Supplemental Information Survival Factor NFIL3 Restricts FOXO-induced Gene Expression in Cancer Megan Keniry 1 , Maira M. Pires 1,2 , Sarah Mense 2 , Celine Lefebvre 1,3 , Boyi Gan 4 , Karen Justiano 1 , Ying-Ka Ingar Lau 1 , Ben Hopkins 1 , Cindy Hodakoski 1,2 , Susan Koujak 1 , Joseph Toole 1 , Franklyn Fenton 1 , Ashley Calahan 1 , Andrea Califano 1,3 , Ronald A. DePinho 5 , Matt Maurer 1, 6 , and Ramon Parsons 2* 1 Institute for Cancer Genetics and Herbert Irving Comprehensive Cancer Center, Columbia University, 1130 St. Nicholas Avenue, NY, NY 10032, USA. 2 Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1470 Madison Ave HCSM 6-117, New York, NY 10029, USA. 3 Department of Biomedical Informatics Columbia University, 1130 St Nicholas Ave, ICRC, New York, NY, 10032, USA. 4 Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 5 Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 6 Department of Medicine, Columbia University Medical Center 630 W. 168th Street, NY, NY 10032, USA. Street, NY, NY 10032, USA. *Correspondence: [email protected](R.P.) *Contact: Ramon Parsons email: [email protected]1
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Supplemental Information
Survival Factor NFIL3 Restricts FOXO-induced Gene Expression in Cancer
Megan Keniry1, Maira M. Pires1,2, Sarah Mense2, Celine Lefebvre1,3, Boyi Gan4, Karen Justiano1, Ying-Ka Ingar Lau1, Ben Hopkins1, Cindy Hodakoski1,2, Susan Koujak1, Joseph Toole1, Franklyn Fenton1, Ashley Calahan1, Andrea Califano1,3, Ronald A. DePinho5, Matt Maurer1, 6, and Ramon Parsons2*
1Institute for Cancer Genetics and Herbert Irving Comprehensive Cancer Center, Columbia University, 1130 St. Nicholas Avenue, NY, NY 10032, USA. 2Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1470 Madison Ave HCSM 6-117, New York, NY 10029, USA. 3Department of Biomedical Informatics Columbia University, 1130 St Nicholas Ave, ICRC, New York, NY, 10032, USA. 4Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 5Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 6Department of Medicine, Columbia University Medical Center 630 W. 168th Street, NY, NY 10032, USA. Street, NY, NY 10032, USA.
Itemized List of Supplemental InformationSupplemental Figures 1-8:
Supplemental Figure 1 accompanies main text Figure 1. Supplemental Figure 2 accompanies main text Figure 2. Supplemental Figure 3 accompanies main text Figure 3. Supplemental Figure 4 accompanies main text Figure 4. Supplemental Figure 5 accompanies main text Figure 5. Supplemental Figure 6 accompanies main text Figure 6. Supplemental Figure 7 accompanies main text Figure 7. Supplemental Figure 8 accompanies main text Figure 8.
Supplemental Table 1 Legend (Accompanying Word File): Supplemental Table 1 accompanies main text Figure 4.
Supplemental Tables 2-6: Supplemental Table 2 accompanies main text Figure 4. Supplemental Table 3 accompanies main text Figure 4. Supplemental Table 4 accompanies main text Figure 4. Supplemental Table 5 accompanies main text Figure 4. Supplemental Table 6 accompanies main text Figure 6. Supplemental Table 7 accompanies main text Figure 8.
Supplemental Table 8 Legend (Accompanying Word File) Supplemental Table 8 is included as a Word file.
Supplemental Materials and Methods This section includes additional Materials and Methods.
Supplemental References
2
Supplemental Figure 1. Genes Examined in Reporter Screen and Validation of the
TRAIL Reporter as an Indicator of PI3K/PTEN Output.
3
(A) Graph depicts the differential expression of genes in the presence of exogenous
PTEN from previously published microarray studies; more detailed descriptions of these
previously published studies (cell lines utilized and experimental designs) can be found
in the Supplemental Experimental Procedures . Genes from these microarray studies
that were differentially expressed by at least two-fold and had available cDNAs in the
human Orfeome Library 1.1 were chosen as candidates for our screen (in blue on the
graph). (B) Side by side comparison of PTEN-induction of six FOXO-regulated
luciferase reporters in HEK293 cells, * significantly induced by exogenous PTEN. The
TRAIL reporter was most strongly induced. (C) TRAIL reporter assays with exogenous
PTEN, with or without DN-FOXO1, *significantly induced, **significantly lower than
control vectors. PTEN induced reporter activity, except in the presence of DN-FOXO1.
