Fig. 6
SND1 recognizes Nrf2 mRNA and enhances its stability in an m6A-dependent manner. A RIP assays were performed using anti-SND1 and IgG antibodies. The enrichments of Nrf2 mRNA by SND1 or IgG were detected via RT-qPCR. B After actinomycin D (5 μg/ml) treatment for 0, 2, 4, 6 h, RT-qPCR was used to analysis the Nrf2 mRNA stability in U87MG cells with SND1 overexpression and A172 cells with SND1 silence. C RT-qPCR detected the mRNA level of Nrf2 in U87MG cells with SND1 overexpression and A172 cells with SND1 silence. D Western blotting showed the protein levels of Nrf2 after SND1 overexpression or silence under (E) Schematic illustration was used to explain the design of dual luciferase reporter plasmids containing wild-type or mutant m6A sites in Nrf2 3’ UTR sequence. F Wild-type or mutant plasmids of reformed dual luciferase reporters were transfected into U87MG cells with SND1 overexpression and A172 cells with A172 cells with SND1 silence, respectively. The relative luciferase activity was measured and normalized. G m6A levels of U87MG cells with or without three m6A methyltransferases (METTL3, METTL14, and WTAP) silence were detected using the m6A RNA Methylation Quantification Kit and m6A dot blot assays. H U87MG cells with indicated interventions were treated with actinomycin D (5 μg/ml) for 0, 2, 4, 6 h, and Nrf2 mRNA stability was analyzed via RT-qPCR. I In METTL3-silenced or control U87MG cells, MeRIP assays and RT-qPCR were performed to calculate the relative enrichment of m6A modification. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and n.s., not significant