Fig. 2

Inhibition of miR-92a in podocytes upregulates its target p57 and impairs proliferation. a RT-qPCR analysis of the relative abundance of miR-92a in podocytes transfected with an anti-miR-control (anti-miR-ctrl) or an anti-miR-92a. Values are normalized to U6snRNA and are relative to control (non-transfected cells). b, c Representative pictures (b) and quantification (c) of podocyte proliferation assay involving decapsulated mouse glomeruli. Podocyte proliferation was assessed after 4 days. Scale bars, 50 μm. d RT-PCR analysis of Ki67 mRNA abundance in intact and anti-miR-ctrl primary podocytes or in anti-miR-92a primary podocytes. e Dual luciferase assay wild-type or mutated p57 3′UTR in HEK293 cells transfected with pre-miR-ctrl, pre-miR-92a, or pre-miR-126. n = 2 independent experiments (each experiment assayed each condition in triplicate). p57 3′UTR miR-92a-binding sequence binding site is indicated in bold and mutated sequence is labeled in red. ***p < 0.001 vs. 3′UTR +Pre-miR- ctrl. f, g Western blot analysis (f) and quantification of p57 protein abundance (g) in untreated or anti-miR-ctrl primary podocytes vs. in anti-miR-92a primary podocytes. Tubulin is shown as a loading control. h Staining of p57 protein (green) in podocyte outgrowths. DAPI-stained nuclei (blue). Adherent glomeruli are indicated by arrows. Scale bars, 100 μm. Statistical analysis: Kruskal–Wallis one-way analysis of variance followed by Dunn’s multiple comparaison test. Values are means ± s.e.m. (n = 4 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control podocytes (control)