Fig. 3

AntagomiR-23b and -218 stabilize MBNL transcripts and rescue alternative splicing defects in DM1 myoblasts. a Real-time PCR quantification of miR-96, miR-23b, and miR-218 expression in myoblasts and muscle biopsies from DM1 patients. U1 and U6 snRNAs were used as reference genes. b Cell growth inhibition assay by MTS method. Human normal myoblasts were transfected with increasing concentrations of antagomiRs against miR-23b and miR-218 (n = 4). TC10 was obtained using the least squares non-linear regression model. c, d qRT-PCR quantification of MBNL1 (c) and MBNL2 (d) expression relative to GAPDH and ACTB genes in human DM1 myoblasts transfected with the indicated antagomiRs or scrambled control antagomiR (sc). e Semiquantitative RT-PCR analyses of splicing events altered in BIN1 (exon 11), ATP2A1 (exon 22), cTNT (exon 5), INR (exon 11), and PKM isoforms in DM1 cells. GAPDH was used as internal control. Inclusion of DLG1 exon 9, which is not altered in DM1, and CAPZB exon 8, which is CELF1-dependent, were used as additional controls. f, h miRNA real-time PCR determination of available miR-23b (f) or miR-218 (h) in DM1 myoblasts 96 h after transfection with 50 nM of antagomiR-23b or 200 nM of antagomiR-218. U1 and U6 snRNAs were used as reference genes in f and h. g, i qRT-PCR analyses of MBNL1 (g) and MBNL2 (i) expression relative to GAPDH and ACTB genes in human myoblasts 96 h after transfection with 50 nM of antagomiR-23b or 200 nM of antagomiR-218. (n = 3). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 according to Student’s t test