Figure 7.

Downregulation of MSI2 activity by treatment with the small molecule Ro 08-2750 improves DM1-related phenotypes

MSI2 quantification by (A) qRT-PCR, relative to the mean of GAPDH, GPI, and HPRT1 levels, and (B) western blot in 7-day differentiated DM1 TDMs treated for 48 h with 10 or 15 μM Ro 08-2750 or with 0.8% DMSO as control. Bar graph and representative blot images of MSI2 immunodetection are shown. β-actin expression was used as an endogenous control. (A and B) n = 6 for myotubes treated with DMSO and 10 μM Ro 08-2750. (C) Relative expression levels of miR-7 were measured by qRT-PCR. Data were normalized to the mean of U1 and U6 levels. (D) Quantification by qRT-PCR of TGFBR1 after small-molecule treatment relative to the mean of GAPDH, GPI, and HPRT1 expression levels used as an endogenous reference. In all qRT-PCR and western blots, the sample size was n = 3, unless otherwise specifically indicated. Representative confocal images of Desmin-immunostained (green) human DM1 TDMs for 7 days after treatment with (E) DMSO as control or with Ro 08-2750 compound at (F) 10 μM or (G) 15 μM. Quantification of the percentage of myogenic fusion index (H) and myotube diameter (I) of DM1 TDMs with the indicated concentrations of the compound (n = 10−15 images). Quantification by western blot of (J) ATG4A, (K) ATG7, and (L) P62. Representative blots used for quantification are shown below the bar graphs. β-actin was used as an endogenous control to normalize protein levels (n = 3). Representative confocal images of LysoTracker staining (red) of 7-day DM1 TDMs treated with (M) DMSO as control or with the Ro 08-2750 compound at (N) 10 μM or (O) 15 μM. (E−G and M−O). Scale bars correspond to 20 μm, and the nuclei were counterstained with DAPI. The bar graphs show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 according to Student’s t test.