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.

*Correspondence: ramon.parsons@mssm.edu (R.P.)

*Contact: Ramon Parsons

email: ramon.parsons@mssm.edu

1

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

PI3K), added 8 hours prior to assays, *significantly induced. (E) TRAIL reporter assays

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)

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(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.

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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.

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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

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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.

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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.

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Supplemental Figure 8. HDAC2 and NFIL3 Regulate Cell Viability in Basal-like

Breast Cell Lines.

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(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.

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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.

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19

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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

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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.

Gene Set Name ES NES p-valueJECHLINGER_EMT_DN 0.63 2.01 <0.01GERY_CEBP_TARGETS 0.55 2 <0.01AD12_ANY_DN 0.62 2 <0.01WERNERONLY_FIBRO_UP 0.56 1.99 <0.01EMT_DN 0.59 1.96 <0.01STRESS_GENOTOXIC_SPECIFIC_UP 0.66 1.95 <0.01WERNER_FIBRO_UP 0.48 1.93 <0.01AD12_32HRS_DN 0.78 1.93 <0.01HDACI_COLON_TSABUT_UP 0.53 1.91 <0.01CMV_UV-CMV_COMMON_HCMV_6HRS_UP 0.67 1.9 <0.01TAKEDA_NUP8_HOXA9_3D_DN 0.6 1.89 <0.01STAEGE_EFTS_UP 0.57 1.88 <0.01BRENTANI_TRANSCRIPTION_FACTORS 0.54 1.88 <0.01TGZ_ADIP_UP 0.71 1.87 <0.01VEGF_HUVEC_30MIN_UP 0.57 1.87 <0.01ZMPSTE24_KO_UP 0.59 1.85 <0.01CMV_HCMV_6HRS_UP 0.64 1.85 <0.01BROCKE_IL6 0.51 1.84 <0.01KRETZSCHMAR_IL6_DIFF 0.51 1.84 <0.01DNMT1_KO_UP 0.52 1.84 <0.01LVAD_HEARTFAILURE_UP 0.55 1.83 0.01ADIP_DIFF_CLUSTER2 0.68 1.82 <0.01HDACI_COLON_BUT_UP 0.43 1.81 <0.01UVC_LOW_ALL_UP 0.69 1.8 0.02UEDA_MOUSE_SCN 0.46 1.79 <0.01MANALO_HYPOXIA_UP 0.51 1.78 0.01HDACI_COLON_TSA2HRS_UP 0.55 1.78 <0.01JISON_SICKLE_CELL 0.53 1.78 <0.01RORIE_ES_PNET_DN 0.56 1.78 <0.01TSA_PANC50_UP 0.62 1.78 <0.01HYPERTROPHY_MODEL 0.68 1.77 <0.01MARSHALL_SPLEEN_BAL 0.6 1.77 0.01UVC_HIGH_ALL_UP 0.65 1.76 <0.01NI2_MOUSE_UP 0.46 1.76 <0.01

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GATA3PATHWAY 0.68 1.75 <0.01HDACI_COLON_BUT12HRS_UP 0.57 1.75 <0.01HDACI_COLON_TSA_UP 0.43 1.75 <0.01FRASOR_ER_UP 0.57 1.74 <0.01MENSE_HYPOXIA_UP 0.49 1.73 <0.01HSA01510_NEURODEGENERATIVE_DISEASES 0.57 1.73 <0.01HDACI_COLON_BUT48HRS_UP 0.47 1.73 <0.01ST_JNK_MAPK_PATHWAY 0.53 1.72 <0.01AD12_24HRS_DN 0.59 1.72 0.03FLECHNER_KIDNEY_TRANSPLANT_WELL_DN 0.55 1.72 <0.01HSA04520_ADHERENS_JUNCTION 0.48 1.72 <0.01STRESS_ARSENIC_SPECIFIC_UP 0.48 1.71 <0.01UVB_NHEK4_24HRS_DN 0.64 1.71 0.05BRCA_PROGNOSIS_POS 0.55 1.71 <0.01HDACI_COLON_CUR24HRS_DN 0.6 1.7 <0.01HDACI_COLON_BUT2HRS_UP 0.41 1.7 <0.01

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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

