Fig. 5.

Mcl-1 plays a critical role in H89/tetrandrine induced cell death. a Western blot analysis of Bcl-2 family members after treatment for 48 h. b AGS and MDA-MB-231 cells were treated with DMSO or H89/tetrandrine for 24 h; the relative Mcl-1 mRNA expression was determined by qPCR, and β-actin was used as a reference. c The cells stably transfected with Mcl-1 were treated with DMSO or H89/tetrandrine for 72 h and subjected to viability assays. d MDA-MB-231 Mcl-1 overexpression and control cells were treated with DMSO or H89/tetrandrine for 48 h. Apoptosis was evaluated by flow cytometry. e Western blot analysis of PARP, caspase 9 and Mcl-1 in the cells after treatment with DMSO or H89/tetrandrine for 48 h. f The mitochondrial membrane potential was measured by flow cytometry with Rho123 in the cells after treatment with DMSO or H89/tetrandrine for 48 h. g LC3 was determined in the cells after treatment with DMSO or H89/tetrandrine for 24 h. h AGS and MDA-MB-231 cells were pre-treated with NAC for 1 h followed by H89/tetrandrine for 36 h; Mcl-1 levels were subsequently determined by Western blot. i Cells with control or Mcl-1 overexpression were treated with DMSO or H89/tetrandrine for 24 h; intracellular ROS levels were subsequently measured. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; *P < 0.05, **P < 0.01 compared with DMSO control. All images are representative of three independent experiments