Figure 3: Compound 1 induces DM1-like splicing defects in HeLa cells.

HeLa cells were co-transfected with the mini-gene of interest and a plasmid that expresses r(CUG)^exp or an empty vector. Varying concentrations of 1 were added in growth medium post transfection, and total RNA was harvested 16–20 h later. The percentage of each splicing isoform was determined by RT–PCR using a radioactively labelled forward PCR primer. (a, top) Schematic of the pre-mRNA splicing isoforms observed for the IR mini-gene. (a, middle) representative gel image of IR alternative splicing in the presence of varying concentrations of 1. (a, bottom) Quantification of exon inclusion for IR alternative splicing in the presence or absence of r(CUG)^exp and 1 (n≥3). Error bars are the s.d. values in the measurements. (b, top) Schematic of the pre-mRNA splicing isoforms observed for the cTNT mini-gene. (b, middle) representative gel image of cTNT alternative splicing in the presence of varying concentrations of 1. (b, bottom) Quantification of exon inclusion for cTNT alternative splicing in the presence or absence of r(CUG)^exp and 1 (n≥3). Error bars are the s.d. values in the measurements. ‘*’ indicates P≤0.05 and ‘**’ indicates P≤0.01 as determined by a two-tailed Student’s t-test.