Bim is required for hispidulin-induced sensitization of cancer cells to TRAIL-induced apoptosis. (A) Human renal carcinoma Caki and A498 cells were transiently transfected with Bim siRNA or control siRNA. After transfection, cells were exposed to 30 μM hispidulin and 50 ng/mL TRAIL for 24 h; (B) Renal carcinoma (Caki and A498) cells were exposed to 30 μM hispidulin for the indicated time periods; (C) Caki cells were pretreated with compound C for 30 min, and then exposed to 30 μM hispidulin for 24 h; (D,E) Caki cells were transiently transfected control (Cont) or AMPK siRNA. After transfection, cells were exposed to 30 μM hispidulin (D) or 30 μM hispidulin plus 50 ng/mL TRAIL for the 24 h (E). (F,G) Caki and/or A498 cells were transiently transfected control (Cont. siRNA), LKB1, TAK1 siRNA or CaMKKβ siRNA. After transfection, cells were exposed to 30 μM hispidulin for the 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction using flow cytometry. The protein levels were determined by Western blotting. The values in panel A and E represent the mean ± SD from three independent experiments. # p < 0.05 compared to the hispidulin plus TRAIL in control siRNA.