GADD45 Promoter

IgG FOXO1 NFIL3 Histone H3

Acetyl Histone H3

Acetyl Histone H4

Control shRNA 1.0 1.2 2.4 1.3 0.9 56.0NFIL3 KD1 1.2 2.1* 1.2* 0.8* 2.1* 500.0*NFIL3 KD2 0.9 1.6* 1.0* 0.85 1.6* 140.0*

GADD45 Promoter

IgG FOXO1 NFIL3 Histone H3

Acetyl Histone H3

Acetyl Histone H4

Control shRNA 1.1 1.1 1.9 0.65 1.0 2.6NFIL3 KD1 1.0 1.9* 0.7 * 0.5 1.9* 29.3*NFIL3 KD2 0.9 3.3* 0.6* 0.2* 3.2* 10.4*

FAS Promoter IgG FOXO1 NFIL3 Histone H3

Acetyl Histone H3

Acetyl Histone H4

Control shRNA 1.2 1.1 2.8 0.9 1.0 217.3NFIL3 KD1 0.8 1.8* 1.1 * 0.8 2.3* 912.7*NFIL3 KD2 0.9 1.8* 0.4* 0.9 2.2* 456.3* TRAIL Promoter

IgG FOXO1 NFIL3 Histone H3

Acetyl Histone H3

Acetyl Histone H4

Control shRNA 1.1 0.9 4.6 0.7 1.0 1.4NFIL3 KD1 1.0 4.1* 2.5* 0.8 1.7* 3.5*NFIL3 KD2 0.9 1.7* 1.8* 0.9 2.0* 3.7*

GAPDH Promoter

IgG FOXO1 NFIL3 Histone H3

Acetyl Histone H3

Acetyl Histone H4

Control shRNA 1.0 0.8 1.1 1.1 9.8 20.8NFIL3 KD1 0.9 0.6* 0.3* 0.7 5.5* 20.1NFIL3 KD2 0.9 1.0 1.2 1.3 7.8 10.1*

24

Supplemental Table 6 continued:

ChIP qPCR: BT549 cells with NFIL3 shRNA or control

GAPDH Promoter

IgG FOXO1

NFIL3 Histone H3

Acetyl Histone H3

Acetyl Histone H4

Control shRNA 0.9 0.6 0.5 1.3 11.1 5.5NFIL3 KD1 0.9 0.9 0.7 1.0 12.9 2.5*NFIL3 KD2 1.1 0.9 1.6* 1.5 10.5 3.6*

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*

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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.

Cell Line Gene qRT-PCR NFIL3 shRNA (KD1)

qRT-PCR NFIL3 shRNA (KD2)

qRT-PCR +H2O2

MCF10A NFIL3 0.8* 0.4*MCF10A TRAIL 1.9* 3.9*MCF10A GADD45 3.4* 3.8*MCF10A GADD45 8.9* 9.2*MCF10A FAS 25.4* 1.4*MDA-MB-468 NFIL3 0.2* 0.6* 1.3MDA-MB-468 TRAIL 2.9* 5.2* 0.7MDA-MB-468 GADD45 0.5* 0.8* 3.1*MDA-MB-478 GADD45 0.7* 1.0 0.9MDA-MB-468 FAS 2.0* 3.3* 1.2

Cell Line Gene qRT-PCR +H2O2

U87MG TRAIL 0.4*U87MG GADD45 6.2*U87MG GADD45 4.8*U87MG FAS 2.4*

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:

CCGGGCACAGATTATGATGAAGATTCTCGAGAATCTTCATCATAATCTGTGCTTTTT

Clone ID: NM_005384.1-1216s1c1 , used for reporter assays in HEK293 cells, gene

expression analysis and ChIPs in HEK293 and BT549 cells, subcellular fractionation

with BT549 cells, signaling analyses with HEK293 cells and BT549 cells and apoptosis

assays in each cell line utilized, denoted as KD1 in figures; sequence:

CCGGCAGATCAAAGTAGAAGCCTTTCTCGAGAAAGGCTTCTACTTTGATCTGTTTTT

Clone ID: NM_005384.1-290s1c1, used for reporter assays in HEK293 cells, qRT-PCR

and ChIP analysis with BT549 cells, signaling analyses with HEK293 and BT549 cells

and apoptosis assays with each cell line used, denoted as KD2 in figures; target

sequence:

CCGGTGGTCCTTAATTCTGCTTTAACTCGAGTTAAAGCAGAATTAAGGACCATTTT.

