”
“No ... I’d rather be dead.”
Suddenly, with a sob, she fell forward on the table, burying her face in
her hands. “You belong to your mother still, Philip ... you can’t shake off
the hard, wicked things she’s taught you. Oh, God! If she’d only died ...
we’d have been married to each other!”
She began to cry softly, and, at the sound, he stopped the mechanical
business of buttoning his coat, and then, almost as if he were speaking to
himself, he said, “Damn them all! We’ve a right to our happiness. They
can’t take it from us. They can’t....”
He raised her face from the table and kissed it again and again with a
kind of wild, rude passion that astonished her, until she lost herself
completely in its power. Suddenly he ceased, and, looking at her, said, “It
doesn’t matter if to-morrow never comes. I love you, Mary ... I love you.
That’s all that matters.”
They were happy then, for in love and in death all things are wiped out.
There, in the midst of the dead and frozen park, she set him free for a little
time.

18
The morning came quickly in a cold gray haze, for the furnaces, starting
to work one by one as the strike collapsed, had begun again to cover the
Flats with a canopy of smoke. It was Mary who went first, going by the
back drive, which led past the railway-station. And with her departure the
whole world turned dark. While she had been there with him, he was happy
with the sense of security that is born of companionship in adventure, but as
her figure faded presently into the smoke and mist that veiled the deserted
houses of the Flats, the enchantment of the night gave way to a cold, painful
sense of actuality. The whole night had been, as some nights are in the
course of lives that move passionately, unreal and charged with strange,
intangible currents of fire and ice. During that brief hour or two when he
had slept, years seemed to have passed. The figure of his father had become
so remote that he no longer seemed cheap and revolting, but only shallow
and pitiful. Even the memory of McTavish and the two men with the lantern
standing over the dead woman in the snow was dim now and unreal.
It was only the sight of the trampled, dirty snow, the black spot where
the fire had been and the pool of blood at the turn of the drive that made
him know how near had been all these things which had happened during
the night. And the park was no longer beautiful and haunted in the
moonlight, but only a dreary expanse of land filled with dead trees and
decaying arbors. The old doubts began slowly to torment him once more—
the feeling of terror lest Naomi should ever discover what had happened,
and the knowledge that he had betrayed her. There was, too, an odd new
fear that he might become such a man as his father. It was born in that cold,
gray light, of a sudden knowledge that deep inside him lay sleeping all the
weaknesses, all the sensuality, of such a man. After what had happened in
the night, he saw suddenly that he might come like his father to live in a
shallow world that shut out all else. He was afraid suddenly, and ashamed,
for he had been guilty of a sin which his father must have committed a
hundred times.
Yet he had, too, an odd new sense of peace, a soothing, physical, animal
sort of peace, that seemed to have had its beginnings months ago, in the
moments of delirium when he had wanted to live only because he could not
die without knowing such an experience as had come to him in the night. It
was, he supposed, Nature herself who had demanded this of him. And now
she had rewarded him with this sense of completeness. Nature, he thought,
had meant his children to be Mary’s children, too; and now that couldn’t be
... unless ... unless Naomi died.
It was a wicked thought that kept stealing back upon him. It lay in hiding
at the back of his mind, even in the last precious moments before Mary had
left, when she stood beside the stove making the coffee. He had thought
again and again, “If only Naomi died ... we could be like this forever.”
Watching her, he had thought, despite all his will to the contrary, of what
love had been with Naomi and what with Mary. And he had told himself
that it wasn’t fair to think such things, because he had never loved Naomi:
at such moments he had almost hated her. Yet she had loved him, and was
ashamed of her love, so that she made all their life together a sordid misery.
And Mary, who had been without shame, had surrounded her love with a
proud and reckless glory. Yet, in the end, it was Mary who hid, who stole
away through the black houses of the Flats as if she had done a shameful
thing, and it was Naomi who bore his children. For a moment he almost
hated the two helpless little creatures he had come so lately to love, because
a part of them was also a part of Naomi.
As he stood by the window, all wretched and tormented, he saw coming
across the trampled snow the battered figure of Hennery. He was coming
from the house, and his bent old figure seemed more feeble and ancient
than it had ever been before. He entered the stable, and Philip heard him
coming painfully up the stairs. At the sight of Philip, he started suddenly,
and said, “You scared me, Mr. Downes ... my nerves is all gone. I ain’t the
same since last night.” He took off his hat and began fumbling in his
pockets. “I got a letter for you ... that strike feller left it for you ... that ... I
doan’ know his name, but the feller that made all the trouble.”
He brought forth a piece of pale mauve paper that must have belonged to
Lily Shane, but was soiled now from contact with Hennery’s pocket.
“He was in the house all night,” said Hennery, “a-hiding there, I guess,
from the police, and he’s gone now.”
