Fig 2: The inhibitory effect of melatonin on H2O2-triggered autophagic GC death is independent of ROS elimination. (A) Primary cultured GCs grown in medium containing 10 µM melatonin for 24 h were then rinsed in PBS, and exposed to 2 h of H2O2 (200 µM) incubation. The inhibitors of melatonin downstream antioxidants (AOI) were added 1 h prior to H2O2 treatment. The total antioxidation capability (T-AOC) was determined as described in Materials and Methods section. Data represent mean ± S.E; n = 3. *P < 0.05, **P < 0.01; NS, not significant, P > 0.05. (B) GCs were subjected to H2O2 exposure following melatonin and/or AOI treatment as mentioned above. The ROS levels were detected by dichlorofluorescein fluorescence (green), and nuclei were counterstained with DAPI (blue). Bar, 20 µm. (C) The optical density of intracellular ROS was quantified using ImageJ software. ** Represents P < 0.01 compared to control group; # Represents P < 0.05; N, not significant. (D) The immunoblotting detection of MAP1LC3B and SQSTM1 in GCs received the indicated treatments as described above. (E and F) Quantification of the MAP1LC3B-II accumulation and SQSTM1 degradation. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3 in each group. ** Represents P < 0.01 compared to control group. ## Represents P < 0.01 compared to H2O2-only-treated cells. N, not significant. (G) Primary cultured GCs treated with H2O2 at different concentrations as indicated for 2 h were collected for immunoblotting analysis of MAP1LC3B and SQSTM1. (H and I) The expression of MAP1LC3B-II and SQSTM1 were quantified by densitometric analysis. ** Represents P < 0.01 compared to control group; NS, not significant. (J) GCs pretreated with or without melatonin (10 µM) for 24 h were then rinsed in PBS, and incubated with H2O2 in various concentrations. 2 h later, the formation of ROS in GCs was observed under a laser confocal-scanning microscope. Bar, 20 µm. (K) Quantification of ROS levels by calculating the green fluorescence optical density in each GC. Experiments were repeated in triplicate, and three fields of each coverslip were selected at random for counting. Data represent mean ± S.E; n = 3. ** Represents P < 0.01 compared to control group; NS, not significant. (L) GCs cultured with 10 µM melatonin for 24 h were then rinsed in PBS, and exposed to 2 h of H2O2 (200 µM) incubation. For the inhibition of melatonin-induced activation of downstream antioxidative components, cells were treated with AOI 1 h before H2O2 exposure. Cell viability was examined as described above. **P < 0.01; NS, not significant, P > 0.05.

Fig 3: Study of subcutaneous adipose tissue from patients. (A) Perilipin-1 expression in abdominal subcutaneous adipose tissue from patients E-II2 and E-I1 compared with controls and previously described patients with FPLD4 (2). Western blot of whole cell extracts was performed using antibodies directed against the N-terminal and C-terminal parts of wild-type perilipin-1, as previously described (2). The mutant isoform was recognized by the N-terminal antibody as an additional band (arrow) just above the 62-kD molecular weight (MW) marker, which was not detected by the C-terminal antibody. Tubulin (antibody T5168, Sigma-Aldrich, St. Quentin-Fallavier, France) was used as a loading control. (B) Consequences on perilipin-1 protein expression of FPLD4-associated PLIN1 frameshift variants studied in (A). (C) Histological and immunohistological analyses of abdominal subcutaneous adipose tissue from patients E-I1 and E-II-2 compared with controls. Adipose tissue samples from patients displayed disorganized fat lobules of heterogeneous size, with increased fibrosis [Sirius red (SR) staining, 16% to 35% of the total sample surface vs 0.2% to 3% in controls], increased vascularization (density of CD34 staining, 0.13% to 0.44% in adipose lobules from patients vs 0.001% to 0.004% in controls), and increased macrophage infiltration with crown-like structures (assessed by CD163 and CD68 staining, density per 10-5 µm2: 1.9 to 2.2 and 3.6 to 4.3 in patients vs 0.0 and 0.0 to 0.01 in controls, respectively).

Fig 4: The integrated stress response (ISR) is required for aggresome formation.(A) Schematic of aggresome formation assay. During recovery from proteotoxic stress, aggresome formation is assessed by anti-p62/SQSTM1 immunofluorescence, which appears as a well-defined, perinuclear puncta. (B) Time course of aggresome formation in control and ISR-null cells indicates diminished aggresome formation in cells with an ablated ISR. White arrows indicate aggresomes. Quantification of the percentage of aggresome-positive cells. N = 2 independent experiments with at least 145 cells quantified per condition. Error bars represent 95% confidence interval calculated by Wilson’s method for confidence interval of a proportion. ****p<0.001 using two-sample Z-test for a proportion (comparisons: control vs. ISR-null at 4 and 8 hr). Scale bar 40 µm. (C) Aggresomes visualized at the ultrastructural level by transmission electron microscopy. In control cells, aggresomes characteristically appear as a perinuclear cluster of electron-dense granules and partially degraded organelles that often deform the nuclear membrane. Fewer aggresomes form in ISR-null cells, and those that do are smaller, more diffuse and rarely indent the nucleus. White arrow indicates nuclear envelope deformation. Scale bar 4 µm. Yellow shade in the zoomed-in images at the lower left highlights aggresomes and protein aggregates. Nu, nucleus. Cy, cytoplasm, Agg, aggresome. *Lipid droplet. Scale bar 1 µm. (D) Confocal immunofluorescence microscopy and 3D reconstruction to visualize the location of the aggresome in relation to the microtubule-organizing center (MTOC) (?-tubulin, TUBG1) and cytoplasmic microtubules (a-tubulin, TUBA1A). In control cells, aggresomes appear as dense p62-positive structures directly adjacent to the MTOC puncta. Representative cells from 8 hr post-bortezomib washout are shown. White arrow indicates MTOC. Scale bar 4 µm.

Fig 5: Deacetylation of FOXO1 via the melatonin-SIRT1 signaling inhibits FOXO1-dependent autophagy in GCs. (A) GCs were cultured with 10 µM melatonin for 24 h, washed in PBS, and then exposed to H2O2 incubation for 1 or 2 h. The protein level of acetylated FOXO1 (Ac-FOXO1), total FOXO1 and the deacetylase SIRT1 was determined by western blotting. (B and C) The relative expression of Ac-FOXO1 and SIRT1 were quantified using densitometric analysis. Data represent mean ± S.E; n = 3. ** Represents P < 0.01 compared to control group. # Represents P > 0.05 compared to control group. (D) GCs transfected with FOXO1N208A,H212R plasmid for 24 h were grown in medium containing 10 µM melatonin. 24 h later, cells were cultured for another 2 h in the presence or absence of Sirtinol (100 µM) or SRT1720 (100 µM). The expression of Ac-FOXO1, total FOXO1, MAP1LC3B and SQSTM1 was then detected by western blotting. (E–G) Quantification of FOXO1 acetylation, MAP1LC3B-II accumulation and SQSTM1 degradation. TUBA1A served as a loading control. Data represent mean ± S.E; n = 3. (H) 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) treatment. Cells were then collected for TEM imaging of the autophagic structures. Bar, 0.8 µm. Enlarged images (below) show clearer autophagic vacuoles (red arrows). (I) Number of autophagic vacuoles per cell section in GCs. Bar graphs are mean ± S.E of results from 10 cell sections. **P < 0.01 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).