(D) TRAIL reporter assays were performed with 100 nM wortmannin (an inhibitor of
performed with exogenous Myr-AKT, which encodes constitutively membrane localized
AKT, *significantly lower than control. (F) TRAIL eporter assays with exogenous NFIL3
and/or exogenous FOXO1, ** significantly less induced than exogenous FOXO1 alone
sample. (G) U87MG cells with control vector or a retroviral PTEN vector were treated
with control or NFIL3 shRNA; endogenous TRAIL expression was measured by qRT-
PCR, *significantly different than control shRNA, ** significantly different than induction
observed with both control vectors and control vector with exogenous PTEN. (H) ChIP
analysis with FOXO1 antibody. DNA was subjected to quantitative PCR for proximal region
of INSR promoter or control -actin; FOXO1 associated with the INSR promoter. Data
are means ± SEM of three experiments.
4
5
Supplemental Figure 2. Sixty Base Pairs of the TRAIL Promoter are Sufficient for
PTEN and NFIL3 Regulation
(A) Luciferase assays were performed using the reporters TRAIL-165 and TRAIL-60
that contain 165 and 60 base pairs of the TRAIL promoter, respectively, *significantly
different than control, ** significantly less well induced by exogenous PTEN. Both of
these reporters were regulated by PTEN and NFIL3. (B) Predicted NFIL3 and FOXO
binding sites (underlined) based on published consensus sequences and mutant
luciferase reporter sequences (used in Fig. 2C-D) are shown; the bold letters for the
NFIL3 consensus are given a higher weight . (C) Putative FOXO and NFIL3 binding
sites were mutated in TRAIL reporter as indicated and were tested for regulation by
FOXO1 and NFIL3; FOXO1 induction was reduced with the mutant FOXO site,
*significantly less induced by exogenous FOXO1 in comparison to the wildtype TRAIL
reporter. Deletion of the NFIL3 site led to a significant (*) reduction in regulation by
NFIL3. Data are means ± SEM of three experiments.
6
Supplemental Figure 3. Role of HDACs in NFIL3/FOXO1 Transcriptional
Regulation
7
(A-B) NFIL3 and HDAC2 physically associate. Co-immunoprecipitations were performed
with HEK293 cell extracts that expressed FLAG-HDAC2 and V5-NFIL3. Faint background
bands are present in input samples due to pre-clearing reactions with normal mouse IgG. (C)
TRAIL reporter assays with samples that were treated with or without 2 M SAHA for 24
hours. HDAC inhibition reduced the NFIL3-mediated repression of the TRAIL reporter
in the presence of exogenous PTEN. (D) TRAIL reporter assays were performed with
the FOXO1 acetylation deficient mutant (6KR) or the acetylation mimetic mutant (6KQ)
with or without exogenous NFIL3. The acetylation mutants had no effect on NFIL3-
mediated regulation of reporter activity, *significantly different than control vector
sample, **significantly less well induced than with exogenous FOXO1 or 6KR mutant,
respectively. Data are means ± SEM of three experiments.
8
Supplemental Figure 4. GSEA with Microarray Data from BT549 cells with NFIL3
shRNA and HDAC Inhibition in the Burkitt’s Lymphoma Cell Line (RJ225)
9
(A-C) BT549 cells with lentiviral GFP or Nfil3 (not targetable by human NFIL3 shRNA)
were treated with human NFIL3-targeting shRNA or control; q-RT-PCR analysis was
performed for GADD45, TRAIL and NFIL3 expression,*significantly different than
control shRNA, ** significantly different than induction observed with GFP. (D) GSEA
was performed with microarray data obtained from BT549 samples that had NFIL3
expression diminished by shRNA. Gene sets induced by exogenous FOXO1, treatment
with LY294002, and exogenous PTEN were significantly enriched in the NFIL3
knockdown samples. (E) Gene Set Enrichment Analysis (GSEA) was performed with
data from the Burkitt’s lymphoma cell line RJ225 that was treated with the HDAC
inhibitor Trichostatin A (TSA) and PTEN pathway gene sets. Gene sets induced by the
PI3K inhibitor LY294002, by exogenous FOXO1 and by exogenous PTEN were up-
regulated by HDAC inhibition in a Burkitt’s lymphoma cell line. Abbreviations: ES=
enrichment score and NES= normalized enrichment score. Data are means ± SEM of
three experiments.
