5-Fluorouracil (5-FU) hardly inhibited ERK phosphorylation in MHCC97-H cells. (A-D) Immunocytochemical staining of pERK (200×) after treatment with 5-FU for 48 hours in MHCC97-H cells. (A) Culture medium without 5-FU was used as control. (B) 5-FU 10 mg/l. (C) 5-FU 20 mg/l. (D) 5-FU 50 mg/l. (E) pERK expression was not reduced after 5-FU treatment. 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 represenr means of six samples in each group; bars indicate standard deviation; *, P < 0.05, when compared to the control. (F) Western blot analysis of pERK protein in MHCC97-H cells after treatment with 5-FU for 48 hours.