PIAS1 Negatively Regulates EMT Figure 2. PIAS1 inhibits the ability of TGFbto suppress E-cadherin promoter activity. A) PIAS1 decreases TGFb inhibition of E-cadherin promoter activity in a SUMO E3 ligase-dependent manner. Lysates of NMuMG cells transfected with the E-cadherin promoter driven luciferase (E- cadherin-p-luciferase) reporter and the b-galactosidase (b-gal) expression construct together with an empty expression vector (2), or one encoding wild type PIAS1 (WT) or SUMO E3 ligase mutant PIAS1 (CS), and left untreated or incubated with 100 pM TGFb for 48 h, were subjected to luciferase and b-galactosidase assays (see MATERIALS and METHODS). Ai) Each column is the mean (6 SEM, n = 5) of b-galactosidase normalized luciferase data relative to that of the basal (2 TGFb) vector control. Aii) Each column is the mean (6 SEM, n = 5) of the percent repression of luciferase activity by TGFb relative to that of the basal (2 TGFb) of the corresponding transfection as shown in Ai (see MATERIALS and METHODS). B) PIAS1 reversal of TGFb-inhibition of the E-cadherin promoter activity is dose-dependent. Lysates of untreated or TGFb-treated cells transfected with E-cadherin-p- luciferase reporter and b-gal plasmid together with an empty vector or increasing concentrations of plasmids containing PIAS1 (WT) or PIAS1 (CS) were subjected to luciferase and b-galactosidase assays. Each column is the mean (6 SEM, n = 6) of percent suppression of E-cadherin luciferase activity by TGFb. C) Endogenous PIAS1 knockdown enhances TGFb suppression of E-cadherin reporter expression. Lysates of untreated or TGFb treated NMuMG cells transiently transfected with the E-cadherin-p-luciferase reporter and b-gal construct together with a control vector (control) or one encoding short hairpin RNAs to knockdown endogenous PIAS1 (PIAS1i), were subjected to luciferase and b-galactosidase assays. Each column is the mean (6 SEM, n = 6) of percent suppression of E-cadherin luciferase activity by TGFb from six independent experiments. * indicates statistical significant difference (P,0.05) as compared to the corresponding TGFb treatment vector control. doi:10.1371/journal.pone.0013971.g002 going TGFb-induced EMT, the SnoN (EdA) mutant failed to inhibit the TGFb response (Figures 6F and 6G). Collectively, our findings suggest that sumoylation contributes significantly to the ability of SnoN to inhibit TGFb-reduction of E-cadherin and hence induction of EMT. In turn, TGFb decreases the level of sumoylated SnoN to facilitate EMT. PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e13971