Fig 1: 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 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-mediated lymphangiogenesis via the METTL3/IGF2BP2-m6A pathway. sh-METTL3 or sh-IGF2BP2 and pcDNA-VEGF-C were cotransfected into ADSCs. sh-NC served as the negative control for sh-METTL3 or sh-IGF2BP2, and the pcDNA-empty vector served as the negative control for pcDNA-VEGF-C plasmids. Subsequently, transfected ADSCs were cocultured with LECs. A RT-qPCR was performed to assess the levels of VEGF-C. B An ELISA was conducted to assess the levels of VEGF-C. C Transwell assays were carried out to evaluate LEC migration. D A tubule formation assay was performed to assess the tubule formation ability of LECs. Data represent the mean of three independent experiments. Error bars represent SD. *P < 0.05, **P < 0.01 or ***P < 0.001
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