10
Supplemental Figure 5. FOXO Subcellular Localization in Cell Lines and MEFs
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(A) Subcellular fractionations with HEK293, MDA-MB-468 (labeled as 468) and BT549
cell lines probed with additional FOXO1 (C29H4) and FOXO3 (75D8) antibodies. MAX
and tubulin are markers for the nuclear (N) and cytoplasmic (C) fractions respectively;
the MAX and tubulin panels for HEK293 and MDA-MB-468 cells are same as those
depicted in the main text Figure 5A. FOXO1 and FOXO3 were in the nucleus and
cytoplasm. (B) Western blot analysis of subcellular fractionations with Large T antigen-
immortalized MEFs (floxed/floxed, Rosa26CreERT2, FoxO1, FoxO3 and FoxO4 that
were treated with Ad-GFP or Ad-Cre); samples were probed for FOXO1, FOXO3, and
control antibodies. The detected FoxO bands were diminished in Ad-Cre treated
samples showing the specificity of these antibodies. (C) Subcellular fractionations with
control or Pten -/- MEFs; residual FoxO is detected in the nuclear fraction of Pten -/-
MEFs. (D-E) The indicated FOXO transcription factor was targeted with siRNA in
HEK293 cells; western blots are shown, indicating the specificity of each antibody
(molecular weights in kD are indicated). (F) The expression of NFIL3 was diminished
with shRNA hairpins (KD1 or KD2) in BT549 cells; nuclear and cytoplasmic fractions
were prepared. Fractions were probed for indicated antibodies. NFIL3 diminishment did
not alter FOXO localization.
12
Supplemental Figure 6. NFIL3 transcription is induced by H2O2 and NFIL3
Regulates Transcription through FOXO.
(A) NFIL3 expression was assessed with qRT-PCR from BT549 extracts that were either
treated with 250M H2O2 for 18 hours or water; NFIL3 was induced by H2O2. (B) Primary
MEFs were treated with Ad-GFP or Ad-Cre; qRT-PCR was performed to confirm the loss
of FoxO expression in Cre treated samples; the PCR only detects recombinants. (C)
Primary MEFs (Rosa26CreERT2, floxed/floxed FoxO1, FoxO3 and FoxO4) of the
indicated genotype were infected with lentivirally delivered control or Nfil3-targetting
13
shRNA; qRT-PCR was performed with samples. Data are means ± SEM of three
experiments.
Supplemental Figure 7. NFIL3 and HDAC2 are Expressed in Poor Prognosis
Cancers and a Series of FOXO1 Targets are Associated with Poor Breast Cancer
Prognosis.
14
The Oncomine database was queried for NFIL3 expression . (A) NFIL3 and HDAC2
expression were elevated in basal-like breast cancer and GBM . (B-D) Kaplan Meier
analysis with NFIL3/FOXO target genes in breast cancer using NKI 295 data from
luminal A and basal subtypes; NFIL3 expression is associated with poor prognosis. (E-
H) Kaplan Meier analysis was done with previously published FOXO targets using NKI
data. Twenty-seven known FOXO targets that showed no evidence of NFIL3 regulation
in our microarray studies were tested for their relationship to breast cancer prognosis
and 11 trended towards poor prognosis and 4 were significantly associated to poor
prognosis with p value < 0.05. (I) The ratio of expression of indicated FOXO target
genes plus or minus the standard deviation of NFIL3 KD samples versus control shRNA
samples is shown. Data are extracted from 293 microarray data from Figure 4, (left
column) and ChIP for FOXO1 with the promoters of these genes in HEK293 cells (right
column), *significantly different than rabbit IgG control binding, ^ChIPs were performed
in BT549 cells for this gene.
