Figure 5.

OXPHOS-derived ROS maintain autophagy and ROS resistance in quiescent cells. (A) OXPHOS-deficient QCs lack respiratory complexes and are therefore expected to produce less ROS from the ETC, possibly affecting autophagy. (B) Baseline ROS levels in EA.hy926 QCs assessed using DCF-DA fluorescent probe by flow cytometry (mean ± S.E.M., n ≥ 6, **p < 0.01, unpaired two-tailed t test). (C) Pharmacological (NAC) and genetic (catalase overexpression) reduction of endogenous ROS in parental QCs was used to recapitulated effects of OXPHOS deficiency on autophagy. (D and E) Representative WB image (D) and quantification (E) of activated LC3B (LC3B-II) in parental or ρ⁰ EA.hy926 QCs treated or not with NAC (1 mM) for 6 h and/or with Baf A (50 nM) for 4 h (mean ± S.E.M., n = 3, ^n.s.p > 0.05, *p < 0.05, unpaired two-tailed t test). (F and G) Representative WB image (F) and quantification (G) of activated LC3B (LC3B-II) protein levels in parental and ρ⁰ EA.hy926 QCs with or without overexpression of catalase in the presence or absence of Baf A (50 nM) (mean ± S.E.M., n = 3, ^n.s.p > 0.05, ***p < 0.001, unpaired two-tailed t test). (H) Treatment with low levels of exogenous ROS (H₂O₂) in OXPHOS deficient QCs was used to restore autophagy and cell death resistance. (I) Representative WB image (top) and quantification (bottom) of the activated LC3B (LC3B-II) protein expression in EA.hy926 parental and ρ⁰ QCs treated or not with H₂O₂ (1 mM) for 4 h in the presence or absence of Baf A (50 nM) (mean ± S.E.M., n = 3, ^n.s.p > 0.05, *p < 0.05, unpaired two-tailed t test). (J) PEITC-induced cell death in parental and ρ⁰ EA.hy926 QCs pre-treated or not with Baf A (10 µM), with or without pre-conditioning by H₂O₂ as shown in the scheme, 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).