Human insulin

Chemicals and reagents were obtained from the following manufacturers: DMEM/low glucose media and bovine calf serum (HyClone Laboratories, Thermo Scientific, Logan, Utah, U.S.A.); fetal bovine serum (Wisent Inc., Saint-Bruno, Quebec, Canada); human insulin (Roche Diagnostics, Indianapolis, Indiana, U.S.A.); 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Oakville, Ontario, Canada). PBDEs were obtained as follows: the technical mixture DE-71 was generously provided by Dr. Doug Arnold (Health Canada), from stock provided by Chemtura (Lawrenceville, Georgia, U.S.A.); DE-79 (Wellington Laboratories, Guelph, Ontario, Canada); purified BDE-47, BDE-28, BDE-100, and BDE-154 (AccuStandard Inc., New Haven, Connecticut, U.S.A.); purified BDE-209 (Sigma-Aldrich, Oakville, Ontario, Canada). Antibodies were purchased as follows: fatty acid binding protein 4 (aP2) (R&D Systems, Minneapolis, Minnesota, U.S.A); perilipin and β-actin (Cell Signaling Technology, Danvers, Massachusetts, U.S.A).

The cells were transferred to a low-attachment 24-well flat plate (Prime surface, Sumitomo) in serum-free DMEM containing 10 ng/ml bFGF (Wako), 10 μg/ml human insulin (CSTI), 100 μg/ml human transferrin (Roche) and 100 μg/ml BSA (Nacalai Tesque) and incubated at 37 °C in a 5% CO₂ incubator. In Fig. 1A, 1.0 × 10⁴ cells were seeded per well and cultured for 10 days. The spheres that were larger than 100 μm were counted. In Fig. 1I, 4.0 × 10⁵ cells were seeded per well and cultured for 6 days.

We utilized three well-characterized HNSCC cell lines: UDSCC1, UDSCC5, and UDSCC6 for our experiments. Cells were cultured in both 2D and 3D models. For 2D culture, cells were seeded in Gibco DMEM medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Bio&SELL GmbH, Feucht, Germany), 1% cell shield (Minerva Biolabs, Berlin, Germany), and 1% NEAA (100×, ThermoFisher Scientific, Waltham, MA, USA) in regular culture flasks. For 3D culturing, the floating sphere formation approach using Ultra-Low Attachment (ULA) surface plates (Corning, New York, NY, USA) was applied: cells were resuspended in medium containing DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) with B27 Supplement (50×, Sigma-Aldrich), supplemented with 0.4% BSA (Sigma Aldrich), 20 ng/mL EGF (Sigma Aldrich), 10 ng/mL bFGF (Thermo Scientific), and 5 µg/mL human Insulin (Roche, Basel, Switzerland) at a density of 20,000 cells per well on 6-well ULA surface plates and incubated for 72 h at 36 °C.

Male TRAIL^-/-ApoE^-/- and ApoE^-/- mice were used for all studies [10] (link). Six week old mice were placed on a HFD (Specialty Feeds; Perth, Australia) for 20 w in specific pathogen-free conditions with 12∶12 h light-dark cycles, and free access to water and food. To minimise stress, mice were monitored daily and handled frequently. Body weights were measured and blood was sampled via tail vein throughout the study. At 18 and 19 w into the HFD, glucose (overnight fasted) and insulin (non-fasted) tolerance tests were performed. Either D-Glucose (1 g/kg body weight; Sigma-Aldrich, Sydney, Australia) or 1 U/kg of body weight of human insulin (Roche, Sydney, Australia) was injected into mice intraperitoneally, followed by plasma glucose measurements over 2 h using a glucometer (Accuchek Performa, Roche, Sydney, Australia) [10] (link). At the end of the diet period, and after overnight fasting, mice were anaesthetised by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and culled by cardiac exsanguination. Kidneys were quickly harvested, weighed and fixed in 10% formaldehyde for immunohistochemistry (IHC) analyses or snap frozen for expression studies.