Then he was silent while Philip opened the note and read in the
powerful, sprawling hand of Krylenko:
 “I’ve had to clear out. If they caught me now, they’d frame something
and send me up. And I’m not through fighting yet. The strike’s bust, and
there’s no good in staying. But I’m coming back. I’ll write you from where I
go.
“Krylenko.”
He read it again and then he heard Hennery saying, “It was a turrible
night, Mr. Downes ... I guess it was one of those nights when all kinds of
slimy things are out walkin’. They’re up and gone too ... both of them ... the
girls, Miss Lily and Miss Irene. And they ain’t comin’ back, so Miss Lily
says. She went away, before it was light, on the New York flier. Oh, it was a
turrible night, Mr. Downes ... I’ve seen things happenin’ here for forty
years, but nothin’ like last night ... nothin’ ever.”
He began to moan and call on the Lord, and Philip remembered suddenly
that the half-finished drawing of Lily Shane had disappeared. She had
carried it off then, without a word. And slowly she again began to take
possession of his imagination. For a moment he tried to picture her house in
Paris where his drawing of her would be hung. She had gone away without
giving him another thought.
Hennery was saying over and over again, “It was a turrible night ...
something must-a happened in the house too. The Devil sure was on the
rampage.”
He stood there, staring out of the window, suffering from a curious, sick
feeling of having been deserted. “By what? By whom?” he asked himself.
“Not by Lily Shane, surely, on whom I had no claims ... whom I barely
knew.” Yet it was Lily Shane who had deserted him. It was as if she had
closed a door behind her, shutting him back into the world of Elmer and his
mother and Jason Downes. The thing he had glimpsed for a moment was
only an illusion....

19
When Hennery had gone off muttering to himself, Philip put on his coat
and went out, for the room had become suddenly unendurable to him. He
did not know why, but all at once he hated it, this room where he had been
happy for the first time since he was a child. It turned suddenly cold and
desolate and hauntingly empty. Running down the stairs, he hurried across
the soiled snow, avoiding the dark stain by the decaying arbor. He went by
that same instinct which always drove him when he was unhappy towards
the furnaces and the engines, and at Hennessey’s corner he turned toward
the district where the tents stood. They presented an odd, bedraggled
appearance now, still housing the remnant of workers who had fought to the
end, all that little army which had met the night before in the park of
Shane’s Castle. Here and there a deserted tent had collapsed in the dirty
snow. Piles of rubbish and filth cluttered the muddy field on every side.
Men, women and children stood in little groups, frightened and helpless and
bedraggled, all the spirit gone out of them. There was no more work for
them now. Wherever they went, no mill would take them in. They had no
homes, no money, no food....
Lost among them, he came presently to feel less lonely, for it was here
that he belonged—in this army of outcasts—a sort of pariah in the world
that should have been his own.
At the door of one of the tents, he recognized Sokoleff. The Ukranian
had let his beard grow and he held a child of two in his arms—a child with
great hollow eyes and blue lips. Sokoleff, who was always drunk and
laughing, was sober now, with a look of misery in his eyes. Philip shook his
free hand in silence, and then said, “You heard about Krylenko?”
“No, I ain’t heard nothin’. I’ve been waitin’ for him. I gotta tell him a
piece of bad news.”
“He’s gone away.”
“Where’s he gone?”
Philip told him, and, after a silence, Sokoleff said, “I suppose he had to
beat it. I suppose he had to ... but what are we gonna do ... the ones that’s
left. He’s the only one with a brain. The rest of us ain’t good for nothin’. We
ain’t even got money to get drunk on.”
“He won’t forget you.”
“Oh, it’s all right for him. He ain’t got nobody ... no children or a wife.
He ain’t even got a girl ... now.”
For a moment the single word “now,” added carelessly after a pause,
meant nothing to Philip, and then suddenly a terrible suspicion took
possession of him. He looked at Sokoleff. “What d’you mean ... now?”
 “Ain’t you heard it?”
“What?”
“It was his girl, Giulia ... that was killed last night.”
Philip felt sick. In a low voice he asked, “And he didn’t know it?”
“I was to tell him, but nobody’s seen him. I’m damned glad he’s went
away now. I won’t have the goddamned dirty job. He’ll be crazy ... crazy as
hell.”
And then Philip saw her again as he had seen her the night before, lying
face down in the snow ... Krylenko’s Giulia.
“She oughtn’t to have went up there,” Sokoleff was saying. “But she was
nuts on him ... she thought that he was the best guy on earth, and she
wanted to hear his speech....” The bearded Slovak spat into the snow. “I
guess that was the last thing she ever heard. She musta died happy.... That’s
better than livin’ like this.”
And Krylenko had been hiding in Shane’s Castle all night while Giulia
lay dead in the snow outside.
The sick baby began to cry, and Sokoleff stroked its bare head with a
calloused paw covered by black hair.
