Fig. 6

CircMET accelerates the decay of CDKN2A mRNA by recruitment of YTHDF2. (A) The circMET-protein complex was pulled down by circMET junction probe with protein extracts from UOK109 and 786-O cells. The enrichment of CDKN2A mRNA was detected by real-time PCR (upper), and the enrichment of YTHDF2 was detected by western blot (lower). (B-C) MS2-RIP and RIP assays were performed to confirm the association of YTHDF2 with CDKN2A mRNA. The relative enrichment of CDKN2A mRNA associated with YTHDF2 was detected by real-time PCR (upper), and IP efficiency of YTHDF2/GFP-antibody was showed in western blot (lower). IgG antibody served as a control. (D) RIP assay was performed with UOK109 and 786-O cells transfected indicated lentivirus, and the relative enrichment of CDKN2A mRNA associated with YTHDF2 was detected by real-time PCR. IgG antibody served as a control. (E) Luciferase activity of CDKN2A-3’-UTR was measured after co-transfected with circMET and sh-YTHDF2. (F) Abundance of YTHDF1/2 among MS2-RIP with anti-GFP antibody from cells transfected with indicated plasmid was measured by western blot. (G) The stability of CDKN2A mRNA in 786-O cells transfected with indicated lentivirus after treatment with α-amanitin. (H-I) The mRNA and protein level of CDKN2A were measured after transfected with indicated gRNA and dCas13 fusions. (J-M) The stability of CDKN2A mRNA in cells transfected with indicated lentivirus after treatment with α-amanitin. (N) Cell viability of UOK109 and 786-O cells was determined using CCK-8 assays after transfection for 48 h. (O-P) A colony formation and tumor sphere formation assay were used to determine the colony and tumor sphere formation ability of UOK109 and 786-O cells co-transfected with indicated lentivirus. The data are presented as the mean ± SD, n.s. = non-significant, *P < 0.05, **P < 0.01, ***P < 0.001