Western-blot analyses of p27 and cyclin D1 expression levels after sorafenib and RNAi treatment. (a) Inhibition of BRAF by BRAF-C2 siRNA decreased the levels of cyclin D1 in all the cell lines and increased the levels of p27Kip1 in 8505C and TPC1 cells, but not in C643 cells as demonstrated in the Western-blot analysis and by quantification of the levels of cyclin D1 and p27 relative to actin (data not shown). (b, c) Sorafenib treatment induced alterations in cyclin D1 and p27Kip1 at different time points depending on the cell line. The graphs represent the quantification of the levels of cyclin D1 and p27Kip1 proteins relative to actin and compared with the DMSO control. (b) Treatment with 4 μM of sorafenib led to decreased expression of cyclin D1 at different time points and at different levels in all the cell lines. (c) Concerning the levels of p27Kip1 after treatment with sorafenib: in 8505C cells there was an increase in all the time points except after 12 h; in TPC1 there is an increase after 12 h, 24 h and 48 h; in C643 there is an increase after 48 h. All values and figures presented are the mean of at least three independent experiments.