Legends to Supplementary Figures
Sup. Figure 1: Prognostic value of FOSL2 and FOSB expression for breast cancer
patients. (a) Correlation of the levels of FOSL2 and FOSB expression and prognosis in
human breast cancer patients using the KM plotter integrative data analysis tool
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www.kmplot.com. Shown are a Kaplan-Meier survival plots for patient samples
classified as having high (red) or low (black) FOSL2 (a, b) or FOSB (c, d) median
expression to assess metastasis-free (DMFS: a-c) or overall (OS: b-d) survival. Hazard
ratio (HR) with 95% confidence intervals and logrank P value are displayed and the
number of patients at risk in the high and low groups indicated for each time point. The
total number of patients with available clinical data is DMFS: n=1609, OS: n=1105.
Sup. Figure 2: Fra-1 expression in EpH4 cells leads to EMT in vitro. (a) AP-1 protein
expression in EpH4, Epras and EpRasXT. Heterochromatin Protein 1a (Hp1) is used to
control loading. (b) qRT-PCR analysis of c-fos and fra-1/2 expression. Bars=mean±sd,
n=2, expression in EpH4 is set to 1. (c) Phalloidin visualization of actin, IHC analysis of
E-cadherin, AJ proteins and fibronectin (Fn) in EpH4-derived cells. Bar=20µM. (d) qRTPCR analysis of epithelial and mesenchymal associated genes in EpH4-derived cells.
Bars=mean±sd, n=2, expression in EpH4 is set to 1. (e) Western blot analysis of 5 and
6 integrins in EpH4-derived cells. Tubulin and actin are used to control the loading.
Sup. Figure 3: Effects of Fra-1 knock-down in EpFra1 cells. (a) Representative phase
contrast photographs of EpFra1 cells after Fra-1 knock-down. (b) Analysis of Fra-1
protein after Fra-1 knock-down. Hdac3 is used to control loading. qRT-PCR analysis of
epithelial (c) and mesenchymal (d) genes after Fra-1 knock-down Bars=mean±sd, n=2,
expression in EpH4 is set to 1. (e) Western blot analysis of epithelial and mesenchymal
proteins after Fra-1 knock-down. Tubulin and -Adaptin are used to control loading. (f)
Western blot (anti-Fra-1, top) and qRT-PCR (cdh1, encoding for E-cadherin, bottom)
analysis of mouse NMuMG mammary epithelial cell pools transduced with GFP or
Fra1ER expressing retroviruses and treated for the indicated time points with 4hydroxyTamoxifen (Tmx). Hdac3 and Gapdh are used to control loading and nuclear
enrichment. Bars=mean±sd, n=2, expression in untreated NMuMG-GFP is set to 1.
Sup. Figure 4: Global gene expression profile and cell death –related gene
expression in Fra-1-expressing EpH4 cells. (a) Heat-map for differentially expressed
genes in EpFra1 cells. Annotated genes with at least 2-fold change in expression in both
lines and a p-value<0.05 were considered significant. The M-value represents the log2fold change between each cell line and the parental EpH4. (b) Expression profiling of
genes related to cell death and/or the p53 pathway revealed by the bioinformatics analysis
assessed by qRT-PCR in EpFra1 cells. Bars=mean±sd, expression in EpH4 is set to1 and
the results of the two EpFra1 lines are pooled.
Sup. Figure 5: Analysis of TGF in Fra-1-expressing EpH4 cells. qRT-PCR analysis
of tgfb1 after Fra1 knock-down (a) and c-myb and tgfb receptors in EpFra1 cells (b).
Bars=mean±sd, n=2, expression in EpH4 is set to 1. (c) Gapdh and Hdac3 western blot in
nuclear from EpH4-derived cells. Comparable amounts of the corresponding non-nuclear
(cytoplasmic) fractions and whole cell extracts (WCE) from one EpFra1 line were also
blotted. (d) ChIP analysis of the tgfb1 promoter in EpH4-derived cells using -Fra-1 or
IgG: Representative agarose gel pictures of qPCR products. (e) Effects of TgfbR inhibitor
treatment (5 days) on mesenchymal genes assessed by qRT-PCR in EpFra1 cells.
Bars=mean±sd, expression in EpH4 is set to1 and the results of the two EpFra1 lines are
pooled.
