Fig. 1

Trifolirhizin induced autophagy-mediated CRC apoptosis in vitro and in vivo. a The diagram of autophagy mechanism. b Images were taken by scanning electron microscope after cells were treated with trifolirhizin (20 μM) for 6 h. Arrow indicates autophagosome including the lamellar structure. (Scale bar: 1 μm). c Immuno-blotting assay of LC3B-I, LC3B-II, and SQSTM1 protein expression. GADPH was used as the loading control (n = 3). d Cells were treated with 20 μM trifolirhizin in the presence or absence of 10 mM 3-MA or 20 μM CQ or 10 nM BafA1. Cells were mixed with Ad-mCherry-GFP-LC3B and then incubated with trifolirhizin for 6 h, and the mCherry (red) and GFP (green) were analyzed by a laser scanning confocal microscope (LSCM) (n = 3). e HCT116 and SW620 cells were treated with either different concentrations or time durations of trifolirhizin. Western blot assay of p-AMPK, AMPK, p-mTOR, and mTOR protein expression (n = 3). f The cells were exposed to different concentrations of trifolirhizin for 48 h, and the cell viability was measured by CCK-8 assay (n = 10). g Expression levels of cleaved caspase-9, cleaved caspase-8, cleaved caspase-3, cleaved PARP, and cytochrome c were determined by western blot (n = 3). h Early apoptosis of the cell was analyzed by flow cytometry with AnnexinV-FITC/PI staining (n = 6). i Tumor-bearing mice were treated with trifolirhizin, Oxaliplatin (positive drug), or with vehicle control. Tumor size was measured every 3 days, and tumors were harvested and weighed after 4 weeks of treatment (n = 8). j Western blot assays of p-AMPK, AMPK, p-mTOR, mTOR, Atg5, Atg7, cleaved-caspase-8, and cleaved-caspase-3 expression in tumor section (n = 3). **P < 0.01 versus model