Determination of fatty acid uptake

Lipid uptake was measured using fluorescently-labeled palmitate (BODIPY® FL C16, Cat. No. D3821 Thermo Fischer Scientific, Waltham, MA, USA). Differentiated cells were starved for 4 h in FBS-free medium. Subsequently, cells were washed and incubated for 3 h under normoxic or hypoxic conditions with or without the previously mentioned pharmacological compounds, in PBS (Dulbecco's Phosphate Buffered Saline, Cat. No. D8662, Sigma-Aldrich, St. Louis, MO, USA) with 1 μM of fluorescently-labeled palmitate (BODIPY FL® C16, stock prepared in DMSO) and 0.1% fatty acid free BSA (bovine serum albumin, Cat. No. A7030 Sigma-Aldrich, St. Louis, MO, USA). After incubation, cells were washed twice with 1 mL PBS and lysed in 150 μL of T-PER (Tissue-Protein Extraction Reagent, Cat. No. RL243205 Thermo Fischer Scientific, Waltham, MA, USA). Fluorescence of intracellular BODIPY-labeled palmitate was measured with an Infinite® 200 PRO (Tecan Trading AG, Switzerland) microplate reader with excitation/emission wavelengths of 470/503 nm. Data were normalized to protein concentration in each well, which was measured using a BCA assay (bicinchoninic acid assay, Cat. No. 23225 Thermo Fischer Scientific, Waltham, MA, USA).