Fig 2: ADSCs regulate VEGF-C-mediated lymphangiogenesis and macrophage infiltration via the METTL3/IGF2BP2-m6A pathway in DFU mice. Sh-METTL3 or sh-IGF2BP2 plasmids were transfected into ADSCs for METTL3 or IGF2BP2 knockdown. sh-NC served as the negative control for sh-METTL3 or sh-IGF2BP2. Subsequently, untreated ADSCs or transfected ADSCs were injected into DFU mice. (N = 5) A The wound closure statistics of DFU mice. B Western blotting was conducted to detect the protein levels of VEGFR3, VEGF-C and LYVE-1. Data represent the mean of five independent experiments. Error bars represent SD. *P < 0.05, **P < 0.01 or ***P < 0.001

Fig 3: ADSCs regulate VEGF-C expression via the METTL3/IGF2BP2-m6A pathway. sh-METTL3 and pcDNA-METTL3 plasmids were transfected into ADSCs for METTL3 knockdown and overexpression, respectively. sh-NC served as the negative control for sh-METTL3, and the pcDNA-empty vector served as the negative control for pcDNA-METTL3 plasmids. A MeRIP-qPCR was performed to assess the m6A levels of VEGF-C mRNA. B Western blotting was conducted to measure the levels of VEGF-C proteins. C An ELISA was carried out to assess the levels of VEGF-C in the supernatant of ADSCs. D, E RNA pulldown and RIP assays were performed to evaluate the interaction between VEGF-C and IGF2BP2. Immunoblotting of IGF2BP2 after RNA pulldown assay with cell lysate (Ly) and full-length biotinylated VEGF-C (FL); NC indicates the beads. sh-IGF2BP2 plasmids were transfected into ADSCs for IGF2BP2 knockdown, and sh-NC served as the negative control for sh-IGF2BP2 plasmids. F RT-qPCR was performed to assess the levels of IGF2BP2. G Western blotting was conducted to measure the protein levels of IGF2BP2 and VEGF-C. Data represent the mean of three independent experiments. Error bars represent SD. *P < 0.05, **P < 0.01 or ***P < 0.001

Fig 4: Let-7 repression is insufficient to explain LIN28A, TERT, and sh-p53 (LTS) rejuvenation of adult muscle progenitors. (A) Quantitative RT-PCR for let-7 microRNAs in TS and LTS myoblasts, relative to young adult human skeletal muscle (HSKM) myoblasts, after control (CTRL), let-7a, let-7b, or combined let-7a and let-7b (let-7a + b) mimic transfection (N = 3 wells of cells for each group). (B) Cell counts after control RNAi (CTRL), let-7a, let-7b, or combined let-7a and let-7b (let-7a + b) mimic transfection in equal initial numbers of TS and LTS myoblasts, relative to young adult HSKM myoblasts (N = 3 wells of cells for each group). (C) Quantification of senescence-associated ß-galactosidase (SA-ß-gal)+ cells in TS and LTS myoblasts, relative to young adult HSKM myoblasts, after control RNAi (Ctrl RNAi), let-7a, let-7b, or combined let-7a and let-7b (let-7a + b) mimic transfection (N = 5 wells of cells for each group). (D) Western blot analysis for the let-7 targets IGF2BP1/2/3 (IMP1/2/3) and HMGA2 protein, relative to Gapdh protein, in young adult HSKM, TS, and LTS myoblasts, after control (CTRL), let-7a (a), let-7b (b), or combined let-7a and let-7b (A,B) mimic transfection. The quantification of WB bands is shown in Figure S2. (E) Representative gene set enrichment analysis (GSEA) enrichment profiles for RNAseq analysis of TS myoblasts. (F–K) Representative GSEA enrichment profiles for RNAseq analysis of LTS myoblasts. *p < 0.05, **p < 0.01, ***p < 0.001.