Fig 2: Overexpression of WTAP promoted bone formation in OVX mice. (A) Schematic diagram illustrating the experimental design. (B) Verification of the efficiency of WTAP overexpression at the mRNA level on day 7 after lentivirus injection in mice. (C) Live animal fluorescence imaging analysis of luciferase expression in the WTAP-overexpressing group and empty vector-carrying group. (D) qRT-PCR analysis of ALP, COL1, and OCN mRNA levels in bone specimens from mice at 7 weeks post-injection. (E) Representative images showing the 3-dimensional trabecular architecture in distal femurs determined by micro-CT reconstruction. (F) Micro-CT measurements of BS/BV, Tb. Sp, BMD, BV/TV, Tb. N, and Tb. Th in the distal femurs of mice after treatment with WTAP-overexpressing lentivirus or empty vector-carrying lentivirus. (G) H&E staining indicated trabecular density. (H) Masson trichrome staining indicated trabecular density and collagen. Data are expressed as the mean ± SEM, *P < .05, **P < .01, ***P < .005.

Fig 3: WTAP contributes to increased m6A levels in EPCs. (A) mRNA expression levels of m6A-related enzymes during EPC differentiation, as detected by quantitative PCR. After differentiation, the expression levels of METTL14, WTAP, FTO, ALKBH5, IGF2BP1-3 and YTHDF1-2 were significantly higher than in EPCs before differentiation. **P<0.01, ***P<0.001 vs. 1D. (B) Western blotting was used to verify the protein expression levels of WTAP, which were also increased after cell differentiation. (C) WTAP expression was validated by western blot analysis following overexpression and knockdown of WTAP. Compared with the control group, the overexpression group exhibited an increase in WTAP protein expression, whereas the knockdown group showed a decrease in WTAP protein expression (with the exception of EPC-shWTAP-1). (D) Immunofluorescence assays were performed to validate alterations in m6A following the successful overexpression and knockdown of WTAP in HPB-EPCs. Overexpression of WTAP in HPB-EPCs increased m6A levels, whereas knockdown of WTAP resulted in a decrease in m6A levels. *P<0.05 vs. EPC-NC; ***P<0.001 vs. EPC-scr. D, day; EPC, endothelial progenitor cell; m6A, N6-methyladenosine; NC, negative control; scr, scrambled; sh, short hairpin; WTAP, Wilms' tumor 1-associated protein.

Fig 4: m6A regulators anticipate in the development and progression of NPC. (a) Knocking down of WTAP, METTL3 and METTL14 in NPC cell line HK-1. (b) WTAP, METTL3, and METTL14 promoted the migration ability of HNE2 cells transfected with siRNA assessed using by wound-healing assay. Images were acquired at 0 h to 24 h. Data have been represented as represent with mean ± SD. *P < 0.05; ***P < 0.001. (c) WTAP, METTL3, and METTL14 promoted the invasive ability of S18 cells after knockdown, as assessed using by Transwell® invasion assays. Images were acquired at 24 h and are representative of and presented from three independent experiments. Data have been represented as represent with mean ± SD. **P < 0.01; ***P < 0.001. (d) Representative cryo-electron microscopy images of exosomes in four groups (scale bar = 200 nm). (e) Western blotting analysis of exosome markers CD9, CD63, and TSG101 in four groups. (f) ELISA showed that knockdown of m6A writers would reduce exosome in NPC microenvironment. (g) Immunofluorescence showed that knockdown of m6A writers can induce uptake of exosome in NPC cells. (h) Represent image of γH2AX IRIF in 5-8F cells were quantified after 2 Gy IR at indicated recovery time in siControl, siMETTL3 and siMETTL14 group. METTL3: methyltransferase 3; METTL14: methyltransferase 14; WTAP: WT1 associated protein; ELISA: enzyme-linked immunoassay; IR: ionizing radiation; IRIF: IR-induced foci.

Fig 5: Knockdown of WTAP inhibits the differentiation of EPCs. (A) Cell Counting Kit 8 was used to analyze the proliferation of EPC-scr, EPC-WTAP-sh2 and EPC-WTAP-sh3 at different times. EPC proliferation was decreased after 72 h of culture following WTAP knockdown. (B) Invasion and (C) tube formation were determined using Transwell and tube formation assays, respectively. In contrast to EPC-scr, cell invasion, and tube formation were decreased after WTAP knockdown. ***P<0.001 vs. EPC-scr. EPC, endothelial progenitor cell; scr, scrambled; sh, short hairpin; WTAP, Wilms' tumor 1-associated protein.