Fig. 8

YTHDF1 identified with m6A-meditated GLIS1 mRNA and participated the translation process of GLIS1 protein. A The expression of YTHDF1 in the 24-month-old and 6-month-old group by IHC assay, and its semi-quantitative analysis (n = 6), scale bar = 50 μm; B immunostaining for YTHDF1 (green), with DAPI (blue) counterstaining by IF staining in the 24-month-old and 6-month-old group (n = 6), scale bar = 50 μm; C, D protein level of YTHDF1 in the 24-month-old and 6-month-old group by western blot, and its semi-quantitative analysis (n = 6); E mRNA level of YTHDF1 by RT-qPCR in HK-2 cells within vector or siYTHDF1 (n = 3); F protein level of GLIS1 in HK-2 cells within vector or siYTHDF1, and its semi-quantitative analysis (n = 3); G double immunostaining for GLIS1 (red) and YTHDF1 (green) by IF staining in HK-2 cells (n = 3), scale bar = 50 μm; H, I RIP and RT-PCR assays confirmed the interaction between YTHDF1 and GLIS1 mRNA, and its semi-quantitative analysis (n = 3); J the expression of GLIS1 with RPL22-FLAG label by ribosomal immunoprecipitation in HK-2 cells (n = 3); K the mRNA level of GLIS1 by RT-qPCR in HK-2 cells within vector or siYTHDF1 (n = 3); L, M relative luciferase activity of the GLIS1-WT or GLIS1-Mut 3′UTR luciferase reporter in the vector and siMETTL3 group, n = 3. The data are expressed as the mean ± SD of three independent experiments. **P < .01 or ***P < .001 versus the 6-month-old group, or vector group, or IgG group by Student’s t-test