Figure 3.

Atrophic muscle markers ameliorate in DM1 TDMs upon MSI2 reduction

(A) Cell growth inhibition assay by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) method. Human control (CNT) myoblasts were transfected with a range of concentrations of ASO1, -2, and -3 (n = 4). TC10 (33.1 nM, 158.3 nM, and 40.6 nM for ASO1, -2 and -3, respectively) was obtained using the least-squares non-linear regression model. Vertical dashed lines indicate 30 and 150 nM concentrations used in the subsequent experiments. MSI2 quantification by (B) qRT-PCR relative to GAPDH and GPI and (C) by western blot relative to β-actin expression. (D) Quantification of miR-7 relative to U1 and U6 in DM1 TDMs transfected with the indicated ASOs. (E) Quantification by qRT-PCR of P21 and TGFBR1 expression relative to the mean of GAPDH and GPI levels after treatment of the muscle cells with the indicated concentrations of ASOs. Relative expression of control myotubes 1.412 ± 0.068 and 0.643 ± 0.042 for P21 and TGFBR1, respectively. (D and E) The sample size was three biological replicates. (F) Analysis of myogenic fusion index and (G) myotube diameter of DM1 myoblasts transfected with the indicated concentrations of ASOs or scramble (n = 10−15 images analyzed). (H−J) Representative confocal images of Desmin-immunostained (green) human TDMs for 7 days after ASO or scrambled control transfection at 150 nM. Nuclei were counterstained with DAPI (blue). Scale bar, 40 μm. The bar graphs show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, according to Student’s t test. In all cases, the statistical analysis compares the values of ASO and scrambled-treated cells (black dashed line or gray bar).