Fig. 5: ML-323-induced increased AMPK phosphorylation triggers protective autophagy and reduces cell apoptosis.

A, B Flow cytometry analysis of apoptosis was using Annexin V-FITC /PI double-staining kits. Blocking AMPK with the inhibitor CC promoted ML-323-induced apoptosis. HCCLM3 and SMMC-7721 cells were treated with ML-323 alone or in combination with DMSO or CC. Statistical analysis was conducted. C Cell-counting kit-8 (CCK-8) showing the cell viability of CC and ML-323-treated HCC cells. Blocking AMPK phosphorylation enhances the effect of ML-323 on HCC cell viability. HCCLM3 and SMMC-7721 cells were treated with ML-323 alone or in combination with DMSO or CC. D Western blot analysis of HCCLM3 and SMMC-7721 cells treated with ML-323 alone or in combination with DMSO or CC. Data represent at least three independent experiments (n = 3; error line, SD). E Western blot analysis of L3CB in HCCLM3 and SMMC-7721 cells treated with ML-323 alone, or in combination with CQ or BafA1. CQ or BafA1 inhibits LC3B degradation. F CCK-8 showing the cell viability of HCCLM3 and SMMC-7721 cells treated with ML-323 alone, or in combination with CQ or BafA1. Autophagy inhibition increases the ML-323 induced growth inhibition of hepatoma cells. G Annexin V-FITC/ PI double-staining showing the effect of the inhibition of autophagy in HCCLM3 and SMMC-7721 cells treated with ML-323 alone, or in combination with CQ or BafA1. H Western blot analysis of cleaved-PARP and caspase 3 in HCCLM3 and SMMC-7721 cells treated with ML-323 alone, or in combination with CQ or BafA1. Data are representative of at least three independent experiments (n = 3; error bars, SD).