Fig. (4) CLCN1-luc minigene reporter construct splicing and expression in immortalized DM1 cell lines. (A) Schematic of CLCN1 luciferase minigene reporter (CLCN1-luc). A genomic segment of human CLCN1 containing exon 2 to 3 and the intervening intron (solid line) was cloned downstream of the CMV promoter (CMVpr) and in-frame with the luciferase coding sequence in the pLenti6/V5-D-TOPO vector. Dotted lines represent correct splicing (as in WT cells). Intron 2 retention (as in DM1 cells) results in a nonsense mutation, as indicated by TGA. (B) RT-PCR analysis with primers spanning CLCN1 exon 2 to the luciferase coding region, demonstrating complete (+ intron 2) and partial (+ partial intron 2) intron 2 retention of the CLCN1-luc construct in immortalized DM1 cells compared to WT cells (- intron 2), which was partially rescued upon treatment of CLCN1-luc DM1 myoblasts with 10 µM AKT inhibitor Triciribine (C). GAPDH was amplified as a control for equivalent cDNA synthesis. (D) CLCN1-luc activity (RLU = relative luciferase units) 24 h after addition of 10 µM Triciribine to CLCN1-luc WT and DM1 myoblasts (in 384-well format). *** represents a p-value = 1.1 x 10^-5 and * represents a p-value = 2.4 x 10^-3 in two-tailed t-tests. Data points are an average of 3 replicates and error bars represent standard deviation.