Fig 2: 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.