Fig. 2

H89 in combination with tetrandrine synergistically induced concomitant apoptosis and autophagy. a FACS analysis of apoptosis following treatment with H89 (6 μM) and tetrandrine (4 μM) alone or combination on Hep3B, AGS, MDA-MB-231, and LOVO for 48 h. b Western blot analysis of PARP, caspase 9, caspase 3 and cytochrome c in the cells after treatment with H89 and/or tetrandrine for 48 h. c The cells were pretreated with z-VAD-fmk (50 μM) for 1 h, followed by treatment with H89 and/or tetrandrine for 72 h; cell viabilities were subsequently evaluated. Z-VAD, z-VAD-fmk. d Western blot analysis of the level of the autophagy-related protein LC3 in the cells after treatment with H89 and/or tetrandrine for 24 h. e Cancer cells were transiently transfected with GFP-LC3 plasmid and subsequently treated as in panel (d); the percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells, and 150–160 cells per condition were counted. Representative images are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (× 40 magnification). f Fluorescence microscopy analysis of AGS cells expressing tandem mRFP-GFP-LC3 reporter treated with H89/tetrandrine, rapamycin (500 nM) and chloroquine (10 μM) for 36 h; the ratio of mRFP vs GFP puncta was used to quantify the autophagy flux. Scale bars, 5 μm. g Cell viability was determined in cells pretreated with 3-MA (2 mM) or bafilomycin A1 (100 nM) for 1 h following H89/tetrandrine combination treatment for 48 h. Baf, Bafilomycin A1. 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. All images are representative of three independent experiments