Figure 8.
ALKBH5 upregulates the mRNA stability of YTHDF1 in turn promotes the translation of YAP. (A) m6A-meRIP-qPCR of YTHDF1 in CTL or ALKBH5 OE P1 CMs (***P < 0.001, n = 4). (B) RT-qPCR analysis of YTHDF1 in cultured P1 cardiomyocytes transfected with control or ALKBH5 OE plasmid (**P < 0.01, n = 4). (C) RT-qPCR analysis of YTHDF1 in cultured P1 cardiomyocytes transfected with control siRNA or ALKBH5 siRNAs (*P < 0.05, n = 4). (D) Western blot analysis of ALKBH5 and YTHDF1 in cultured P1 cardiomyocytes transfected with control or ALKBH5 OE plasmid. (E) RT-qPCR of YTHDF1 transcripts in Act D-treated control and ALKBH5 siRNA P1CMs (n = 4). (F) Western blot analysis of YTHDF1 in Act D-treated control or ALKBH5 siRNA P1 CMs (G) Western blot assay of YTHDF1 in hearts from P1, P7, P14, P21 and 8W mice. (H) RT-qPCR analysis of YTHDF1 in hearts from P1 and P7 cardiomyocytes (***P < 0.001, n = 4). (I) Western blot analysis of YTHDF1 in cultured P1 cardiomyocytes transfected with control, YTHDF1 siRNA or YTHDF1 OE plasmid. (J) Cardiomyocytes isolated from P1 mice were transfected with CTL-siRNA or YTHDF1-siRNA and immunostained against EdU, phospho-histone H3 (pH3) and α-actinin (marks cardiomyocytes). DAPI was used for nuclear staining. n = 5 per group. *P < 0.05. The arrows point to EdU/pH3-positive signal. Scale bar, 50 μm. (K) Western blot analysis of YAP in cultured P1 cardiomyocytes transfected with control or YTHDF1 OE plasmid. (L) Western blot analysis of YAP in cultured P1 cardiomyocytes transfected with control or YTHDF1 siRNA. (M) RIP-qPCR analysis of the interaction of YAP and YTHDF1 in hiPSC-CMs. Enrichment of YAP was normalized to input (***P < 0.001, n = 4). (N) Western blot analysis of YAP and YTHDF1 in CTL, ALKBH5 OE, YTHDF1 siRNA and ALKBH5 OE+YTHDF1 siRNA P1 CMs. (O) Neonatal P1 cardiomyocytes in ALKBH5 OE and ALKBH5 OE+YTHDF1 siRNA group were immunostained against EdU and pH3 (*P < 0.05, **P < 0.01). n = 5 per group.