Effect of silencing Keap1 on the cell viability, ROS level and protein expression of Nrf2 and ERK signaling. ACHN cells were transfected with siKeap1 and divided into five groups, which were the control, Ax (1 µM Ax), NC+Ax, siKeap1+Ax and siKeap1 groups. (A) Cell viability was determined by a Cell Counting kit-8 assay. (B) ROS levels are presented in a bar graph. (C) Flow cytometry was used to detect the ROS level. (D) Nrf2 protein expression was detected by western blotting. Lamin B1 served as an internal control. (E) Keap1 protein expression was detected by western blotting. GAPDH served as an internal control. (F) NQO1, HO1, p-ERK and ERK protein expressions were detected by western blotting. GAPDH served as an internal control. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P<0.01. Keap1, Kelch-like ECH-associated protein 1; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; ERK, extracellular signal-regulated kinase; Ax, Axitinib; NC, negative control; si, small interfering; NQO1, anti- NAD(P)H dehydrogenase [quinone] 1; HO1, heme oxygenase 1; p-, phosphorylated.