Actrapid penfill insulin

All cell lines were from American Type Culture Collection (ATCC, Manassas, VA, USA). All media and supplements were from Thermo Scientific (Waltham, MA, USA) unless stated otherwise. Cells were grown in RPMI-1640 except for HeLa and Cama-1 grown in DMEM low glucose, and KCN-69 and SK-N-BE2C grown in MEM/EBSS. All media were supplemented with 10% fetal bovine serum (Biosera, Boussens, France), 100 IU/ml penicillin, and 100 μg/ml streptomycin. In addition, MDA-MB-468 and MDA-MB-321 media were supplemented with 1 mM sodium pyruvate; BT-549 medium was supplemented with 0.023 IU/ml Actrapid Penfill insulin (Novo Nordisk AS, Bagsvaerd, Denmark); and Cama-1 medium was supplemented with 1% MEM non-essential amino acids.
When indicated, cells were treated with etoposide, paclitaxel, TNF-α, TPA (all Sigma-Aldrich, St. Louis, MO, USA), anti-hTNF-α-hIgG2 (InvivoGen, Toulouse, France), GF109203X (Calbiochem, San Diego, CA, USA) and zVADfmk (Enzo Life Sciences, Farmingdale, NY, USA). LBW242 and LCL161 were kindly provided by Novartis (Basel, Switzerland).

For GTTs, mice received a glucose bolus i.p. (2 g/kg body weight, Braun) after 6 h of fasting. Blood glucose was measured after 0, 15, 30, 60, 90 and 120 min using a freestyle lite glucometer (Cat#7091870, Abbott). Blood was collected at time points 0, 15 and 30 min for insulin measurements.
For ITTs, mice were fasted 3 h and injected with 1U/kg body weight insulin (Actrapid Penfill Insulin 100 IU/mL, Novo Nordisk). Glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after injection.
For GLP-1 measurements, mice were i.p. injected with 25 mg/kg body weight Sitagliptin (Cat# sc-364620, Santa Cruz) 30 min prior to oral glucose administration and blood collected in diprotein A (Cat# I9759, Bachem). To block GLP-1 signaling, synthetic exendin (9–39) 25 nM/kg body weight (Cat# H-8740, Bachem) was injected i.p. 1 min prior to GTT.