Figure 3.
Furamidine rescues mis-splicing in DM1 and DM2 fibroblasts
(A–C) RT-PCR splicing analysis of common alternate exon events INSR eon11 (A), SYNE1 exon65 (B), and FLNB exon31 (C) in control (black), DM1 (blue), and DM2 (green) fibroblasts untreated (−) or treated with 2 or 4 μM furamidine (see insert in A for structure). Furamidine rescues INSR exon11 splicing across all the DM1 and DM2 fibroblast lines (A); FLNB exon31 splicing in all DM lines except DM2-B line which does not show significant mis-splicing versus control (B); and SYNE1 exon65 splicing only in DM2 lines (C).
(D) Quantification of fluorescence in situ hybridization (FISH) analysis (D) of CCUG RNA foci in DM2 fibroblasts. DM2 fibroblast untreated (−) or treated with 2 or 4 μM furamidine for a period of 4 days followed by FISH analysis using a Cy3-(CAGG)6 probe. The average number of RNA foci per nuclei within a given field was calculated and expressed as a relative value versus untreated cells.
(E) MBNL1 expression relative to GAPDH was determined via RT-qPCR (E) and displays significant increases for both DM1-B (blue) and DM2-A (green) fibroblasts when expressed as a percentage relative to untreated fibroblast.
(F) MBNL1 protein levels via western blot (F) show an increasing trend in DM1-B (blue) line and a significant increase with 2 μM furamidine treatment in DM2-A (green) fibroblasts relative to GAPDH. All experiments were done in at least triplicate using biological replicates (two-tailed t-test ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p <0.001).