15
Supplemental Figure 8. HDAC2 and NFIL3 Regulate Cell Viability in Basal-like
Breast Cell Lines.
16
(A) HDAC2 was diminished in MCF10A cells using shRNA (KD1 or KD2). A western
blot is shown. (B) The MCF10A samples with and without HDAC2 knockdown were
subjected to apoptosis analysis as described in the Supplemental Materials and
Methods. HDAC2 shRNA induced cell death. (C-D) Cell lines with NFIL3 reduced by
shRNA were analyzed by western analysis and these cells were examined for cell
death/apoptosis as described in the Supplemental Materials and Methods. The percent
of apoptotic cells equals the propidium iodide and Annexin V single and double positive
cells divided by the total number of cells. (E) MCF10A cells with either lenti-viral GFP or
mouse Nfil3 (not targetable by human NFIL3 shRNA) were treated with control or
human NFIL3-targeting shRNA. Cells remaining after treatment were stained with
crystal violet, plates were scanned with UMAX PowerLook 1100 scanner and Image J
was utilized to quantify cell density. Mouse Nfil3 partially rescued the cell death
induced by human NFIL3 shRNA. (F-G) qRT-PCR with MCF10A samples treated with
NFIL3 shRNA or control, *significantly different than control shRNA, ** significantly
different than induction observed with GFP, *** significantly lower detected expression
than samples with lentivirally delivered mouse Nfil3; note that the control samples only
have human NFIL3. (H) Colony assays in presence of NFIL3 shRNA with or without
TRAIL shRNA). The TRAIL KD1 had 41% of endogenous TRAIL by q-RT-PCR with
1.5% error and the KD2 for TRAIL had 63% of endogenous TRAIL with 1.0% error; both
TRAIL KDs were significant. Data are means ± SEM of three experiments.
17
Supplemental Table 1 Legend (See accompanying Microsoft Word file):
Supplemental Table 1. Differentially Expressed Genes in NFIL3 Knockdown
Samples
Gene expression profiling was performed with NFIL3 knockdown samples and scramble
shRNA controls (HEK293 cells). One-Way ANOVA analysis was used to detect 399
differentially expressed genes with an FDR=0.05 and at least a two-fold change in gene
expression. 289 of these genes were up regulated.
18
19
20
Supplemental Table 4. Gene Set Enrichment Analysis with Curated Broad Institute Molecular Signatures Database Sets and PTEN Pathway Sets
Gene set enrichment analysis was performed with 1,679 gene sets that are curated by the Broad Institute Molecular Signatures Database and the NFIL3 knockdown microarray data from HEK293 cells. The experimentally derived PTEN-pathway gene sets were included in this analysis for comparison. Of these gene sets tested, 1403 were enriched to some degree in the NFIL3 knockdown samples. Enriched gene sets were ranked based on the normalized enrichment score (NES). The PTEN pathway gene sets and corresponding rankings are shown; three of these are in the top tenth percentile of enriched gene sets.
Gene Set Name SIZE ES NESp-
value
NES Rank (out of 1403 gene
sets)TERRAGNI- GENE SET INDUCED BY LY294002 21 0.64 1.70 0.03 49RAMASWAMY- FOXO1 INDUCED SET [CLASS 2A] 31 0.46 1.60 <0.001 94MATSUSHIMA-NISHIU- GENE SET INDUCED BY PTEN 84 0.45 1.54 0.03 134GOMIS GENE SET INDUCED BY FOXO-SMAD 10 0.70 1.43 0.08 234MATSUSHIMA-NISHIU- GENE SET REPRESSED BY PTEN 54 0.29 1.01 0.43 1038RAMASWAMY- FOXO1 REPRESSED SET [CLASS 3] 25 0.29 0.86 0.68 1229TERRAGNI- GENE SET REPRESSED BY LY294002 42 0.28 0.82 0.69 1279RAMASWAMY- FOXO1 INDUCED SET [CLASS 1] 22 0.17 0.54 0.95 1395
21
Supplemental Table 5. The Top 50 Gene Sets that were Enriched in NFIL3 Knockdown Microarray Data
GSEA was performed with microarray data from HEK293 cells that were treated with NFIL3 shRNA as described in the Supplemental Experimental Procedures. The gene sets utilized were curated on the Molecular Signatures Database, Broad Institute, and were rank ordered by Normalized Enrichment Score (NES). The top 50 most enriched gene sets (out of over 1400 examined gene sets) are included in this table. Abbreviations: ES=Enrichment Score and NES=Normalized Enrichment Score. Eight of these gene sets (genes induced by HDAC inhibition) are also included in Figure 4G.
