Figure 4.

The P62 Enhanced the METTL3 Occupancy on IGFII mRNA 3′ UTR in the Mesenchymal Stem Cells with TNF-α Treatment

(A) RIP with anti-METTL3 followed by RT-PCR with CUDR mRNA primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). IgG RIP as negative control is shown. CUDR mRNA as INPUT is shown. (B) Anti-CTCF co-IP followed by western blotting with anti-METTL3 primers in the mesenchymal stem cells treated with TNF-α or CUDR knockdown and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. IgG IP as negative control is shown. INPUT refers to western blotting with anti-CTCF. (C) Biotin-CUDR cDNA pull-down followed by western blotting with anti-MELLT3 and anti-CTCF primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. Biotin as INPUT and histone as internal control are shown. (D) Western blotting with anti-METTL3 and anti-FTO in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. β-actin as internal control is shown. (E) RIP with anti-METTL3 followed by RT-PCR with IGFII mRNA primers in the mesenchymal stem cells treated with TNF-α or CUDR knockdown and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. IgG RIP as negative control is shown. IGFII mRNA as INPUT is shown. (F) Super-EMSA (gel-shift) with biotin-IGFII cRNA probe and anti-METTL3 antibody in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The intensity of the band was examined by western blotting with anti-biotin. (G) The super-METTL3 bands were quantified by calculating the band gray density value (n = 3). Each value was presented as mean ± SEM. **p < 0.01.