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. 2018 Jul 7;18:138–157. doi: 10.1016/j.redox.2018.07.004

Fig. 10.

Fig. 10

Melatonin prevents autophagic death by antagonizing the interaction of acetylated FOXO1 and ATG7 in H2O2-treated GCs. (A) GCs pretreated with 10 μM melatonin for 24 h were then washed in PBS, and grown in medium supplemented with H2O2 (200 μM). 2 h later, the cell lysates were processed for coimmunoprecipitation with anti-FOXO1, followed by probing with anti-BECN1, MTOR, SQSTM1, ATG3, ATG5, ATG7, ATG12, SIRT1 and EP300. WCL, whole-cell lysates. IP, immunoprecipitation. (B–D) The amount of coimmunoprecipitated ATG7, SIRT1 and EP300 for each IP reaction was normalized to TUBA1A content in the whole-cell lysates (input). Data represent mean ± S.E; n = 3. **P < 0.01. (E–H) GCs were cultured with 10 μM melatonin for 24 h, rinsed in PBS, and exposed to 200 μM H2O2 for 2 h. Sirtinol (100 μM) or SRT1720 (100 μM) was added 1 h prior to H2O2 incubation. After the indicated treatments, cells were collected for coimmunoprecipitation (E–G) or CCK-8 assay (H). For immunoprecipitation, the cell lysates were precipitated with anti-FOXO1, and probed with anti-Ac-FOXO1 or anti-ATG7. The protein levels of Ac-FOXO1 and ATG7 in the immunoprecipitates were quantified as described above. Data represent mean ± S.E; n = 3. ** Represents P < 0.01 vs. control group. ## Represents P < 0.01 vs. H2O2-only-treated cells. && Represents P < 0.01 vs. melatonin+H2O2 group. NS, not significant, P > 0.05. (I) GCs transfected with FOXO1N208A,H212R plasmid for 24 h were cultured for another 24 h in the presence or absence of 10 μM melatonin before 2 h of Sirtinol (100 μM) or SRT1720 (100 μM) treatment. Cell lysates were then extracted for co-immunoprecipitation with anti-FOXO1, followed by probing with anti-Ac-FOXO1 or anti-ATG7. (J and K) The amount of Ac-FOXO1 and ATG7 in the immunoprecipitates was quantified as mentioned above. **P < 0.01 vs. non treatment control. ##, P < 0.01; N, not significant, P > 0.05. (L) The detection of cell viability by CCK-8 assay. GCs were treated as above. Data represent mean ± S.E; n = 3 in each group. ** Represents P < 0.01 compared with the FOXO1N208A,H212R (DBD) group. ## Represents P < 0.01 compared with the FOXO1N208A,H212R (DBD)+melatonin group. NS, not significant, P > 0.05.