Supplemental Table 6. ChIP qPCR: HEK293 and BT549 cells with NFIL3 shRNA
ChIPs were performed in HEK293 and BT549 cells that were treated with a control shRNA or NFIL3 targeting shRNA (KD1 or KD2). Promoter binding was quantified by q-PCR using -actin sequence as a control (promoter of interest/-actin control); these samples are also normalized to the IgG control. * The difference between the control shRNA and NFIL3 targeting shRNA was significant by Student’s t test. All ChIPs were performed at least twice and qPCR samples were analyzed in triplicate.
ChIP qPCR: HEK293 cells with NFIL3 shRNA or control
ChIP qPCR: BT549 cells with NFIL3 shRNA or control
GADD45 Promoter
IgG Histone H3
Control shRNA 1.2 2.1NFIL3 KD1 0.8 2.3NFIL3 KD2 1.2 0.66*
TRAIL Promoter
IgG Histone H3
Control shRNA 1.2 2.4NFIL3 KD1 0.9 0.8*NFIL3 KD2 1.0 1.7*
25
Supplemental Table 7. FOXO Target genes were differentially expressed in MCF10A, MDA-MB-468 and U87MG cells.
NFIL3 expression was diminished in MCF10A and MDA-MB-468 cells via shRNA; the expression of indicated genes was analyzed by qRT-PCR. Genes were also analyzed in samples that were treated with or without hydrogen peroxide (250 M for 18 hours). The numbers presented are normalized gene expression units (gene of interest/GAPDH control); these samples are also normalized to controls (the shRNA samples are normalized to a control shRNA and the hydrogen peroxide is normalized to water control). Empty boxes were not determined. The * mark means that the difference in expression was statistically significant by Student’s t test. Data are means of three experiments.
Supplemental Table 8 Legend (See accompanying Microsoft Word file):
26
Supplemental Table 8. Gene-specific PCR primers
The primers used for gene expression analysis, ChIP analysis (qPCR), PCR primers for ABCD assays, and mutagenesis primers for TRAIL reporter mutants are detailed.
Supplemental Materials and Methods
Plasmid Constructs
The TRAIL (1523 bp and 165 bp) reporters were from J. Millbrant. The TK-Renilla
control reporter was from Promega (Madison, WI). The pCEP4 PTEN vector was
previously described . D. Accili donated the DN-FOXO1 vector and FOXO1AAA. Myr-
AKT was obtained from T. Franke. The pCDNA3-FOXO1 was from W. Sellers. The
pME18S-FLAG-HDAC2 vector, used in co-immunoprecipitations, was from R. Dalla-
Favera. Expression vectors for candidate regulators of the PTEN pathway were
prepared by using the LR Clonase and pDEST40 destination vector (Invitrogen,
Carlsbad, CA) with intermediate vectors from the human Orfeome collection1.1 (Open
Biosystems, Huntsville, AL). The pBABE-hygro p53 DD vector for MEF immortalization
was a gift from M. Oren. MSCVpuro-NFIL3 was made as follows: the NFIL3 gene was
amplified using the primers (P170: 5’-
GCGCGGATCCACATGCAGCTGAGAAAAAATGCAG-3’) and (P171:5’-
CGCGCGCTCGAGTTACCCAGAGTCTGAAGCAGA); the corresponding PCR product
was cut with BamHI and XhoI and was ligated into the BglII/XhoI sites of MSCVpuro.
MSCVneo-PTEN vector was previously described . The HDAC2 expression vector
utilized in reporter assays was prepared by amplifying the full-length cDNA from
HEK293-derived cDNA with the primers: 5’ATGCGCTCACCTCCCTGCGG and
27
3’CGCGGATCCGCGGTCAGGGGTTGCTGAGCTGT. The HDAC2 PCR product was
cloned into the TOPO TA vector pcDNA3.1/V5-HIS TOPO (Invitrogen, Carlsbad, CA)
according to the Invitrogen protocol. The FOXO1 acetylation mutants were obtained
from Addgene (plasmid numbers 17562, 12148, and 17560). The lentiviral Nfil3 vector
and GFP control lenti-vector were gifts from S.E. Plevy.
