WTAP-mediated N6-methyladenosine modification of circ_PVT1 promotes proliferation and tenogenic differentiation of tendon stem/progenitor cells

Figure 4 An N6-methyladenosine writer, WTAP, interacted with circular RNA plasmacytoma variant translocation 1. A and B: An N6-methyladenosine (m⁶A) dot blot assay showing the m⁶A levels in the total RNA of mouse tendon stem/progenitor cells (TSPCs); C: SRAMP (http://www.cuilab.cn/sramp) software showing the potential m⁶A modification sites of circular RNA plasmacytoma variant translocation 1 (circ_PVT1) and the genomic sequence GGACU with very high probability; D: Methylated RNA immunoprecipitation polymerase chain reaction assay indicates that the m⁶A modification level of circ_PVT1 was upregulated in the transforming growth factor-β1-induced TSPCs; E and F: Silver staining of the gel after sodium-dodecyl sulfate gel electrophoresis separation of the proteins revealed that they immunoprecipitated with the 3’-biotin-labeled probe for circ_PVT1 and with the biotinylated antisense probe in RNA pull-down assays. The line shows the position of WTAP. Western blot analysis following the RNA pull-down assay indicates the interaction between circ_PVT1 and WTAP protein; G and H: Quantitative polymerase chain reaction analysis following the RNA immunoprecipitation assay confirmed the interaction between the WTAP protein and circ_PVT1. All data are presented as the mean ± SD. ^bP < 0.01, ^cP < 0.001. NS: No significance; NC: Negative control; TGF-β1: Transforming growth factor-β1; circ_PVT1: Circular RNA plasmacytoma variant translocation 1; m⁶A: N6-methyladenosine.