All at once Philip was happy again; even in the midst of all the misery
about him, he was gloriously, selfishly happy, because he knew that,
whatever happened, he had known what Krylenko had lost now forever. He
thought suddenly, “The jungle at Megambo was less cruel and savage than
this world about me.”

20
To Jason Downes the tragedy in the park of Shane’s Castle had only one
significance—that it tarnished all the glory of his astonishing return. When
the papers appeared in the morning, the first pages were filled with the
news of “the riot precipitated by strikers last night.” It recounted the death
of a Pole and of Giulia Rizzo, and announced triumphantly that the strike
was broken at last. And far back, among the advertisements of Peruna and
Lydia Pinkham’s Compound, there appeared a brief paragraph or two
announcing the return of Jason Downes, and touching upon the remarkable
story of his accident and consequent loss of memory. There were, doubtless,
people who never saw it at all.
 But he made the most of his return, walking the round of all the cigar-
stores and poker-rooms which he had haunted in his youth. He even went to
Hennessey’s saloon, beginning to thrive again on the money of the strike-
breakers. But he found no great triumph, for he discovered only one or two
men who had ever known him and to the others he was only Emma
Downes’ husband, whom they barely noticed in the excitement of
discussing the riots of the night before. Even his dudishness had dated
during those long twenty-six years: he must have heard the titters that went
up from poolroom loafers at the sight of the faun-colored vest, the waxed
mustaches and the tan derby. He was pushed aside at bars and thrust into the
corner in the poolrooms.
Half in desperation, he went at last to find an audience in the group of
old men who sat all day about the stove of McTavish’s undertaking-parlors.
They were old: they would remember who he was. But even there the
clamor of the tragedy drowned his tale. He found the place filled with
Italians—the father and the seven orphaned brothers and sisters of Giulia
Rizzo. The father wept and wrung his hands. The older children joined him,
and the four youngest huddled dumbly in a corner. It was Jason’s own son,
Philip, who was trying to quiet them. He nodded to his father, gave him a
sudden glance of contempt, and then disappeared with McTavish into the
back room where the undertaker had prepared Giulia for her last rest. For a
moment Jason hung about hopefully, and then, confused and depressed by
the ungoverned emotions of the Italians, he slipped out of the door, and up
the street toward the Peerless Restaurant. He was like a bedraggled bantam
rooster which had lost its proud tail-feathers, but as he approached the
restaurant he grew a bit more jaunty: there was always Em who thought him
wonderful....
Behind the partition of the undertaking-rooms, Philip and McTavish
stood looking down at Giulia. The blood had been washed away and her
face was white like marble against the dark coil of her hair. She was clothed
in a dress of black silk.
“It was her best dress,” said McTavish. “The old man brought it up here
this morning.”
Philip asked, “Are they going to bury her in the Potters Field? Old Rizzo
hasn’t got a cent, with all these children to feed.”
 “No, I’ve arranged that. I fixed it up with the priest. She had to be buried
in consecrated ground ... and ... and I bought enough for her. I ain’t got any
family, so I might as well spend my money on something.”

21
Philip saw his father at the restaurant, but there was little conversation
between them, and Emma kept talking about the riot of the night before,
observing that, “now that the police had tried something besides coddling a
lot of dirty foreigners, the strike was over in a hurry.”
At this remark, Philip rose quietly and went out without another word to
either of them. At home he found the druggist’s wife sitting with the twins.
Naomi, she said, was out. She had gone to see Mabelle. Mrs. Stimson
wanted more details of his father’s return, and also news of what had
happened at Shane’s Castle. After answering a dozen questions, he went
away quickly.
At four o’clock his father came and saw the twins, diddling them both on
his feet until they cried and Mrs. Stimson said, with the air of a snapping-
turtle, “I’m going to leave them with you. Naomi ought to have been home
two hours ago, and I’ve got a household of my own to look after.” (Even for
her poor Jason appeared to have lost his fascination.)
At seven when Philip came in to sit with the twins while Naomi went to
choir practice, he found little Naomi crying and his father asleep in the
Morris-chair by the gas stove. Jason had removed his collar and wrapped
himself in a blanket. With him, sleeping was simply a way of filling in time
between the high spots in existence: he slept when he was bored, and he
slept when he was forced to wait.
Holding the baby against him, and patting its back softly, Philip
approached his father and touched him with the toe of his shoe. “Pa!” he
said. “Pa! Wake up!”
Jason awakened with all the catlike reluctance of a sensual nature,
stretching himself and yawning and closing his eyes. He would have fallen
asleep a second time but for the insistence of Philip’s toe, the desperate
crying of the child, and Philip’s voice saying, “Wake up! Wake up!” There
was something in the very prodding of the toe which indicated a contempt
or at least a lack of respect. Jason noticed it and scowled.