Modulation of MBNL-mediated Splicing in Muscle Cells

Human HeLa and mouse C2C12 cells were grown in high-glucose DMEM medium (Lonza) supplemented with 10% fetal bovine serum (Sigma) and 1x antibiotic/antimycotic (Sigma). Human skeletal myoblast (HSkM) cells were grown in HAM F-10 medium (Sigma) supplemented with 20% FBS, 1x antibiotic/antimycotic, 0.39 µg/ml dexamethasone (Sigma) and 10 ng/ml epidermal growth (Sigma). All cells were grown at 37 °C and in a 5% atmosphere of CO₂. HeLa, C2C12 or HSkM cells plated in 12-well plates were transfected at 50–60% confluence with Lipofectamine® 2000 (Invitrogen™) according to the manufacturer’s protocol. Single transfection was conducted with an siRNA mix against MBNL1 and MBNL2 (25 nM each) (FUTURE synthesis and RiboTask™, respectively^{67 (link),68 (link)}), 50 nM AllStars negative control siRNA (Qiagen), or a specific AON at 125 nM, in which AON-Ctrl was 2′OMe-PS unspecific to a tested transcript. Co-transfection was conducted with 200 ng of the minigene and 500 ng (or as indicated in the figures) of the MBNL1, SRSF1 or GFP expressing vector. For verification of the MBNL-binding regions and inhibition of the MBNL/(CUG)^exp interaction, the co-transfection was followed by a 4-hour incubation and transfection with selected AONs. Erythromycin was added directly to the medium. The cells were harvested 48 hours after transfection.