Figures & data
Figure 1. FTO was down-regulated in myocardial IRI mice and H/R-induced cardiomyocytes. (a) m6A RNA methylation colorimetric quantification analysis detected the m6A content in total RNA of myocardial IRI mice and control (Sham) mice. (b) m6A RNA methylation colorimetric quantification analysis detected the m6A content in total RNA of H/R and control cardiomyocytes. (c) Western blot analysis detected the FTO protein level in myocardial IRI mice and control (Sham) mice. (d) Western blot analysis detected the FTO protein level in H/R-induced and control cardiomyocytes. *p < 0.05, **p < 0.01.
Figure 2. FTO mitigated the apoptosis and inflammatory reaction induced by H/R. (a, b) RT-PCR and Western blot assay were performed to detected the FTO mRNA and protein levels in H/R-induced cardiomyocytes transfected with FTO overexpression plasmids and shRNA-FTO. (c, d) Apoptosis analysis using flow cytometry revealed the apoptotic rate of H/R-induced cardiomyocytes transfected with FTO overexpression plasmids and shRNA-FTO. (e, f) Cell cycle analysis using flow cytometry detected the cellular distribution at G0/G1, S, G2/M phase. (g) IL-1β, (h) TNF-α, and (i) IL-6 were detected with ELISA. *p < 0.05.
Figure 3. YAP1 mRNA acted as the target of FTO, and FTO uninstalled the m6A modification of YAP1. (a) Online predictive tool (SRAMP, http://www.cuilab.cn/sramp) revealed the potential m6A modification sites in the 3’-UTR with very high probability. (b) The binding sites or motif of FTO were AUGGACU. (c) The m6A modification sites of Yap1 mRNA were identified in the genomic sequence (ATGGACT). (d) RNA immunoprecipitation (RIP)-PCR analysis revealed the Yap1 mRNA level enriched by the anti-FTO antibody. (e) MeRIP-PCR assay indicated the m6A modification level of H/R-induced cardiomyocytes upon FTO overexpression or FTO knockdown. *p < 0.05.
Figure 4. FTO enhanced the stability of YAP1 mRNA through uninstalling the m6A modification of YAP1 mRNA. (a) RT-PCR assay detected the YAP1 mRNA level in H/R-induced cardiomyocytes or control cells. (b) RNA immunoprecipitation (RIP) -PCR assay detected the YAP1 mRNA enrichment precipitated by anti-m6A antibody. Input acted as the positive control. (c) RNA stability analysis detected the YAP1 mRNA expression in H/R-induced cardiomyocytes treated with Actinomycin D (act D). Cardiomyocytes were transfected with FTO overexpression (FTO OE) and FTO knockdown (sh-FTO). (d) Western blotting assay indicated the Yap1 protein in FTO overexpression or FTO knockdown. *p < 0.05.
Figure 5. FTO enhanced the stability of YAP1 mRNA through uninstalling the m6A modification of YAP1 mRNA.
Supplemental Material
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