Fig. 5

Downregulation of Twist1 is required for A20-mediated inhibition of proliferation and migration of HCC cells. a The total mRNA of indicated cells was extracted and the relative mRNA expression levels of EMT related genes were determined using qPCR methods. Results were normalized by β-actin. *, p < 0.05. b Protein levels of Twist1 in indicated cells were determined by Western blot assays. c Cells were transfected with pBabe-puro-flag-twist1 or empty vector for 24 h and then cell lysates were subjected to Western blot assay and probed with the indicated antibodies. d The proliferative properties of the cells treated as described for panel C were determined by the Brdu cell proliferation assay at indicated times. Plots are mean ± SEM of data from three independent experiments. *, p < 0.05. e The migratory properties of the cells treated as described for panel C were analyzed by transwell migration assay. Representative results are shown in the upper panel. Plots in the lower panel are mean ± SEM of data from three independent experiments. *, p < 0.05. f Cells were transfected with Twist1 siRNA (siTwist1, 20nM) or scrambled siRNA (si con, 20nM) for 60 h and then cell lysates were subjected to Western blot assay and probed with the indicated antibodies. g The proliferative properties of the cells treated as described for panel F were determined by the Brdu cell proliferation assay at indicated times. Plots are mean ± SEM of data from three independent experiments. *, p < 0.05. h The migratory properties of the cells treated as described for panel F were analyzed by transwell migration assay. Representative results are shown in the upper panel. Plots in the lower panel are mean ± SEM of data from three independent experiments. *, p < 0.05