Figure 4

TGFβ-mediated LIF upregulation regulates p21 expression at the transcriptional level. A, WM278 cells were treated or not with TGFβ and LIF for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. B, WM278 cells transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by qPCR. Data is graphed as the mean of the fold induction of p21 gene expression in response to TGFβ for at least 3 biological replicates. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the mock and scrambled siRNA treated conditions (*p < 0.05, **p<0.01). C, WM278 cells were transfected with a scrambled or a LIF siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin content was used as a control. D, WM278 and WM793B cells transfected with a scrambled or a LIF siRNA 24 h earlier were transfected with the p21 luciferase reporter and the Renilla luciferase constructs, treated or not with TGFβ for 24 h, lysed and assessed for luciferase activity. Data is graphed as the arithmetic mean of relative luciferase units normalized to Renilla luciferase activity for 3 independent experiments. The error bars are the standard errors of the mean. For statistical analysis the t-test was performed compared to the non-treated control (***p<0.001). E, WM278 cells were treated with LIF (100 ng/mL) or F, with TGFβ for different period of times or G, with scrambled or LIF siRNA in the presence or absence of TGFβ, and phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 content was used as a control.