Figure 3

TGFβ mediates its effects through p21 regulation. A, WM278 and WM793B were treated or not with TGFβ for 24 h and expression of LIF, p21, p15, and c-MYC was analyzed by Western blot(left panel). β-tubulin was used as control. Right panel: Densitometry of LIF and p21 protein levels. Error bars are standard errors of mean and t-test was performed compared to non-treated control (***p<0.001). B, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and cell cycle distribution was analyzed by propidium iodide staining. Data is graphed as mean of percentages of cells in G1 phase for 3 independent experiments. Error bars are standard errors of mean and t-test was performed compared to non-treated control (**p < 0.01, *p < 0.05). C, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and p21 expression was analyzed by Western blot. β-tubulin was used as control. D, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and apoptosis was determined by caspase3/7 activity. Data is graphed as geometric mean of relative luciferase units normalized to non-treated control for at least 3 independent experiments. Error bars are the standard errors of mean and z-test was performed compared to non-treated control (*p < 0.05). E, WM278 cells transfected with scrambled or p21 siRNA 48 h earlier were treated or not with TGFβ for 24 h and gene expression was analyzed by qPCR. Data is graphed as mean of fold induction of gene expression in response to TGFβ for at least 3 replicates. Error bars are standard errors of mean and t-test was performed compared to mock and scrambled siRNA treated conditions (*p < 0.05).