Fig. 7

CircKEAP1 expression is modulated by m6A methylation. (A). MeRIP assays for m6A-modified circKEAP1 in HOS and U2OS cell lines, as well as cells over-expressing circKEAP1 with or without the m6A site mutated. (B). RNA pulldown assay was performed using a circKEAP1 or empty control probe and YTHDF2, METTL3, and FTO were detected by western blotting. (C). RIP assay and PCR analysis for circKEAP1 in U2OS cells using FTO, METTL3, YTHDF1/2 antibodies. (D). Expression of circKEAP1 in HOS and U2OS cells transfected with the indicated siRNAs under actinomycin D (5 µg/ml) treatment for 0, 6 and 12 h by qPCR. (E). Expression of KEAP1 was detected by western blotting in HOS and U2OS cells transfected with the indicated siRNAs. (F). QPCR analysis of circKEAP1 in HOS and U2OS cells transfected with control, si-METTL3 or si-FTO. (G). MeRIP assays for m6A-modified circKEAP1 in U2OS cells transfected with control, si-METTL3 or si-FTO. Error bars represent three independent experiments. *, **, *** indicates significant differences compared with 0 h or control group at a p value < 0.05, < 0.01, < 0.001, respectively