Clone ID: NM_017373.2-395s1c1, was utilized to diminish Nfil3 in MEFs, denoted as

KD1 in figures; sequence:

28

CCGGGACGCCATGTATTGGGAGAAACTCGAGTTTCTCCCAATACATGGCGTCTTTT

TG.

Clone ID: NM_017373.2-694s1c1, was utilized to diminish Nfil3 in MEFs, denoted as

KD2 in figures; sequence:

CCGGCGGAAGTTGCATCTCAGTCATCTCGAGATGACTGAGATGCAACTTCCGTTTT

TG.

Mission lentiviral shRNA vectors targeting HDAC2 were obtained from Sigma:

Clone ID: NM_001527.1-1678s1c1, denoted as KD1 in figures, sequence:

CCGGCAGTCTCACCAATTTCAGAAACTCGAGTTTCTGAAATTGGTGAGACTGTTTTT

Clone ID: NM_001527.1-508s1c1 , denoted as KD2 in figures, sequence:

CCGGCCAGCGTTTGATGGACTCTTTCTCGAGAAAGAGTCCATCAAACGCTGGTTTT

T.

Variations of the TRAIL luciferase reporters were made by performing site directed

mutagenesis using the vectors and primers described in Supplemental Table 7 with the

Stratagene Quikchange XL site directed mutagenesis kit (La Jolla, CA).

Cell culture and Chemicals

Cell lines were obtained from ATCC and propagated under suggested conditions.

SAHA and TSA were purchased from Sigma (St. Louis, MO). FOXO1, FOXO3 and

FOXO4 were diminished in HEK293 cells with the respective siRNA sequences (GCC

29

CUG GCU CUC ACA GCA AUU, CGAAUCAGCUGACGACAGUUU, and

GGACUGGACUUCAACUUUGUU) that were transfected with Lipofectamine 2000

(Invitrogen, Carlsbad, CA). FOXO1AAA was transfected into HEK293 and BT549 cells

using the Amaxa nucleofector technology kit V (program P20 for BT549 cells and X-001

for HEK293 cells) with one million cells/transfection in order to prepare RNA for gene

expression analysis (Lonza, Cologne, Germany).

Reporter Screen Design

The goal of the reporter screen was to identify which (if any) of the PTEN regulated

genes from previously published microarray studies were novel components/regulators

of the PI3K/PTEN pathway. We assembled a list of PTEN-regulated genes from four

published gene expression microarray experiments . Each microarray study was set-up

in a slightly different manner: Simpson et al. infected the PTEN mutant breast cancer

cell line MDA-MB-468 with Ad-PTEN and collected time points for microarray analysis.

PTEN was greatly overexpressed in this setting, leading to growth arrest and cell death.

Stolarov et al. employed a retroviral-mediated ecdysone-inducible system in the

glioblastoma cell line (U87MG, PTEN null) to exogenously express PTEN; this level of

expression was enough to halt the growth of U87MG cells. Matsushima-Nishiu et al.

infected the PTEN mutant endometrial cancer cell line HEC-151 with Ad-PTEN;

samples were collected 24 hours after infection and expressed high levels of PTEN by

western blot that induced cell death. Hong et al. transfected the human lung

adenocarcinoma cell line CL1-5 with pCEP4-PTEN to obtain stable clones that

overexpressed PTEN; these clones expressed 20 fold higher PTEN and were utilized

30

for microarray analysis. In order for a gene to be utilized in our screen, it needed to be

differentially expressed by at least two fold upon the addition of exogenous PTEN in the

published microarray study and it needed to be included in the Human Orfeome 1.1

Library (Open Biosystems, Huntsville, AL) in order to easily prepare a cDNA expression

vector for screening. The prepared expression vectors were utilized in TRAIL luciferase

reporter assays as described in the Experimental Procedures in the main text. The

amount of PTEN expression vector transfected into the log phase HEK293 cells was

enough to hinder cellular growth (data not shown).

Isolation of Pten KO mouse embryonic fibroblasts (MEFs)

Ptenflox/+ mice in a mixed 129S4 backcrossed to C57B1/6 background were mated to

obtain Ptenflox/flox embryos . Genotypes were determined by PCR using DNA from either

the tail or yolk sac. PCR primer sequences were obtained from the Jackson Laboratory

(primer stock number 006440) and are as follows: forward oIMR6260 5’-ACT CAA GGC

AGG GAT GAG C-3’ and reverse oIMR6261 5’-AGC TGT GGT GGG TTA TGG TC-3’.

Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 mouse embryos.

Pregnant mice were sacrificed by exposure to CO2 and embryos were surgically

harvested. For MEF isolation the head and visceral organs were removed, and the

remaining tissue was minced and then digested using 0.05% trypsin at 37°C. MEFs

were cultured in 5% CO2 at 37°C in DMEM supplemented with 10% fetal bovine serum

and 2 mM glutamine. MEFs were immortalized by infection with a retrovirus expressing

dominant negative p53 (pBABE-hygro p53 DD), and infected MEFs were selected with

100 μg/ml hygromycin. Immortalized Ptenflox/flox MEFs were infected with adenovirus

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expressing either Cre recombinase or GFP (catalog numbers 1045 and 1060, Vector

Biolabs). Infections were performed overnight in MEF growth medium containing 2 x 107

PFU/ml adenovirus and 4 μg/ml polybrene. Western blot confirmed loss of Pten in Ad-

Cre infected MEFs and normal Pten expression in Ad-GFP infected MEFs.

Infections

Retroviruses (MSCVpuro-NFIL3 and MSCVneo-PTEN) were prepared using Phoenix-

HEK293 cells transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Titers

were collected 48 hours post-transfection, were filtered with a 0.45 M filter and were

then utilized with growth media along with a final concentration of 5 g/mL polybrene

(hexadimethrine bromide) (Sigma, St. Louis, MO). Lentiviral shRNA viruses were

obtained by transfecting HEK293T cells with a ratio of 1.3:1:0.01 of shRNA vector to

CMV8.9 to VSV-G using Lipofectamine 2000. Titers were collected 48 hours post-

transfection, were filtered using a 0.45 M filter and were utilized with growth media

along with a final concentration of 12 g/mL polybrene. Infected cell lines were selected

accordingly with puromycin and/or neomycin. Samples from shRNA experiments were

collected 96 hours post-infection with the exception of those samples derived from

Clone ID: NM_005384.1-1746s1c1 for NFIL3, which were collected 48 hours post-

infection (along with proper control samples) due to cell death induction.

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Subcellular Fractionation

Log phase cells were scraped from 10 cm plates into ice cold PBS with 1X protease

inhibitor (PI), (from 100 X mammalian protease inhibitor cocktail, Sigma, St. Louis, MO).

Cells were washed twice with ice cold PBS (containing 1 X PI). Then cells were

incubated in Buffer A (10 mM Hepes pH 7.4, 10 mM KCL, 100 M EDTA, 1 X PI) for 15

minutes on ice. After this, 10% NP-40 was added to the Buffer A mixture at a 1:20

dilution. Samples were vortexed and rotated end over end for 2 minutes; then samples

were centrifuged at 1000 x g for 5 minutes. The cytoplasmic fraction is comprised of the

supernatant. The pellet was washed with Buffer A and then extracted with Buffer C (20

mM Hepes pH 7.4, 400 mM NaCl, 1 mM EDTA, 1 X PI) to obtain the nuclear fraction.

Avidin-Biotin Complex DNA (ABCD) Assays

Nuclear lysates were prepared from transfected HEK293 cells as detailed above in the

subcellular fractionation section. Lysates were diluted 1:3 in Buffer M (25 mM Hepes

pH 7.5, 20% glycerol, 0.4% Triton X-100, 0.5 mM EDTA and 1X mammalian protease

inhibitor cocktail) and incubated for one hour with biotinylated PCR products

corresponding to the TRAIL promoter that were bound to M-280 streptavidin-coupled

dynabeads (Invitrogen, Carlsbad, CA). Primer sequences for biotinylated DNA can be

found in the Table S8. Isolated DNA-protein samples were washed four times in Buffer

M, eluted with 2X sample buffer, and used for western blotting.

33

Immunoprecipitations

Epitope-tagged proteins:

Nuclear protein lysates were prepared as described for subcellular fractionations,

except transfected cells were treated with 5 M bortezomib for 18 hours prior to

harvesting cells. Lysates were diluted 1:4 in buffer M. Samples were pre-cleared with 2

g of normal mouse IgG and 25 L ProteinA/G agarose (Santa Cruz Biotechnology,

Santa Cruz, CA). Input samples were collected and pre-cleared lysates were incubated

with either 20 L of anti-V5 agarose affinity gel (antibody produced in mouse (Sigma, St.

