Fig. 2.

Melatonin represses H₂O₂-induced autophagy in cultured GCs. (A) GCs received 24 h of melatonin (10 μM) treatment were then rinsed in PBS, and incubated with 200 μM H₂O₂ for 2 h. The acidic vesicular organelles (AVOs, red) were detected using acridine orange staining. Bar, 10 µm. (B) The formation of autophagic vacuoles was quantified by calculating the amount of AVOs per cell. Experiments were repeated in triplicate, and 3 fields of each coverslip were selected at random for counting. Data represent mean ± S.E; n = 3 in each group. **P < 0.01. (C) GCs cultured with or without 10 μM melatonin for 24 h were then washed in PBS, and treated with H₂O₂ for 0–2 h. The expression of MAP1LC3B and SQSTM1 in GCs was determined by western blotting. (D–F) The MAP1LC3B-II accumulation, conversion of MAP1LC3B-I to MAP1LC3B-II and SQSTM1 degradation were quantified by densitometric analysis. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. * *P < 0.01; NS, not significant, P > 0.05. (G) GCs transfected with GFP-MAP1LC3B plasmid for 24 h were cultured for another 24 h in the presence or absence of 10 μM melatonin before 2 h of H₂O₂ (200 μM) incubation. For the inhibition of autolysosome formation, pepstatin A (10 μg/ml) and E64 (10 μg/ml) were added 1 h prior to H₂O₂ (200 μM) exposure. Bar, 10 µm. (H) The formation of autophagosomes was assessed by quantifying the GFP-MAP1LC3B puncta per cell. Experiments were repeated in triplicate, and 3 fields of each coverslip were selected in random for counting. Data represent mean ± S.E; n = 3 in each group. *P < 0.05, **P < 0.01; N, not significant, P > 0.05. P.E., pepstatin A and E64. (I) GCs pretreated with 10 μM melatonin for 24 h were then rinsed in PBS, and exposed to 200 μM H₂O₂ for 2 h. To block the autophagic flux, pepstatin A (10 μg/ml) and E64 (10 μg/ml) were added 1 h before H₂O₂ exposure. Western blotting showed expression levels of MAP1LC3B and TUBA1A. (J) Quantification of immunoblot signals for MAP1LC3B-II accumulation. Data represent mean ± S.E; n = 3. *P < 0.05, **P < 0.01; N, not significant, P > 0.05. (K) GCs were grown in medium containing 10 μM melatonin for 24 h, washed using PBS, incubated with 200 μM H₂O₂ for 0–2 h, and then processed for determining cell viability using the CCK-8 assay. Data represent mean ± S.E; n = 3. *P < 0.05, **P < 0.01; NS, not significant, P > 0.05.