Mouse embryonic fibroblast (MEFs) culture and differentiation into adipocytes.

Primary MEFs cultures were established from postnatal day 0 (PND0) control and AnkB-RW mice following a described protocol (^{30, 31}). In brief, after removal of all internal organs, bodies were minced and digested in 1X HBSS (Genesee) containing 0.25% trypsin (Life Technologies) and 0.2 mg/ml DNAse (Sigma) for 30 min at 37°C. Tissue was washed three times with 1X HBSS and dissociated in MEF media (Dulbecco’s modified Eagle’s medium (DMEM, Genesee) supplemented with 10% fetal bovine serum (FBS, Genesee), non-essential amino-acids (MEM, Life Technologies), 2 mM glutamine (Life Technologies), and penicillin/streptomycin (Life Technologies), and vigorously agitated for 30 seconds. Dissociated cells were then passed through a 100 μm disposable cell strainer to remove any residual non-dissociated tissue, pelleted by centrifugation at 1,500 rpm for 5 minutes, plated on a 100-mm tissue culture dishes containing MEF media, and incubated at 37°C and 5% of CO₂ until the culture reached 100% confluence. 48-hours post-confluence MEFs were induced to undergo adipogenic differentiation by incubation for two days with induction media (MEF media supplemented with 1.8 uM insulin (Humulin R; Eli Lilly), 5 nM dexamethasone (Sigma), 10 mM rosiglitazone (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Cayman). Cells were then switched to maintenance media (MEF media supplemented with 1.8 nM insulin, 10 mM rosiglitazone) for an additional two days. Differentiated adipocytes were kept in MEF media until use.