Fig. 2. p66Shc regulates APAP-induced mitochondrial dynamics perturbation in hepatocytes.

a AML12 cells were exposed to APAP (5 mM) and the p66Shc, p52Shc, and p46Shc proteins, n = 3. b–f AML12 cells were transfected with si-control or si-p66Shc and then exposed to APAP (5 mM). b H₂O₂ levels, n = 8. c, d p66Shc, p-p66Shc, CYP2E1, OMA1, L-OPA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. e, f OMA1 ubiquitination level, n = 3. g, h AML12 cells were transfected with pcDNA 3.1 or pcDNA-p66Shc and then exposed to APAP and the p66Shc, OMA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. Mitochondrial ROS, cell apoptosis, and mitochondrial fragmentation were determined via representative fluorescence images of MitoSOX- (i), Tunel-(j), and TOM20-stained (k) cells. Scale bar, 200 μm, 100 μm, or 12.5 μm. The colocalization of p66Shc and mitochondria (Mitotracker) (l), OMA1 and mitochondria (Mitotracker) (m) and p66Shc and OMA1 (n). Scale bar, 12.5 μm. (o) AML12 cells were transfected with si-control or si-p66Shc and then treated with APAP and MG132. Mitochondrial fragmentation was determined via representative fluorescence images of TOM20-stained cells. Scale bar, 12.5 μm. ^**p < 0.01 vs. the si-control group; ^##p < 0.01 vs. the APAP group; ^&&p < 0.01 vs. the si-p66Shc group.