Mission lentiviral shRNA vectors targeting NFIL3 were obtained from Sigma:
Clone ID: NM_005384.1-1746s1c1, used for gene expression analysis and ChIPs in
HEK293 cells, denoted as KD2 in figures, sequence:
(06-866) from Upstate/Millipore (Billerica, MA); the total histone H3 antibody (ab1791)
was from AbCam (Cambridge, MA) . Samples were then incubated with Protein G
agarose for one hour and then extensively washed. DNA was then eluted, crosslinks
were reversed and the DNA was purified using phenol: chloroform extraction and
sodium acetate/ethanol precipitation. The purified DNA was subjected to quantitative
real-time PCR. RNA samples were prepared in parallel to chromatin samples to ensure
that target genes were induced by at least five fold.
Prediction of Transcription Factor Binding Sites for ChIPs
TFSEARCH software was utilized to identify putative FOXO and NFIL3 binding sites
using a threshold of 75% for consensus sites to aid in the design of ChIP primers and
search for NFIL3 binding sites in FOXO regulated genes . The consensus for the
forkhead box transcription factor HNF3 was employed to identify FOXO binding sites
as the FOXO consensus was not included in this software and the consensus sites for
these proteins are nearly identical. For comparison the HNF3 site from the
TFSEARCH database is: NNNT(A/G)TTT(A/G)(T /C)T(T/C) compared to the
complement of the FOXO consensus: T(A/G)TTTA(T/C) .
37
Immunofluorescence
Cells were grown on coverslips, washed twice with PBS and fixed in 2%
paraformaldehyde in PBS pH 7.4 for 30 minutes. Samples were washed four times with
PBS and then permeablized with 0.1% Triton X-100 plus 1% goat serum for 15 minutes.
Samples were washed twice with PBS and incubated with primary (1:100) antibody for 2
hours. Samples were washed three times (5 minutes each) with PBS and were then
incubated with Alexa 488–conjugated goat anti–rabbit secondary antibody (Invitrogen)
at 1:600 dilution in PBS for one hour. Samples were washed three times (5 minutes
each) with PBS, were mounted with Gold polymount.
Cell Death Assays
The NFIL3 (or HDAC2) shRNA targeted samples (in MCF10a, MDA-MB-468 and BT549
cells) were collected at 96 hours post infection and were stained with propidium iodide
and Annexin V FITC using the ApoAlert Annexin V Apoptosis Kit (Clontech, Mountain
View, CA) according to manufacturer’s protocol. Cells were sorted with an LSRII flow
cytometer (BD Biosciences, San Jose, CA). 30,000 cells were analyzed per sample and
this experiment was repeated several times. Seventy percent confluent U87MG cells
that were transduced to express NFIL3 and/or PTEN were treated with water or 250 M
H2O2 for 18 hours (St. Louis, MO); samples were analyzed using the ApoAlert Annexin
V Apoptosis Kit as described above for cell death. Data are means ± SEM of three
experiments.
38
The apoptosis assays for MDA-MB-468 and BT549 cells transduced to express NFIL3
were performed by treating seventy percent confluent cells with 250 M H2O2 for 18
hours. After treatment, the remaining cells were stained with 0.5% crystal violet in 10%
buffered formalin solution for 15 minutes. Samples were washed several times with
PBS. The dye remaining on the plate was solubilized into 10% acetic acid solution for
30 minutes with agitation and the OD565 was measured. Data are means ± SEM of
three experiments.
The MCF10A cell death rescue experiments were performed by infecting cells (20,000
cells in a well of a six well plate) that harbor either lentivirally-delivered <GFP> or
<Nfil3> with the indicated lentivirally-delivered shRNA (400L of frozen viral supernatant
per well of a six well plate). 8 days post-infection, the remaining cells were stained with
0.5% crystal violet in 10% buffered formalin solution for 15 minutes. Samples were
washed several times with PBS. Plates were scanned using the UMAX PowerLook
1100 scanner and Image J was utilized to quantify reduced cell numbers.
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