Figure 4: Compound 1 induces an MBNL1-dependent DM1-like splicing shift in normal human fibroblasts.
From: Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules
(a) Representative RT–PCR assays showing splicing changes in MBNL1-dependent exons in normal fibroblasts treated with increasing amounts of 1 (n=2). The DM1-like splicing shift is depicted as alternative exon inclusion (+alt. ex.), while the normal splicing isoform is depicted as alternative exon exclusion (-alt. ex). As a control, untreated and DMSO-treated (DMSO) normal fibroblasts (n=5) and DM1 fibroblasts expressing 2,000 CUG repeats (DM1 2000 CUG) (n=4) were used. No RT lane refers to RT–PCR control without reverse transcriptase. (b,c) Quantification of alternative splicing shift towards the DM1-like phenotype (% of alternative exon inclusion) in normal fibroblasts treated with increasing concentrations of 1 (orange bars; n=2), untreated and DMSO-treated normal fibroblasts (green bars; n=5), siMBNL1-treated normal fibroblasts (red bars; n=2) and DM1-affected human fibroblasts expressing 2,000 r(CUG) repeats (blue bars; n=2). Splicing of MBNL1-dependent exons is shown in b while splicing of MBNL1-independent exons is shown in c. Each sample was subjected to RT–PCR twice. The errors reported are the s.d. values derived from analysis of all samples. ‘*’ indicates P≤0.05; ‘**’ indicates P≤0.01; and ‘***’ indicates P≤0.001 as determined by a two-tailed Student’s t-test.
