Fig. 4

Activation of mitophagy reverses the effects of cirsiliol including the expression of mitophagy proteins, the loss of mitochondrial membrane potential, and ROS production in HCT116 cells. For cirsiliol alone treatment group, HCT116 cells were exposed to 20 μM cirsiliol for 24 h. For UMI-77 group, HCT116 cells were simultaneously treated with 20 μM cirsiliol and 10 μM UMI-77 for 24 h. A The levels of mitophagy proteins were determined by western blot analysis. B–E Quantitative analysis of western blot for PINK1 (B), Parkin (C), BNIP3 (D), FUNDC1 (E). F Representative images of JC-1 staining. Scale bar: 50 μm. G Quantification of the JC-1 fluorescence ratio. H Representative images of H2DCFDA staining. Scale bar: 50 μm. I Quantification of H2DCFDA fluorescence intensity. n = 3 (A–E), and 5 (F–I) per group. Data represent means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs ctrl. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs cirsiliol 20 μM