Effects of reducing basal pERK levels on sorafenib sensitivity. (A, B) Immunocytochemical staining of pERK (200×) in MHCC97-H cells after treatment with 20 μM U0126 for 6 hours. (A) DMSO (0.1%) was used as solvent control. (B) U0126 20 μM. (C) pERK expression was reduced after U0126 treatment in MHCC97-H cells. The expression rate of pERK was calculated from pERK density determined as described in Materials and methods and the rate in the control group was set as the 100% baseline. Columns represent means from six samples in each group; bars indicate standard deviation; *, P < 0.05, Student's t-test was used when compared with solvent control. (D) Effects of U0126 treatment for 6 hours on cell proliferation in MHCC97-H cells. (E) Effects of sorafenib individually and in sequential combination with U0126 on cell proliferation in MHCC97-H cells. In the sequential combination experiments, cells were pre-treated with 20 μM U0126 for 6 hours and then exposed to sorafenib for a further 24 hours. Each value represents the average of six independent determinations with four replicates per experiment; bars indicate standard error.