Fig. 6.

MT suppresses VSMC calcification and aging by inducing ECs derived exosomes secretion in a m6A dependent manner. (A) Expression heatmap of m6A-related genes in ECs before and after MT treatment. (B) qRT-PCR confirmed that the expression level of WTAP, pri-miR-302, miR-302d-5p of ECs treated with Veh, MT, MT plus DMSO, MT plus luzindole respectively. (C) Dot blot identified m6A modification levels of total RNA in ECs undergoing Veh and MT interventions. (D) The m6A content of total RNA from ECs treated with Veh or MT was measured by ELISA using an EpiQuik m6A RNA methylation quantification kit. (E) The expression level of methylated pri-miR-302 was measured by MeRIP-PCR. (F)qRT-PCR confirmed that the expression level of WTAP, pri-miR-302, miR-302d-5p of ECs after knock-down of WTAP. (G) qRT-PCR confirmed that the expression level of HNRNPA2B1, pri-miR-302, miR-302d-5p of ECs after knock-down of HNRNPA2B1. (H) Detection of pri-miR-302 binding to DGCR8 by immunoprecipitation experiments in control and HNRNPA2B1 down-expression ECs. (I)The expression level of Runx2, osteopontin, osteocalcin, P16, P21, P53 in Veh-ECs-Exos, MT-ECs-Exos, MT-ECs-Exos plus siRNA ctrl and MT-ECs-Exos plus WTAP KD treated ECs.(J) The calcium nodules in VSMCs was measured by Alizarin red S staining (upper panel, scale bar = 200 μm), the senescence VSMCs was measured by SA-β-Gal staining (lower panel, scale bar = 200 μm). Results are represented by mean ± SEM. *p < 0.05. **p < 0.01, ***p < 0.001.