Figure 6.

Degradation of MSI2 transcripts enhances MBNL1 levels in 7-day differentiated TDMs

(A) Quantification and representative blots of MBNL1 levels in protein extracts obtained from DM1 TDMs treated with the indicated concentrations of MSI2-targeting or scrambled ASOs. (B and C) The western blots shown are representative of the amounts of MBNL2 and CELF1. β-actin was used as an endogenous control (n = 3). Representative confocal images of (D−I) MBNL1 (green) or (J−O) MBNL2 (red) immunodetection in DM1 TDMs treated for 48 h with scramble (D, J, G, and M), ASO1 (E, K, H, and N), or ASO3 (F, L, I, and O) at the indicated concentrations. Nuclei were counterstained with DAPI (scale bar, 50 μm). (P−R) Semiquantitative RT-PCR analyses and representative agarose gels of splicing events altered in DM1 cells: (P) PKM isoforms represented as the percent of PKM2 and (Q) SERCA1 (exon 22) and (R) NFIX (exon 7) represented as the percent of exon inclusion. GAPDH was used as an internal control (n = 3). In all cases, the statistical analyses were performed comparing values from treated cells with those obtained for cells treated with the scrambled ASO at the same concentration. Green dashed lines indicate values obtained for healthy control TDMs. The bar graphs show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, according to Student’s t test.