Figure 4

Activation of RAF/MEK/ERK and STAT3 pathways and expression levels of cyclin D1 after treatment with sorafenib and 5-fluorouracil (5-FU). Cells were treated with sorafenib (8 μM, 24 h), 5-FU (4 mg/L, 48 h), or combination of the two agents in different treatment sequences. Western blot analysis was performed to detect the expression levels of p-C-RAF, total C-RAF, p-ERK1/2, total ERK1/2, p-STAT3 (Y705), total STAT3, and cyclin D1. Loading controls were carried out by probing the blots for β-actin. Representative western blots (A) and quantification analysis (B and C) are shown. Asterisks (*) indicate significant differences in protein expression (P < 0.05). C, control; S, sorafeinb; F, 5-FU.