Metformin inhibits pronephric cyst formation in pkd2 morphants. Tg(wt1b:GFP) pkd2 morphants were immersed in embryo media (E3) supplemented with 2.5, 5, 10, or 20 mM metformin until 48 hpf. (A) Representative images of the pronephros in the control embryos, pkd2 morphants, and metformin-treated pkd2 morphants are shown. Note the pronephric cysts (arrows) in the Tg(wt1b:GFP) pkd2 morphant. Photographs from in vivo observation through fluorescence microscopy (dorsal view, anterior to the top) were recorded at 48 hpf. The lower panels show overlays of transmission (grey) and fluorescence (green) images of the upper panels. Scale bar, 100 µm. (B) Comparative frequencies of the cystic phenotype in the control embryos and pkd2 morphants treated with different concentrations of metformin as indicated (n = 45–99 per group). Data represent five independent experiments. **P < 0.01, ***P < 0.001 compared with pkd2 knockdown. (C) Transverse histological sections of the embryos at 48 hpf, illustrating the pronephric cyst (indicated by the mark *) in a pkd2 morphant and the rescue effect of metformin treatment. Arrows indicate the position of glomeruli. H&E stain, scale bar, 10 µm.
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