Research Paper Volume 13, Issue 24 pp 25903—25919

Figure 4. METTL14-dependent m6A modification regulated the expression of PTPN6. (A) Heat map of normalized gene expression levels of SONFH BMSCs transfected with OE-NC or OE-METTL14. Red indicates down-regulation of gene expression and Green indicates up-regulation of gene expression. (B) PTPN6 mRNA levels in normal tissues (n = 20) and osteonecrosis tissues (n = 20) were measured by qRT-PCR. (C) IHC assay detected the expression of PTPN6 in femoral head tissues of two group. Scale bar = 100 μm. (D, E) qRT-PCR and western blot quantified the abundance of PTPN6 in normal BMSCs and SONFH BMSCs. (F) The correlation between METTL14 and PTPN6 expression in SONFH BMSCs was analyzed by Pearson’s Correlation Coefficient. (G, H) qRT-PCR and western blot confirmed that PTPN6 expression increased with the up-regulation of METTL14 in SONFH BMSCs. (I) The methylated PTPN6 mRNA level in the METTL14 overexpressed BMSCs was determined by MeRIP-qPCR. (J) The mRNA half-life of PTPN6 in SONFH BMSCs transfected with OE-NC or OE-METTL14. (K) SONFH BMSCs were pre-transfected with wild-type or mutated PTPN6-3′UTR plasmids, and then treated with OE-NC or OE-METTL14. The luciferase activity was normalized to firefly luciferase activity. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s is no significance.