Fig. 3: AOPPs inhibited autophagy and lysosomal biogenesis via the ROS-dependent mTOR pathway in granulosa cells. | Cell Death & Disease

Fig. 3: AOPPs inhibited autophagy and lysosomal biogenesis via the ROS-dependent mTOR pathway in granulosa cells.

From: Advanced oxidation protein products attenuate the autophagy-lysosome pathway in ovarian granulosa cells by modulating the ROS-dependent mTOR-TFEB pathway

Fig. 3

A Representative blots and quantification showing mTOR, p-mTOR, AKT, p-AKT and PI3K expression levels in negative control cells and KGN/COV434 cells treated with 200 μg/mL BSA, 50 μg/mL AOPPs, 100 μg/mL AOPPs, and 200 μg/mL AOPPs for 48 h. B Representative blots and quantification showing p-mTOR, p-AKT and PI3K expression levels in the control group, AOPP group, NAC group, and NAC + AOPP group. C Representative blots and quantification showing p-mTOR, p-AKT and PI3K expression levels in negative control cells, KGN/COV434 cells treated with 200 μg/mL AOPPs for 48 h (AOPP group), KGN/COV434 cells treated with 1 μM rapamycin for 48 h (rapamycin group), and KGN/COV434 cells pretreated with 1 μM rapamycin for 1 h and then treated with AOPPs for 48 h (rapamycin + AOPP group). D Representative blots and quantification showing p-mTOR, p-AKT and PI3K expression levels in negative control cells, KGN/COV434 cells treated with 200 μg/mL AOPPs for 48 h (AOPP group), cells starved in HBSS for 3 h for KGN cells and 1 h for COV434 cells (HBSS group), and cells treated with AOPPs for 48 h and then starved with HBSS for 3 h for KGN cells and 1 h for COV434 cells (HBSS + AOPP group). E Representative images of RFP-GFP-LC3 under a confocal laser scanning microscope in the control group, AOPP group, rapamycin group, rapamycin + AOPP group, HBSS group and HBSS + AOPP group. The GFP + RFP+ (yellow) puncta are autophagosomes. The numbers of autophagosomes were quantified for 10 cells. High-magnification images of the indicated boxed areas are shown in the bottom panels. F Lysosomal biogenesis assessed by Lyso-Tracker Red in the control group, AOPP group, rapamycin group, rapamycin + AOPP group, HBSS group and HBSS + AOPP group. G LAMP1 expression as detected by immunofluorescence in the control group, AOPP group, rapamycin group, rapamycin + AOPP group, HBSS group and HBSS + AOPP group. All experiments were repeated for three times. The results are expressed as the mean ± SD from experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by one-way ANOVA and Tukey tests for post hoc comparisons.

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