Fig 2: Model of PA and OA treatment effects on autophagy during mouse preimplantation development. Autophagy begins when a phagosome is initiated to form an autophagosome. Phagosomes recruit autophagy-related proteins to direct contents into the phagosome as it elongates and seals off to become an autophagosome. After autophagosome formation is completed, autophagosomes are transported to a lysosome for autophagosome maturation. Fusion of autophagosomes and lysosomes creates an autolysosome. Acidic contents of the lysosome degrade the autophagosome, then releasing the degraded contents into the cytosol for recycling. PA treatment of mouse preimplantation embryos increased autophagosome accumulation by elevating autophagosome formation, reducing autophagosome maturation, and reducing autophagosome degradation by disruption of lysosomal activity. In contrast, OA treatment alone had no obvious effect on these events. However, PA + OA cotreatment restricted PA-induced effects on preimplantation embryo autophagy, but only after 40 h of cotreatment time, resulting in the successful reversal of PA-induced effects by 48 h of exposure. Created with BioRender.com. LC3, light chain 3; OA, oleic acid; PA, palmitic acid.

Fig 3: Diane-35 and metformin reduced AR and IGF-1 expression and increased GLUT4 expression in the early-stage tumor tissue of PCOS patients with EC. Immunohistochemistry (A) and PCR analysis (B) for AR, GLUT4, and IGF-1 in endometrial carcinoma tissue, scale bars = 100 µm. Transmission electron microscope analysis of patients’ tissues: autophagosomes: red arrow, autolysosome: white arrow, lysosome: red asterisk, and lipid droplet: LD, scale bars = 2 µm (C,D). Western blotting analysis of LC3 I/II (E) and cleaved caspase-3 (F) expression levels from 7 different patient samples. Data are shown as the mean ± S.D. * p < 0.05 and ** p < 0.01, ANOVA.

Fig 4: Immunofluorescence intensity of p62 in preimplantation embryos after 48 h of NEFA treatment. Relative p62 immunofluorescence intensity per cell (±SD) of 2-cell mouse embryos after exposure to 100 µM PA, 250 µM OA, 100 µM PA and 250 µM OA, or KSOMaa medium alone (control) for 48 h. Only blastocyst embryos were assessed. n = 3, one-way ANOVA with Tukey’s HSD post hoc test. Significant differences are indicated by **P < 0.01. A: after 48 h of NEFA exposure, the PA-alone group resulted in a significantly higher LC3-II puncta count per cell compared with the control group (P = 0.0062), the OA-alone group (P = 0.0014), and the PA + OA combination group (P = 0.0017). B: representative images of p62 immunofluorescence after 48 h of exposure to NEFA treatments. Scale bars = 100 µm. Con, control; HSD, honestly significant difference; KSOMaa, potassium simplex optimization media with amino acid; LC3, light chain 3; NEFA, nonesterified fatty acid; OA, oleic acid; PA, palmitic acid.

Fig 5: Verification of the PI3K/mTORC1 pathway on Diane-35 and metformin-mediated autophagy. (A,B) Transfection efficiency of the CIP2A overexpression system in Ishikawa cells. (C,D) mRNA and protein levels of PP2A, c-Myc, and cleaved caspase-3 in cells subjected to different treatments. CCK-8 (E), colony formation (F), and flow cytometry (G) were performed to measure apoptosis in Ishikawa cells after pretreatment with 3-methyladenine (3-MA, 5mM) or transfection with the CIP2A overexpression vector. All the cells were treated with Diane-35 and metformin (10 mM). (H–J) Cell autophagy was determined using the LC3 level measured by qRT-PCR, Western blotting, and immunofluorescence. Data are shown as the mean ± S.D. n.s. = no statistically significant differences. * p < 0.05, & p < 0.05, and ** p < 0.01, ## p < 0.05, && p < 0.01, ANOVA. Scale bars = 10 µm.