Download Supplementary Figure Legends

Lambert, A.W. et al.
2015
Supplementary Figure Legends
Supplementary Figure S1. POSTN expression is TGF-β dependent in M cells. (A) MII
cells were treated with TGF-β1 (10 ng/ml), or vehicle control, for 24 hours prior to RNA
isolation. POSTN expression was measured by qRT-PCR. (B) POSTN expression was
assessed in MIII cells with stable expression of SMAD7, or an shRNA directed at
SMAD2, and control cells (pB and shGFP, respectively) (21). * P < 0.05, ** P < 0.01;
error bars represent SEM.
Supplementary Figure S2. Co-expression of POSTN and integrin αvβ3 in basal-like
breast cancer cell lines. (A) Western blot analysis for POSTN in media conditioned
from the indicated cell lines. Ponceau staining was used to show protein loading. (B)
Flow cytometry was used to assess the surface levels of integrin αvβ3 in the indicated
cell lines.
Supplementary Figure S3. Knockdown of POSTN and ITGB3 in SUM159 cells. (A)
Left: qRT-PCR of POSTN expression in control (shGFP) and knockdown (shPN)
SUM159 cells; Right: An ELISA assay was used to measure POSTN secretion in the
indicated SUM159 cells. Inset shows western blot analysis for POSTN expression in
the cell lysates from the same cell lines. (B) Flow cytometric analysis of surface levels
of integrin β3 (CD61) in SUM159 shGFP and shBeta3 cells. (C) The proliferation of the
indicated SUM159 cells was assessed by trypan blue exclusion staining at day 0 and
after 72 hours of growth. The relative increase in cell number during this time was
normalized to shGFP cells. (D) The apoptotic population was measured by Annexin-VPI staining. (E) Photomicrographs to show the morphology of the indicated SUM159
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2015
cells. (F) Western blot showing the expression of characteristic EMT markers in the
indicated cell lines. ** P < 0.01, error bars represent SEM.
Supplementary Figure S4. Functional validation of POSTN and ITGB3 function using
additional shRNA hairpins. (A) Left: qRT-PCR of POSTN expression in control (shGFP)
and knockdown (shPN-2) SUM159 cells; Right: qRT-PCR of ITGB3 expression in
control (shGFP) and knockdown (shBeta3-2) SUM159 cells. (B) The indicated control
of knockdown SUM159 cells were grown as mammospheres and quantified, n=3. (C)
Representative ALDH plots of the indicated SUM159 cells. Cells were grown in serumreduced media (0.1% serum) for 48 hours prior to analysis. DEAB, an inhibitor of
ALDH, was used as a gating control. (D) Expression of IL6, and IL8 was measured in
the indicated SUM159 cell lines by qRT-PCR. RNA was isolated from cells grown in
serum-reduced media for 48 hours. (E) Western blot analysis of STAT3 phosphorylation
(tyrosine 705), total STAT3 and actin in the indicated SUM159 cell lines. Protein was
isolated from cells grown in serum-reduced media (0.1% serum) for 48 hours. (F) The
activity of a transfected NF-κB luciferase reporter was measured in the indicated cell
lines after 16 hours of growth in serum-reduced media (0.1% serum). Relative
luminescence units (RLUs) were calculated based on co-transfection of a Renilla
luciferase construct. TNF-α treatment served as a positive control, n=3 * P < 0.05, ** P
< 0.01, error bars represent SEM.
Supplementary Figure S5. Knockdown of POSTN in MIII cells impairs mammosphere
formation. (A) Left: qRT-PCR of POSTN expression in control (shGFP) and knockdown
(shPN) MIII cells; Right: An ELISA assay was used to measure POSTN secretion in the
indicated MIII cells. Inset shows western blot analysis for POSTN expression in the cell
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2015
lysates from the same cell lines. (B) Control (shGFP) or knockdown (shPN) MIII cells
were grown as mammospheres and quantified, n=3. * P < 0.05, ** P < 0.01, error bars
represent SEM.
Supplementary Figure S6. Gene sets repressed in SUM159 shPN cells. (A) Gene set
enrichment analysis (GSEA) of the top gene sets significantly repressed (FDR q < 0.05)
in SUM159 shPN cells compared to shGFP cells, n=3. RNA was isolated from cells
grown in serum-reduced media (0.1% serum) for 48 hours. (B) Leading edge analysis
for the 20 gene sets most strongly downregulated in shPN versus shGFP cells as
determined by GSEA (FDR q < 0.1). Each row indicates a gene set and each column
corresponds to a gene, with a shaded cell indicating that the gene was in the leading
edge of the GSEA and an empty cell indicating that the gene was not present in the
gene set.
Supplementary Figure S7. Conditioned media rescue of the ALDH subpopulation and
inhibition of ERK signaling in SUM159 cells. (A) The indicated SUM159 cells were
treated with serum-reduced media (mock) or conditioned media collected from control
shGFP cells for 24 hours prior to quantification of the ALDH-positive subpopulation,
n=3. (B) Ingenuity Pathway Analysis of genes differentially expressed in SUM159 shPN
cells, compared to shGFP cells, shows that shPN cells exhibit significant activation of a
gene expression profile characteristic of cells treated with PD98059, an ERK pathway
inhibitor (score +4.087, P = 1.58 x 10-8). Red shapes indicate expression is increased
relative to shGFP cells while green shapes indicate repression. Blue lines are
consistent with predicted repression in response to the inhibitor and orange lines are
consistent with predicted induction. Yellow and gray lines depict inconsistent changes.
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2015
(C) Same as in (B) but shows a gene expression profile characteristic of cell treated
with U0126, another ERK pathway inhibitor (score +2.391, P = 2.29 x 10 -8). (D)
SUM159 shGFP cells were treated with DMSO (mock) or an ERK pathway inhibitor
(U0126) for 24 hours prior to RNA isolation. IL1A, IL6 and IL8 mRNA levels were
measured by qRT-PCR. * P < 0.05, ** P < 0.01; error bars represent SEM.
Supplementary Figure S8. POSTN regulates cancer stem cell phenotypes in Hs578T
cells. (A) qRT-PCR of POSTN levels in control (shGFP) and knockdown (shPN) cells.
Inset shows western blot analysis for POSTN in the conditioned media of the same
cells. (B) Control (shGFP) or knockdown (shPN) Hs578T cells were grown as
mammospheres and quantified, n=3. (C) The fold change in the percentage of ALDH+
cells was determined using the ALDEFLUOR assay. The indicated Hs578T cells were
grown in serum-reduced media (0.1% serum) for 48 hours prior to analysis, n=3. (D)
qRT-PCR was used to measure IL6 expression in the indicated Hs578T cells. (E)
Western blot analysis of STAT3 phosphorylation (tyrosine 705) in the indicated Hs578T
cell lines. Protein was isolated from cells grown in serum-reduced media for 48 hours. *
P < 0.05, ** P < 0.01; error bars represent SEM
Supplementary Figure S9. POSTN expression in breast tumors. POSTN expression
in a publicly available dataset of primary breast cancer samples (GSE21653), according
to molecular subtype.
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