The degree of sorafenib's inhibition of ERK phosphorylation was significantly associated with basal pERK levels in hepatocellular carcinoma (HCC) cell lines. (A) Immunocytochemical staining of pERK (200×) after treatment with sorafenib for 24 hours in four HCC cell lines. (B) pERK expression rate in HCC cell lines after sorafenib treatment. pERK levels were reduced after sorafenib treatment in all four cell lines, but the inhibition effect in SMMC-7721 cells, with lower levels of pERK, was significantly weaker when compared to the other three HCC cell lines with relatively higher basal pERK levels (two-way ANOVA, P < 0.0001, n = 6). The expression rate of pERK was calculated from pERK density determined as described in Materials and methods and the rate in each control group was set as the 100% baseline. Columns represent means of six samples in each group; bars indicate standard error.