Fig. 4

Inhibition of reactive oxygen species (ROS) potentiates c-Met-mediated protection of renal cancer cells against sorafenib-induced apoptotic death. a 786-O cells were pre-incubated with the ROS scavenger NAC (10 μM) for 2 h, and then subjected to treatment with sorafenib (10 μM) or vehicle for 24 h. The expression of Bcl-2, cleaved Caspase-3, γ-H2AX in cellular lysates was measured by Western blot. β-Actin was used as the internal control. b 786-O cells were pre-incubated with NAC (10 μM) for 2 h followed by combination treatments with HGF (50 ng/ml), sorafenib (10 μM) or only vehicle for 24 h. The cells were then stained by using oxidative stress detection reagent and checked for endogenous ROS through flow cytometry. The mean fluorescence intensity has been represented as bar graphs. c 786-O cells were subjected to treatment as in b, and then the percent of cell death was studied by using annexin V (APC) and propidium iodide staining through flow cytometry. The percent of total apoptotic cells (early + late) are represented as bar graphs. d Following incubation with the mitochondrial electron transport chain blocker Mitoquinone (MitoQ) (1 μM) for 2 h, 786-O cells were subjected to combination treatments with HGF (50 ng/ml), sorafenib (10 μM), or only vehicle for 24 h. The cellular ROS was then measured as described in b. e 786-O cells were subjected to treatment as in d, and then the percent of cell death was checked through annexin V (APC) and propidium iodide staining as shown in c. f 786-0 cells were subjected to combination treatments with HGF (50 ng/ml), sorafenib (10 μM), or only vehicle for 24 h, and then the expression of SOD-2 in cell lysates was measured by Western blot. β-Actin was checked as an internal control. a–f Results are representative of three different experiments. b–e The columns are the mean ± S.D. of duplicate readings from two independent experiments. *p < 0.05 compared with control cells treated with vehicle alone