Cell Culture and Transfection Protocol

WT and Mbnl1,2 DKO MEFs were gifts from Maurice Swanson. HeLa cells (ATCC), MCF7 cells and WT and Mbnl1,2 DKO MEFs were grown in high-glucose DMEM (Lonza) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich) and 1 × penicillin–streptomycin (Sigma Aldrich). HSkM (Life Technologies) cells were grown in HAM F-10 medium (Sigma Aldrich) supplemented with 20% FBS, 1 × penicillin–streptomycin, 0.39 µg/mL dexamethasone (Sigma Aldrich) and 10 ng/mL epidermal growth (Sigma Aldrich). All cells were grown at 37 °C and an atmosphere containing 5% CO₂. Prior to transfection, the cells were plated in 6-well or 12-well plates and transfected at 50–60% confluence with plasmids using Lipofectamine 3000 (Thermo Fisher) or with siRNA or antisense oligonucleotides using Lipofectamine RNAiMAX (Thermo Fisher) according to the manufacturer’s protocol. Genes were knocked down with siRNAs against MBNL1, MBNL2, DDX5, DDX17 or control siRNA was used (siCtrl) [9 (link), 37 (link)] (Sigma Aldrich) at 50 nM. SSOs with 2′-O-methoxyethyl-phosphorothioate (2′MOE-PS) modification, SSO-e7 and SSO-Ctrl, were used at 25 nM. Cotransfection with 200 ng of the ATP2A1 e22 minigenes and 250 ng of pEGFP-MBNL1-41 expression vector was preceded by siRNA treatment and  4 h of incubation period. The cells were harvested 48 h after transfection. The siRNA and SSO sequences are listed in Suppl. Table S1.