Figure 6.

ETC-derived ROS regulate autophagy during quiescence via ATG4B. (A and B) ROS-sensitive ATG4B both activates (by cleaving pre-LC3) and inhibits (by removing PE) LC3B (A), leading to a complex relationship between ATG4B activity, ROS and autophagy (B). At optimal ROS levels ATG4B activity is partially attenuated, the activating pre-LC3 cleavage can proceed while the inhibitory removal of PE is suppressed, leading to the maximal autophagic flux. (C) The LC3B-PLA2 (phospholipase A2)-based reporter to assess ATG4B function. Active ATG4B liberates PLA2, producing signal in a fluorogenic assay. (D) ATG4B activity in cell lysates from EA.hy926 QCs treated or not with H₂O₂ (1 mM) for 4 h and incubated with increasing concentrations of LC3B-PLA2 fusion protein substrate (mean ± S.E.M., n ≥ 4, *p < 0.05, **p < 0.01, ***p < 0.001, versus parental cells, two-Way ANOVA with Tukey’s multiple comparisons test). (E) ATG4B activity in cell lysates from liver of control or tfam KO mice incubated with increasing concentrations of LC3B-PLA2 fusion protein substrate (mean ± S.E.M., n = 4 mice, **p < 0.01, ***p < 0.001, versus control cells, two-way ANOVA with Tukey’s multiple comparisons test). (F and G) Representative WB image (F) and quantification (G) of the activated LC3B (LC3B-II) protein in EA.hy926 ρ⁰ QCs treated with FMK-9a (1 µM) and NSC185058 (0.1 µM) for 4h (mean ± S.E.M., n = 3, *p < 0.05, unpaired two-tailed t test). (H) PEITC-induced cell death in parental and ρ⁰ EA.hy926 QCs pre-treated or not with FMK-9a (1 µM) and NSC185058 (0.1 µM) for 2 h, measured by ANXA5 and PI. (mean ± S.E.M, n = 4, ^n.s.p > 0.05, **p < 0.01, one-way ANOVA with Sidak’s multiple comparisons test).