Fig. 4

PKA and ERK signaling are involved in H89/tetrandrine induced cell death. a Western blot of p-CREB1-s133 and CREB1 in AGS and MDA-MB-231 cells exposed to H89 and/or tetrandrine for 24 h. b The cells were pre-treated with FSK (10 μM) for 1 h, followed by treatment with DMSO or H89/tetrandrine, and apoptosis was measured by flow cytometry after 36 h of treatment. c GFP-LC3 punctate was determined using a fluorescence microscope after 24 h of treatment. Scale bars, 5 μm. d Western blot of p-ERK1/2, T-ERK and p-MEK in cells incubated with H89/tetrandrine for 0, 2, 4, 8, 12 & 24 h. e MDA-MB-231 was pre-treated with 20 μM PD98059 for 1 h prior to exposure to H89/tetrandrine. Apoptosis was detected by flow cytometry after 36 h, and (f) p-ERK and PARP levels were analyzed by Western blot at 8 h and 36 h, respectively. g After 24 h of treatment, LC3 expression was measured by Western blot. h Cells were pre-treated with PD98059 and FSK alone or in combination for 1 h prior to exposure to H89/tetrandrine for 72 h. Cell viability was determined by trypan blue staining. PD, PD98059. i Analysis of p-CREB1-s133 and p-ERK levels in the cells pretreated with NAC for 1 h prior to exposure to H89/tetrandrine for 8 h. j The cells were pre-treated with FSK or PD98059 for 1 h followed by H89/tetrandrine combination treatment for 24 h; intracellular ROS were subsequently detected by flow cytometry. 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 compared with DMSO control. All images are representative of three independent experiments