Louis, MO)) or 20 L of anti-FLAG M2-agarose from mouse (Sigma, St. Louis, MO)

overnight. After this, samples were washed three times with buffer M and eluted

overnight. V5 agarose purifications were eluted overnight with 750 g/mL V5 peptide

(Sigma, St. Louis, MO) in PBS with 0.1% SDS. FLAG agarose purifications were eluted

overnight with 9 g/mL FLAG peptide (Sigma, St. Louis, MO) in buffer M. After

overnight incubations, samples were centrifuged at 8000 x g for 15 minutes, then

filtered using 0.35 M filters (USB, Cleveland, OH), then centrifuged at 16.1 x g for 15

minutes and were analyzed by western blotting.

Endogenous proteins:

100 million cells were washed with ice cold PBS and lysed in 1mL IP buffer (20mM Tris

pH 7.5, 150mM NaCl, 1mM EDTA, 1% TritonX-100, 1mM EGTA, 2.5mM sodium

34

pyrophosphate, 1mM B-glycerophosphate, 1mM NaVo4 and 1X mammalian PI) on ice

for 30 minutes with several brief intervals of vortexing. Samples were then sonicated for

5 seconds three times using a Fisher Scientific sonic dismembrator 60 at setting 3.5.

Then, samples were centrifuged for 10 minutes and pre-cleared with 7 g of normal

rabbit IgG and 25 L ProteinA/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA).

Input samples were collected and pre-cleared lysates were incubated with 7g of

indicated antibody (NFIL3 antibody (Santa Cruz H300), HDAC2 (Santa Cruz H-54) or

normal rabbit IgG (Santa Cruz)) overnight. Samples were washed three times with lysis

buffer and western blotting was performed using the IP/Western Blot ReliaBlot Kit

(Bethyl Laboratories, Montgomery, TX) according to the manufacture’s protocol. The

HDAC1 antibody was from Santa Cruz Biotechnology (H51).

Microarray Analysis

Total RNA was prepared from HEK293 or BT549 cells that were either treated with

control virus (scramble shRNA) or virus enabling the knockdown of NFIL3. RNA was

labeled using RNA Spike in Kit for Two color v4.0 and analyzed with Whole Human

Genome Oligo Microarray Kit (Agilent, Santa Clara, CA). The microarray data was

imported into the Base database for filtering and normalization . Microarray data was

extracted using Mean FG- Median BG (ch1=G, ch2=R), filtered: log (ch (1)*ch

(2))/2>=.1, and normalized using Lowess normalization. Data was then exported from

the Base database and analyzed using Partek software (St Louis, MO.). Gene set

enrichment analysis (GSEA) was done using the Broad Institute platform and Base-

35

filtered microarray data . Samples were analyzed with the default GSEA settings

(weighted, Signal2Noise), except the Burkitt’s lymphoma samples and the BT549 NFIL3

knockdown samples, which were analyzed using log2 ratio of classes and the

differences or classes respectively due to the number of samples.

Statistical Analysis

Student’s t tests were two sided with the statistical significance set at P < 0.05.

Quantitative Real Time PCR

Total RNA was prepared using the Qiagen RNeasy kit, which was then used to

generate cDNA using Superscript Reverse Transcriptase II (Invitrogen, Carlsbad, CA).

DNA preparations from ChIPs were also analyzed by quantitative PCR. Samples were

analyzed using SYBR green reagents and the AB7500 Real-time system from Applied

Biosystems (Foster City, CA). Expression levels were normalized to GAPDH in gene

expression experiments and -actin for chromatin immunoprecipitations.

Chromatin Immunoprecipitations

Chromatin immunoprecipitation (ChIP) analysis was performed as previously

described . Pre-cleared chromatin preparations (prepared from 10 million cells) were

incubated overnight with 7 g of one of the following antibodies: normal rabbit IgG,

36

FOXO1 antibody (H128), NFIL3 antibody (H300), HDAC2 (H-54), from Santa Cruz

Biotechnology (Santa Cruz, CA); Acetyl histone H3 (06-0599) and acetyl histone H4

(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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