Fig. 7.

WTAP promotes fibroblast proliferation and migration in pulmonary fibrosis through m⁶A methylation. (A) Representative immunofluorescence images of the merged photos in normal and IPF samples. (B) Representative western blot images of WTAP. (C) qRT-PCR result of the transfection efficiency. (D) Knockdown of WTAP significantly reduced the expression of α-SMA and Collagen III in TGF-β–stimulated fibroblasts and reversed their upregulation induced by TGF-β. (E) Representative immunofluorescence images to detect WTAP, Ki-67 and α-SMA expression in TGF-β-induced groups at 48 h. Scale bars, 20 μm. (F) Cell proliferation was measured by EdU staining. (G-H) Fibroblasts migration ability was measured by the wound healing and transwell assay. (I) Dot blot assay using an anti-m⁶A antibody in TGF-β-induced fibroblasts, shWTAP and NC groups. MB staining was included as a loading control. P < 0.05 was considered significant. (J) Western blot images of IGFBP5, KLF6, NR4A3 and MYC levels. (K) MeRIP-qPCR analysis of MYC, IGFBP5, KLF6 and NR4A3 in TGF-β-induced fibroblasts. (L) Putative KLF6 binding site on the promoter region of WTAP. (M) KLF6 binds to the promoter region of WTAP in TGF-β induced fibroblasts. Chromatin-IP was performed using KLF6 antibody or control IgG. Values are percentage of input. (O) KLF6 activates WTAP promoter activity in dual-luciferase reporter assay. (O) Western blotting analysis and correlation analyses of WTAP expression with expression of KLF6 in 8 freshly collected human IPF samples. *p < 0.05; **p < 0.01; ***p < 0.001 compared to the corresponding groups