Fig. 5.
Tet1 and Tet2 control miRNA secretion in BMMSCs through demethylation of P2rX7. a Bradford assay showed exosome secretion in control and Tet DKO BMMSCs. Exosomes from the culture supernatant of 1 × 106 BMMSCs containing proteins were assessed. b Western blotting showed exosomes from control and Tet DKO BMMSCs expressed CD9 and CD81. Exosome proteins from equal volumes of culture supernatant of WT and Tet DKO BMMSCs were loaded for western blotting. c Exosomes volumes derived from control and Tet DKO BMMSCs was detected by EXOCEP exosome quantitation kit. d Immunofluorescent staining showed CD9 and CD81-positive red immunofluorescence labeled exosomes localized in control and Tet DKO BMMSCs. e Transmission electron microscopic (TEM) showed the micro-vesicular in control and Tet DKO BMMSCs. f P2rX7 expression (red) was co-localized with CD146 (green) in BMMSCs, as assessed by immunofluorescent double staining. g, h The expression of P2rX7 in control and Tet DKO BMMSCs, as assessed by western blotting (g) and qPCR analysis (h). i, j Tet1 and Tet2 binding on the promoter of P2rX7 in BMMSCs, as assessed by ChIP-qPCR. IgG was used as a control. k Enrichment of 5-hmC in the P2rX7 promoter in control and Tet DKO BMMSCs, as assessed by hMeDIP-qPCR analysis. IgG was used as a control. l The levels of miR-297a-5p, miR-297b-5p, and miR-297c-5p in control and P2rX7 siRNA-treated BMMSCs, as assessed by qPCR. m Exosome secretion levels in control and P2rX7 siRNA-treated BMMSCs, as assessed by Bradford assay. n Western blotting showed the expression of CD9 and CD81 in exosomes derived from control and P2rX7 siRNA-treated BMMSCs. o, p Mineralized nodule formation and the expression of Runx2, ALP, and OCN in control and P2rX7 siRNA-treated BMMSCs, as analyzed by alizarin red staining (o) and western blot (p). *p < 0.05, **p < 0.01, ***p < 0.001 (mean ± SD). Scale bar, 10 μm (d), 200 nm (e), 50 μm (f). Results are from three independent experiments. p values were calculated using two-tailed Student's t test