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. 2020 Nov 20;7:592550. doi: 10.3389/fcvm.2020.592550

Figure 4.

Figure 4

FTO-mediated mRNA expression of Klf5 depends on its m6A demethylase activity. (A) A sketch map of 0 to +1,728 bp region of the Klf5 mRNA (NM:009769.4) showing the position of 4 KLF5-m6A sites. (B) ELISA assays of total m6A levels in human VSMCs infected with pLV-FTO, FTO mutant (H231A and D233A; pLV-FTO-Mut) or pLV-GFP. (C,D) qRT-PCR (C) and Western blot assay (D) for Klf5 mRNA and protein expressions, respectively. **P < 0.01 vs. pLV-GFP. (E) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of FTO-mediated m6A modifications on Klf5 mRNA in VSMCs infected with the pLV-FTO, pLV-FTO-Mut, or pLV-GFP. (F) Wild-type (WT-Klf5) or m6A consensus sequence mutant Klf5 cDNA (Mut-Klf5) was reconstructed with firefly luciferase reporter. Relative luciferase activity was measured in WT-Klf5 and Mut-Klf5 (A-to-C mutation on m6A site 1,457) after co-infection with pLV-FTO or pLV-GFP in HEK293 cell. Data were mean ± SD, n = 3. *P < 0.05, **P <0.01 vs. pLV-GFP. (G) VSMCs infected with pLV-GFP or pLV-FTO, and then transfected with si-Con or si-Klf5 for 24 h, MTT assay for the treated cell at corresponding time points. *P < 0.05 and **P < 0.01 vs. pLV-GFP group. (H) Wound-scratch healing assay for the treated cell migration at 24 h. *P < 0.05 and **P < 0.01 vs. pLV-GFP.