Sup. Figure 6: Analysis of the Zeb1/2 pathway in EpFra1 cells. (a) Heat-map for zeb1
and zeb2 expression in EpFra1 cells from microarray analysis. M values and fold change
relative to EpH4 are presented. Analysis of Zeb1, Zeb2 and snai2/Slug mRNA (b) and
protein (c) expression after Fra-1 knock-down. Bars=mean±sd, n=2, expression in EpH4
is set to1. Tubulin is used to control loading. (d) Heat-map for differentially expressed
genes in EpFra1 cells that overlap with published Zeb-related microarray data. Left:
Genes down-regulated in EpFra1 cells and potentially repressed by Zeb134 or Zeb235 or
both. Right: Genes down-regulated in EpFra1 cells and potentially repressed by Zeb235.
M values and fold change relative to EpH4 are presented. (e) qRT-PCR analysis of Zeb
target genes after Fra-1 knock-down. Bars=mean±sd, n=2, expression in EpH4 is set to1.
Sup. Figure 7: Effect of TGF inhibition and analyses of EpRas-Fra1 cells. Effects of
TgfbR inhibitor treatment on Zeb1, Zeb2 and snai2/Slug mRNA (a) and protein (b) in
EpFra1 cells. Bars=mean±sd, expression in EpH4 is set to1 and the results of the two
EpFra1 lines are pooled. Gapdh is used to control loading. Analyses of EpRas-Fra1 cells:
(c) Fra-1 mRNA expression relative to parental EpRas cells (set to 1). (d) Representative
phase contrast pictures. qRT-PCR analysis of e-cadherin, vimentin (e) zeb1 and zeb2 (f)
expression in EpRas-Fra1 cells. Bars=mean±sd, n=2, expression in EpRas is set to 1. (g)
Heat-map for differentially expressed zeb target genes in EpRas-Fra1 cells after gene
expression profiling. Annotated genes with at least 2-fold change in expression in two
independent EpRas-Fra1 lines and a p-value<0.05 were considered significant. Fold
change relative to EpRas are presented and genes which are common with EpFra1 cells
are bolded.
Sup. Figure 8: Transcriptional control of Zeb1 and Zeb2 by Fra-1. (a) qRT-PCR
analysis of mir221 and trps1 in EpH4-derived cells. Bars=mean±sd, n=2, expression in
EpH4 is set to 1. (b) Trps1 protein expression in EpH4-derived cells. Hdac3 is used to
control loading. ChIP analysis of zeb1 (c) and zeb2 (d) promoters in EpH4-derived cells
using -Fra-1 or IgG. Representative agarose gel pictures of qPCR products.
Sup. Figure 9: Effects of manipulating zeb1 on zeb2 expression in EpH4-derived
cells. (a) Gapdh and Sp1 western blot in nuclear fractions of EpFra1-zeb-knock down
cells. (b) qRT-PCR analysis of Zeb knock-down cells generated using five independent
shRNA constructs for zeb1 and zeb2 expression. Bars represent the mean±sd results of
the two independent cultures generated with each shRNA. Expression in the parental
EpFra1 cell line is set to 1. EpFra1-sZ1.1 and EpFra1-sZ1.2 used in main figures
correspond to zeb1-sh1 and zeb1-sh4, respectively. (c) Representative phase contrast
pictures of EpFosER cell clones stably expressing GFP (EpGFP) or Zeb1 (EpdEF1GFP)
cultivated in the absence of tamoxifen40. (d) qRT-PCR analysis of zeb1, zeb2, cdh1
(encoding for E-cadherin) and vimentin (vim) in EpdEF1GFP cells. Bars=mean±sd, n=2,
expression in EpH4 is set to 1.
Sup. Figure 10: Effects of Knock-down of zeb1 or zeb2 in Fra-1-expressing EpH4
cells on additional Zeb & EMT targets/regulators. qRT-PCR analysis of Zeb knockdown clones for epithelial genes (a), mir200/trps1 (b) and mesenchymal genes (c). Bars
represent the mean±sd results of at least two EpFra1-sZ1 and two EpFra1-sZ2 clones.
Expression in EpH4 is set to 1. (d) Western blot analysis of 5 and 6 integrins in Zeb
knock-down clones. -Adaptin and Hdac3 are used to control the loading.
Sup. Table 1: Gene Set Enrichment Analysis (GSEA) of EpFra1 cells for canonical
transcription factor motifs. Enriched putative transcription factor binding sites motifs in
the gene sets upregulated (A) or downregulated (B) in EpFra1 cells compared to EpH4
cells. The most significantly enriched motifs are highlighted and the annotated
transcription factor and p-value indicated.
Sup. Table 2: Reagents list and catalogue numbers.
Sup. Table 3: Primers sequences and description.