Fig. 5
circPLPP4 is modulated by m6A methylation. A Predicted m6A sites in circPLPP4 from a N6-methyladenosine (m6A) modification predictor (SRAMP), which based on sequence. B Flow diagram of m6A-specific immunoprecipitation (MeRIP) assays. C MeRIP assays for detecting m6A-modified circPLPP4 in A2780 CDDP cells and SKOV3 CDDP cells. D m6A RIP-qPCR analysis of circPLPP4 in A2780, SKOV3, A2780CDDP and SKOV3CDDP cells. Error bars represent the mean ± SD of three experiments. E Heat map profiling the expression of m6A WERs in 40 OC tissues (including 20 platinum resistant OC tissues and 20 platinum sensitive OC tissues). F, G METTL3 and IGF2BP1 were examined by RNA pulldown assays and western blotting using circPLPP4 probe. H METTL3 expression was evaluated by western blotting in the indicated OC cells. GAPDH acted as the loading control. I m6A RIP-qPCR analysis of the m6A level in circPLPP4 in the indicated cells. Error bars represent the mean ± SD of triplicate experiments. J qRT-PCR analysis of circPLPP4 expression in the indicated cells. Error bars represent the mean ± SD of triplicate. K Correlation analysis demonstrated the correlation between METTL3 and circPLPP4 in OC tissues obtained from SYSUCC. Statistical analyses were performed by Spearman correlation coefficient. L, N Control or METTL3-knockdown or IGF2BP1-knockdown A2780CDDP cells were treated with actinomycin D (5 mg/mL) for the indicated times. Total RNA was extracted and then analyzed using RT-qPCR to assess the half-lives of circPLPP4. Error bars represent the mean ± SD of three experiment. M, P RIP analysis indicating that the enrichment of circPLPP4 on IGF2BP1 in the indicated cells. Error bars represent the mean ± SD of triplicate experiment. P The putative wild- type m6A sites and designed mutant m6A sites in circPLPP4. Q qRT-PCR analysis of circPLPP4 expression in the circPLPP4 Wt or circPLPP4-Mut A2780CDDP cells with or without METTL3 or IGF2BP1 silencing. Error bars represent the mean ± SD of triplicate experiments. R The luciferase activities of different mutated circPLPP4 reporter in the indicated groups. Error bars represent the mean ± SD of three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns indicates no significance