Transient Transfection of Lipid Metabolism Genes

Simian SV-40 transformed monkey kidney cells (COS-7 ATCC CRL-1651) were cultured in DMEM (Invitrogen) containing 10% FCS (Sigma) under standard conditions (95% humidified atmosphere, 37 °C, 5% CO₂). Cells were transiently transfected with pcDNA4/HisMax plasmid coding for mAtgl, mPnpla6, mPnpla7, mHsl, mMgl, or β-galactosidase (lacZ) using Metafectene^TM (Biontex GmbH, Munich, Germany) as described (7 (link), 43 (link), 44 (link)). Cells were harvested 48 h after transfection. For the preparation of cell lysates, cells were resuspended in sucrose solution (see above) and disrupted by sonication (SONOPLUS ultrasonic homogenizer HD 2070) on ice. Nuclei and unbroken cells were removed by centrifugation at 1,000 × g for 10 min at 4 °C. Protein concentrations were determined as described below.