Insulin

Human lung carcinoma A549 cells (ATCC, Manassas, VA, USA) were grown in RPMI-1640 medium with L-glutamine and 10 mM D-Glucose prepared following the manufacturer’s instructions. Human osteosarcoma stably transfected U2foxRELOC cells (a gift from Dr Wolfgang Link), human osteosarcoma U2OS cells, human large cell lung cancer NCI-H460 cells, human ovary adenocarcinoma OVCAR3 cells, human embryonic kidney 293 (HEK293) cells and adipocyte-like differentiated 3T3-L1 cells (ATCC) were grown in DMEM with L-glutamine and 25 mM D-Glucose. Human colorectal carcinoma HCT116 cells (ATCC) were cultured in DMEM:HAM F12 (1:1) with L-glutamine and 12.5 mM D-Glucose. Human breast adenocarcinoma MCF7 cells (ATCC) were cultured in MEM medium without phenol red (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10 mM D-glucose, 2 mM L-glutamine, 1 mM pyruvate (Biological Industries), 0.01 mg mL⁻¹ insulin and 1% non-essential aminoacids (Biological Industries). Media were supplemented with 10% heat-inactivated FBS, penicillin (50 U mL⁻¹) and streptomycin (50 μg mL⁻¹). U2foxRELOC cells, which express a resistance to Geneticin, were incubated with G418 (Gibco) at 100 μg mL⁻¹. 3T3-L1 pre-adipocyte cells were grown in DMEM with 0% FBS, 10% NCS and 0.5% streptomycin/penicillin. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.