Fig. 4

Increase of MBNL1 and MBNL2 upon silencing of miR-23b or miR-218 in human myoblast. a–f Western blot quantification of MBNL1 (a, d), MBNL2 (b, e), and CELF1 (c, f) expression levels in DM1 human myoblast 96 h after transfection with 50 nM of antagomiR-23b, 200 nM of antagomiR-218 or a scrambled control antagomiR (sc). β-ACTIN expression was used as endogenous control (n = 3). Data are mean ± SEM. **p < 0.01, ***p < 0.001 in Student’s t test. g–n Representative confocal images of MBNL1 (green) and MBNL2 (red) staining in healthy controls (control cells) and DM1 human myoblast 96 h after transfection with antagomiRs against miR-23b (50 nM) or miR-218 (200 nM) and a scrambled control antagomiR (DM1 cells). Nuclei were counterstained with DAPI (blue). In DM1 cells, endogenous MBNL1 (h) and MBNL2 (l) were in nuclear aggregates (green and red puncta) and the total amount of both was reduced compared to control cells (g) and (k), respectively. In contrast, DM1 cells treated with antagomiRs against miR-23b or miR-218 showed a robust increase in cytoplasmic and nuclear MBNL1 (i, j) and MBNL2 (m, n) levels compared to DM1 cells. b–q Representative fluorescence of FISH images showing (CUG)n RNA foci (red) in DM1 human fibroblasts transfected with antagomiRs against miR-23b (50 nM) or miR-218 (200 nM) and a scrambled control antagomiR. Nuclei were counterstained with Hoechst (blue). AntagomiRs did not significant change the number of ribonuclear foci in DM1 fibroblasts (r). Scale bar = 20 μm