Were stimulated with 2.5 ng/mL of TGF-b 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 = 36SD). doi:10.1371/journal.pone.0053238.gcontrol cell lines stably transduced with merely the ER-hormone binding domain (Fig. 2B). The ER:Sox4 fusion construct thus allows for conditional and robust activation of Sox4 by 4-OHT. We subsequently used this system to test whether SOX4 induced gene expression changes could contribute to EMT. To this end we generated polyclonal HMLE lines expressing ER:Sox4. Similar to U2OS cells, ER:Sox4 localization in HMLE cells was dependent on 4-OHT resulting in sustained translocation from the cytoplasm to the nucleus, and robustly activated the Sox4 responsive promoter in luciferase assays (Fig. 2C and Fig. 2D). Subsequently, ER:Sox4 HMLE cells were treated with 4-OHT as indicated 18325633 after which mRNA was isolated and analyzed by qRTPCR for expression changes in both epithelial and mesenchymal markers. Sox4 activation alone was Bromopyruvic acid sufficient to induce expression of mesenchymal markers including CDH2 (N-cadherin), VIM (vimentin) and FN1 (fibronectin), whereas expression of CDH1 (Ecadherin) remained unaltered (Fig. 3A). No expression changes in epithelial and mesenchymal markers were observed in the ER HMLE cells (Fig. S2A). Since the SOX-family member SOX9 has been demonstrated to directly regulate N-cadherin expression inchondrocytic CFK2 cells, we investigated SOX4 SIS3 biological activity mediated activation of the CDH2 promoter in more detail [18]. In order to test SOX4 mediated activation of the CDH2 gene we performed luciferase assays in HEK293 cells using a CDH2-promoter luciferase construct (kindly provided by Dr. David Goltzman). Co-transfection of Sox4 and the CDH2-promoter luciferase construct resulted in a potent induction of luciferase expression compared to control transfected cells. Only a minor effect of Sox4 was observed on the control pGL3 reporter lacking the CDH2promoter (Fig. 3B). Moreover, Sox4 mediated activation of the CDH2-promoter could be inhibited by overexpression of a dominant negative Sox4 construct (Sox4 1-135aa), consisting of the N-terminal region and DNA-binding domain thereby blocking Sox4 DNA-binding (Fig. 3B) [19]. These findings indicate that Sox4 expression is sufficient to induce CDH2 expression and most likely depends on its DNA-binding. Next, we wished to determine whether SOX4 could bind to the CDH2 promoter [18]. Bioinformatic analysis using the ContraV2 software revealed several highly conserved SOX4 binding motifs in the promoter region and the first intron of the CDH2 gene (Fig. 3C) [20]. WeSOX4 Affects Mesenchymal Genes in TGFb Induced EMTFigure 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 optima.Were stimulated with 2.5 ng/mL of TGF-b 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 = 36SD). doi:10.1371/journal.pone.0053238.gcontrol cell lines stably transduced with merely the ER-hormone binding domain (Fig. 2B). The ER:Sox4 fusion construct thus allows for conditional and robust activation of Sox4 by 4-OHT. We subsequently used this system to test whether SOX4 induced gene expression changes could contribute to EMT. To this end we generated polyclonal HMLE lines expressing ER:Sox4. Similar to U2OS cells, ER:Sox4 localization in HMLE cells was dependent on 4-OHT resulting in sustained translocation from the cytoplasm to the nucleus, and robustly activated the Sox4 responsive promoter in luciferase assays (Fig. 2C and Fig. 2D). Subsequently, ER:Sox4 HMLE cells were treated with 4-OHT as indicated 18325633 after which mRNA was isolated and analyzed by qRTPCR for expression changes in both epithelial and mesenchymal markers. Sox4 activation alone was sufficient to induce expression of mesenchymal markers including CDH2 (N-cadherin), VIM (vimentin) and FN1 (fibronectin), whereas expression of CDH1 (Ecadherin) remained unaltered (Fig. 3A). No expression changes in epithelial and mesenchymal markers were observed in the ER HMLE cells (Fig. S2A). Since the SOX-family member SOX9 has been demonstrated to directly regulate N-cadherin expression inchondrocytic CFK2 cells, we investigated SOX4 mediated activation of the CDH2 promoter in more detail [18]. In order to test SOX4 mediated activation of the CDH2 gene we performed luciferase assays in HEK293 cells using a CDH2-promoter luciferase construct (kindly provided by Dr. David Goltzman). Co-transfection of Sox4 and the CDH2-promoter luciferase construct resulted in a potent induction of luciferase expression compared to control transfected cells. Only a minor effect of Sox4 was observed on the control pGL3 reporter lacking the CDH2promoter (Fig. 3B). Moreover, Sox4 mediated activation of the CDH2-promoter could be inhibited by overexpression of a dominant negative Sox4 construct (Sox4 1-135aa), consisting of the N-terminal region and DNA-binding domain thereby blocking Sox4 DNA-binding (Fig. 3B) [19]. These findings indicate that Sox4 expression is sufficient to induce CDH2 expression and most likely depends on its DNA-binding. Next, we wished to determine whether SOX4 could bind to the CDH2 promoter [18]. Bioinformatic analysis using the ContraV2 software revealed several highly conserved SOX4 binding motifs in the promoter region and the first intron of the CDH2 gene (Fig. 3C) [20]. WeSOX4 Affects Mesenchymal Genes in TGFb Induced EMTFigure 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 optima.
