Figure 3: ROS-dependent autophagy induction requires Ca²⁺ and TRPML1.

(a) In HeLa cells stably expressing mRFP–GFP–LC3, CCCP treatment (5 μM for 3 h) increased the formation of autophagosomes, ‘visualized’ as mRFP⁺ GFP⁺ puncta. Co-treatment with NAC, BAPTA-AM, or ML-SI3 abolished the increases. Scale bar, 10 μm. (b) Quantification of various treatment conditions on CCCP-induced autophagosome formation (mean±s.e.m., n≥30 randomly-selected cells for each treatment). *P<0.05, ANOVA. (c) NAC did not affect ML-SA5-induced autophagosome formation. Means are shown with s.e.m. (n≥40 randomly-selected cells for each treatment). *P<0.05, ANOVA. (d) Western blot analysis of LC3-I and -II (arrows) protein expression in CCCP (10 μM, 3 h) -treated WT and ML-IV human fibroblasts. Torin 1 (1 μM) was used as a positive control to induce autophagy, and bafilomycin A1 (Baf, 0.5 μM) was used to inhibit lysosomal degradation. (e) Quantitative analysis of LC3-II levels under various experimental conditions shown in d. Data are presented as mean±s.e.m. (from at least three independent experiments); *P<0.05, paired t-tests.