Table 1.
qRT-PCR primers.
Table 2.
Antibodies conditions for western blot analysis.
Table 3.
qRT-PCR primer sequences used in the biotinylated oligonucleotide pull down assay.
Table 4.
Gene Ontology category ''positive regulation of transcription, DNA-dependent (GO:0045893) genes significantly regulated and greater than 2 fold upregulated during TGF-β-induced EMT.
Figure 1.
SOX4 expression is increased by TGF-β during EMT.
(A) MARA analysis predicts Sox activity during EMT in HMEC and NMuMG cells (see text for details) (B) Public microarrays databases generated in non-transformed HMEC cells treated with TGF-β or left untreated were analyzed and SOX4 expression was assessed. (C) HMLE cells were stimulated with 2.5 ng/mL of TGF-β as indicated, lysed and mRNA expression of CDH2 (N-cadherin), VIM (vimentin), CDH1 (E-cadherin) were analyzed by qRT-PCR. (D) HMLE cells were stimulated with 2.5 ng/mL of TGF-β as indicated and lysed. The protein lysates were visualized by Western-Blotting using anti-N-cadherin, anti-SOX4, anti-Tubulin and anti-E-cadherin antibodies. Western blot data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).
Figure 2.
Generation of a Sox4 conditional activation system.
The hormone binding domain of the ER was fused to the N-terminus of Sox4. (A) ER:Sox4 was stably transduced in U2OS cells. Immuno-fluorescence analysis of ER:Sox4 localization using an anti-ER antibody after stimulation with 4-OHT (100 nM) for the time points indicated. (B) U2OS cells expressing ER and ER:Sox4 were transfected with an optimal Sox4 luciferase reporter construct and treated overnight with 4-OHT (100 nM) after which luciferase activity was measured. (C) ER:Sox4 localization in HMLE cells in presence and absence of 4-OHT. (D) HMLE cells expressing ER and ER:Sox4 were transfected with an optimal Sox4 luciferase reporter construct and stimulated overnight with 4-OHT (100 nM) after which luciferase activity was measured. Confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).
Figure 3.
Sox4 activation induces upregulation of mesenchymal markers.
(A) HMLE cell lines expressing ER:Sox4 were stimulated with 4-OHT (100 mM) as indicated. Cells were lysed and mRNA expression of CDH2 (N-cadherin), VIM (vimentin), FN1 (fibronectin) and CDH1 (E-cadherin) was analyzed by qRT-PCR. (B) HEK293T cells were transiently transfected with Flag-tagged Sox4 Wt or Flag-tagged Sox4 1-135aa and co-transfected with a CDH2 luciferase reporter construct as indicated. After 48 hours luciferase activity was measured. Protein expression was assayed by Western blotting using anti-Flag antibody. (C) Schematic representation of the CDH2 promoter region and predicted Sox4 binding sites. (D) Chromatin Immunoprecipitation (ChIP) assay using IgG and SOX4 antibodies in MDA-MB-231 cells. Real time PCR was performed using CDH2 promoter-specific primers to test SOX4 occupancy at this region. (E) HEK293T cells were transiently transfected with the empty vector pcDNA3 or Flag-tagged Sox4 Wt. After 48 hours cells were harvested and nuclear fraction was extracted. Nuclear extracts were used to perform a biotinylated oligonucleotide pull down assay in which three CDH2 promoter sites and two sites localized in the first intron of CDH2 were included. Lysates were assessed by western blotting using anti-Flag antibody. (F) HMLE cell lines expressing ER:Sox4 were stimulated with 4-OHT (100 nM) as indicated or left untreated. Cells were lysed and lysates were analyzed by Western blotting using anti-N-cadherin, anti-Tubulin, anti-E-cadherin and anti-ER antibodies. (G) HMLE cells expressing ER:Sox4 were treated with 4-OHT (100 nM) as indicated. Cells were fixed, permeabilized and the expression of N-cadherin and E-cadherin was assessed (green and red respectively). Blue = DAPI. Western blot and confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).
Figure 4.
SOX4 knockdown delays TGF-β-induced EMT.
HMLE cells line were either transduced with a control shRNA (Scr shRNA) or with a shRNA targeting SOX4 (SOX4 shRNA). (A) Scr shRNA and SOX4 shRNA cell lines were either treated with 2.5 ng/mL of TGF-β for 7 days or left untreated. Cells were lysed and analyzed by Western Blotting using anti-SOX4 and anti-Tubulin antibodies (B) HMLE cell lines expressing Scr shRNA and SOX4 shRNA were stimulated with 2.5 ng/mL of TGF-β as indicated. Cells were lysed and mRNA expression of CDH2 (N-cadherin), VIM (vimentin) and CDH1 (E-cadherin) were assessed. (C) Cell lysates of HMLE cell lines expressing Scr shRNA and SOX4 shRNA stimulated with 2.5 ng/mL of TGF-β as indicated and analyzed by western blotting using N-cadherin, anti-Tubulin and anti-E-cadherin antibodies. (D) Scr shRNA and SOX4 shRNA cell lines were stimulated with 2.5 ng/mL of TGF-β as indicated. Cells were fixed, permeabilized and N-cadherin expression was assessed (green). Blue = DAPI; red = phallodin. Western blot and confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).