SupFigure 1:
A, Rbfox2 expression changes are reversible upon removal of TGFβ. Real time RT-PCR
assays of Rbfox2 expression in NMuMG and PY2T cell lines. Cells were treated with 2 ng/µl
TGFβ1 for up to 4-5 weeks. Then, TGFβ was removed for up to 7 days (NMuMG) or for up
to 17 days (PY2T). Total RNA was isolated from NMuMG and PY2T cells as indicated and
was reversed transcribed to cDNA using random hexamer oligos. The expression of Rbfox2
was measured by real time RT-PCR, and mRNA expression was normalized to Tbp
expression. B, splicing changes in Rbfox2 targets are reversible upon removal of TGFβ.
Alternative splicing of Rbfox2 target exons at different time points after induction of the EMT
in NMuMG and PY2T cells. EMT was induced by adding 2 ng/µl of TGFβ1 to the culture
medium for up to 4-5 weeks. Then, TGFβ was removed for up to 3 days (NMuMG) or for up
to 22 days (PY2T).
SupFigure 2:
A, alternative Rbfox2 isoform expression in TGFβ-dependent EMT. Real Time RT-PCR
assays of Rbfox2 isoform expression in NMuMG, PY2T and MTflEcad/MTdelEcad cells.
NMuMG and PY2T cells were treated with 2 ng/µl TGFβ1 for up to 4-5 weeks. Total RNA
was isolated from NMuMG and PY2T cells as indicated and was reversed transcribed to
cDNA using random hexamer oligos. The expression of Rbfox2 isoforms was measured by
real time RT-PCR, and mRNA expression was normalized to Tbp expression.
B, upregulation of Rbfox2 in MTdeltaEcad cells. Immunoblotting analysis of cell lysates
obtained from MTflEcad and MTdeltaEcad was performed with antibodies that specifically
detect Rbfox2. Immunoblotting for actin was used as the loading control.
SupFigure 3:
A, alternative splicing of CD44 in TGFβ-dependent EMT. Alternative splicing of CD44
exons v8-v10 at different time points after induction of the EMT in NMuMG and PY2T cells.
EMT was induced by adding 2 ng/µl of TGFβ1 to the culture medium. Shown are changes in
mRNA expression (left) and changes in alternative exon inclusion (right). B, Rbfox2
depletion does not alter splicing of CD44 exons v8-v10. The cells were treated with 2 ng/µl of
TGFβ1 for 0 or 3 days or 4-5 weeks and then transfected with the indicated siRNAs. Fortyeight hours after transfection, total RNA was isolated, and cDNA synthesis and real time PCR
were performed.
SupFigure 4:
Expression of Esrps in NMuMG and PY2T cells. A, B, RT-PCR assay of Esrps expressed in
NMuMG and PY2T cells at different times after EMT was induced by adding 2 ng/µl TGFβ1
to the culture medium. Samples were prepared as described in the legends to Figure 1.
SupFigure 5:
A, B, Rbfox2-depleted cells maintain a mesenchymal phenotype. NMuMG (A) or PY2T cells
(B) were treated with 2 ng/µl TGFβ1 for 4-5 weeks and then plated on coverslips and
transfected with control or Rbfox2-specific siRNAs. Forty-eight hours after transfection, cells
were processed for immunofluorescence analysis using an Rbfox2-specific antibody to
control for knockdown efficiency, a vinculin-specific antibody to analyze the formation of
focal adhesions and Phalloidin488 to visualize actin stress fibers.