Fig. 7: The protective effect of METTL14 knockdown in injured podocyte relied on promoting Sirt1 mRNA m6A modification and degradation. | Cell Death & Disease

Fig. 7: The protective effect of METTL14 knockdown in injured podocyte relied on promoting Sirt1 mRNA m6A modification and degradation.

From: METTL14 aggravates podocyte injury and glomerulopathy progression through N6-methyladenosine-dependent downregulating of Sirt1

Fig. 7

A Representative images of immunohistochemical staining for Sirt1 on renal biopsies from normal subjects (n = 4) and patients with MCD (n = 6), FSGS (n = 6) and DN (n = 8). Scale bars, 20 μm. B The relative mRNA levels of Sirt1 in podocytes transfected with SiNC or SiMETTL14 under ADR (0.4 μg/ml) or AGE (50 μg/ml) condition for 24 h (n = 6). C Western blot analysis showing the upregulation of Sirt1 expression in ADR-treated podocytes (n = 6). D Western blot analysis showing METTL14 knockdown weakened AGE-induced inhibition of Sirt1 expression in podocytes (n = 6). E, F Representative confocal microscopic images and quantification of immunofluorescence co-staining for METTL14 and Sirt1 in podocytes transfected with SiNC or SiMETTL14 under ADR or AGE condition. Scale bar, 40 μm (n = 6). G, H MeRIP-qPCR analysis in podocytes with different treatment. The results indicated ADR or AGE intervention promoted m6A antibody binding with Sirt1 mRNA. METTL14 silencing ameliorated the upregulation of methylated Sirt1 mRNA levels induced by ADR or AGE treatment (n = 5). I, J Luciferase assays were performed in podocytes transfected with wild-type or mutant Sirt1 reporter plasmids under different condition. AGE or ADR administration attenuated the luciferase activity of podocytes transfected with wild-type Sirt1 reporter plasmids. METTL14 knockdown in podocytes relieved the reduction of luciferase activity induced by AGE or ADR treatment. However, ADR or AGE intervention had no effect on the luciferase activity of podocytes transfected with mutant Sirt1 reporter plasmids (n = 5). K, L Podocytes with different treatment were followed by incubation with actinomycin D for 0, 2, 4, 6 h. The decay curves of Sirt1 mRNA were shown. All values are normalized as fractions of the original mRNA levels (n = 6). *P < 0.05, **P < 0.01 vs. Con/BSA + SiNC group, #P < 0.05, ##P < 0.01 vs. ADR/AGE + SiNC group. Data are presented as mean ± SEM.

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