Ham f 10 medium

This study used the Vero cell lineage to characterize polymeric biomaterials’ cytotoxic behavior and adhesion characteristics (Adolfo Lutz Institute, São Paulo, SP, Brazil). This fibroblast-like cell line was established from African green monkey kidney epithelial cells (Cercopithecus aethiops) [101 (link)]. Cells were seeded at a high density, 10 × 10⁴ cells/ml, and cultured in Ham F-10 medium (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂.

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.

The murine C2C12 myoblast cell line (ATCC, Manassas, VA, USA) was cultured in DMEM (Dulbecco’s modified Eagle’s medium, Eurobio, Courtaboeuf, France) supplemented with L-Glutamin, 10% (v/v) fetal calf serum (Eurobio), 50 units/mL penicillin and 50 μg/ml streptomycin. Cells were grown to 80% confluence and differentiated into myotubes in DMEM supplemented with 5% (v/v) horse serum (Invitrogen, Carlsbad, CA, USA). The medium was changed every 48 hours.
Murine satellite cells were extracted from posterior leg muscles of C3H mice as previously described [68 (link)]. Cells were cultured in “medium A” containing HAM F10 medium (Sigma) supplemented with 5 mM L-Glutamin, 20% (v/v) horse serum, 50 units/mL penicillin, 50 μg/mL streptomycin and 5 ng/mL Basic-Fibroblast Growth Factor (Sigma). At 70% confluence cells were differentiated into myotubes with HAM F10 medium supplemented with 10% (v/v) horse serum or trans-differentiated into fat storage cells when 50 mM glucose were added to “medium A”.