Ng ER:Sox4 were treated with 4-OHT (100 nM) as indicated. Cells

Ng 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 = 36SD). doi:10.1371/journal.pone.370-86-5 0053238.gand E-cadherin expression and localization was analyzed by immuno-fluorescence microscopy after activation of Sox4 for the indicated time-points. Sox4 activation again resulted in increased expression of N-cadherin without affecting the levels of E-cadherin expression (Fig. 3G). Since TGF-b-mediated downregulation of CDH1 may only occurs after three days [21], we investigated whether prolonged activation of Sox4 by 4-OHT for four days could result in reduced CDH1 expression. As expected, qRT-PCR analysis revealed that continued Sox4 activation increased expression of the mesenchymal markers CDH2, VIM and FN1, but did not result in downregulation of the epithelial markers CDH1 and CTNNB1 (b-Catenin) (Fig. S2C, left panel). No changes were observed in the ER HMLE cells (Fig. S2C, right panel). Consistent with the qRT-PCR results, western blot analysis demonstrated that prolonged 4-OHT treatment of ER:Sox4 HMLE cells resulted in upregulation of N-cadherin, but did not result in altered expression of the epithelial markers E-cadherin and b-Catenin (Fig. S2D). No alterations in the expression of epithelial and mesenchymal markers was observed in the ER HMLE cells (Fig. S2D). In addition, immuno-fluorescence microscopy showed no change in E-cadherin expression in both the ER:SOX4 and ER HMLE cells after 4 day stimulation with 4OHT (Fig. S2E) Thus, Sox4 activation is sufficient to induce expression of the mesenchymal marker N-cadherin in HMLE cells without altering E-cadherin expression.demonstrated that after 10 days of TGF-b-mediated EMT induction, the SOX4 knockdown HMLE cells expressed less Ncadherin than the scrambled control cells (Fig. 4D). Taken together, these data show that TGF-b-mediated regulation of SOX4 expression and activity is required for efficient induction of N-cadherin and potentially other mesenchymal markers.DiscussionTumor metastasis is the major cause of cancer-related death and in a wide-variety of tumors the epithelial-to-mesenchymal transition has been demonstrated to contribute to this process. EMT is characterized by the loss of epithelial makers and acquisition of mesenchymal traits, confers invasive, chemotherapy resistance and stem cell properties to tumor cell [4,22]. Cancers exploit this developmental process and as a result can acquire a more invasive and metastatic phenotype. The TGF-b signaling pathway is one of the most potent and well-studied inducers of EMT during both embryonic development and cancer progression. Here, we demonstrate that expression of the developmental Lixisenatide web transcription factor SOX4 increases during TGF-b-induced EMT and can contribute to the change in cellular phenotype by controlling the expression of mesenchymal markers in human mammary epithelial cells. In the developing mouse SOX4 is highly expressed in mesenchymal tissues and at variable levels in other tissues [23,24,25]. In combination with SOX11 and SOX12, two additional members of the SOXC group of transcription factors, SOX4 has been demonstrated to be vital for the survival of neuronal and mesenchymal progenitor cells, indicating that also during development SOX4 contributes to acqui.Ng 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 = 36SD). doi:10.1371/journal.pone.0053238.gand E-cadherin expression and localization was analyzed by immuno-fluorescence microscopy after activation of Sox4 for the indicated time-points. Sox4 activation again resulted in increased expression of N-cadherin without affecting the levels of E-cadherin expression (Fig. 3G). Since TGF-b-mediated downregulation of CDH1 may only occurs after three days [21], we investigated whether prolonged activation of Sox4 by 4-OHT for four days could result in reduced CDH1 expression. As expected, qRT-PCR analysis revealed that continued Sox4 activation increased expression of the mesenchymal markers CDH2, VIM and FN1, but did not result in downregulation of the epithelial markers CDH1 and CTNNB1 (b-Catenin) (Fig. S2C, left panel). No changes were observed in the ER HMLE cells (Fig. S2C, right panel). Consistent with the qRT-PCR results, western blot analysis demonstrated that prolonged 4-OHT treatment of ER:Sox4 HMLE cells resulted in upregulation of N-cadherin, but did not result in altered expression of the epithelial markers E-cadherin and b-Catenin (Fig. S2D). No alterations in the expression of epithelial and mesenchymal markers was observed in the ER HMLE cells (Fig. S2D). In addition, immuno-fluorescence microscopy showed no change in E-cadherin expression in both the ER:SOX4 and ER HMLE cells after 4 day stimulation with 4OHT (Fig. S2E) Thus, Sox4 activation is sufficient to induce expression of the mesenchymal marker N-cadherin in HMLE cells without altering E-cadherin expression.demonstrated that after 10 days of TGF-b-mediated EMT induction, the SOX4 knockdown HMLE cells expressed less Ncadherin than the scrambled control cells (Fig. 4D). Taken together, these data show that TGF-b-mediated regulation of SOX4 expression and activity is required for efficient induction of N-cadherin and potentially other mesenchymal markers.DiscussionTumor metastasis is the major cause of cancer-related death and in a wide-variety of tumors the epithelial-to-mesenchymal transition has been demonstrated to contribute to this process. EMT is characterized by the loss of epithelial makers and acquisition of mesenchymal traits, confers invasive, chemotherapy resistance and stem cell properties to tumor cell [4,22]. Cancers exploit this developmental process and as a result can acquire a more invasive and metastatic phenotype. The TGF-b signaling pathway is one of the most potent and well-studied inducers of EMT during both embryonic development and cancer progression. Here, we demonstrate that expression of the developmental transcription factor SOX4 increases during TGF-b-induced EMT and can contribute to the change in cellular phenotype by controlling the expression of mesenchymal markers in human mammary epithelial cells. In the developing mouse SOX4 is highly expressed in mesenchymal tissues and at variable levels in other tissues [23,24,25]. In combination with SOX11 and SOX12, two additional members of the SOXC group of transcription factors, SOX4 has been demonstrated to be vital for the survival of neuronal and mesenchymal progenitor cells, indicating that also during development SOX4 contributes to acqui.