Insulin 91077c

For generation of orospheres, the OSCC cells were seeded at 5 × 10⁴ cells/well in 6-well non-adherent plates (Corning Inc., Corning, NY, USA) in serum-free DMEM/F12 medium (11330057; Thermo Fisher Scientific Inc, Bartlesville, OK, USA.) supplemented with basic fibroblast growth factor (bFGF) (20 ng/mL, Invitrogen, Carlsbad, CA, USA), B27 supplement (Invitrogen, Carlsbad, CA, USA), 5 μg/mL insulin (91077C; Sigma Aldrich Co. (St. Louis, MO, USA)), and epidermal growth factor (EGF) (20 ng/mL, Millipore, Bedford, MA, USA). The cells were then incubated at 37 °C in a 5% humidified CO₂ incubator for 12–15 days, and the formed orospheres were observed and counted using an inverted phase-contrast microscope. After 12 days of culture, primary orospheres consisting of ≥50 μm were counted, and images were taken under the microscope. Secondary orospheres were generated by dissociating primary orospheres using the trypsinization method, and the dissociated primary orospheres were then pipetted through a 22G needle to obtain a single-cell suspension (Thermo Fisher Scientific Inc, Bartlesville, OK, USA). After dissociation of the primary orospheres, cell seeding was done as for primary tumorspheres. After 12–15 days of culture, secondary orospheres consisting of ≥50 µm were counted, and the images were taken under the microscope.

AN3CA cells (1 × 10⁴ cells) suspended in 2 mL sphere formation medium (DMEM/F12 medium containing 20 ng/mL epidermal growth factor (PeproTech Asia, Rehovot, Israel), 20 ng/mL basic fibroblast growth factor (PeproTech Asia, Rehovot, Israel), 4 μg/mL heparin (H3194, Sigma, St. Louis, MO, USA), 1 µg/mL Hydrocortisone (H0888, Sigma, St. Louis, MO, USA), 0.4% bovine serum albumin (15260037, Invitrogen, Paisley, UK), 5 μg/mL insulin (91077C, Sigma, St. Louis, MO, USA), and 1% methylcellulose (M0512, Sigma, St. Louis, MO, USA)) were seeded into a well of an ultra-low-attachment 6-well plate (Corning Inc., Corning, NY, USA). The cells were incubated for 10 days for sphere formation as the 1st sphere. Then, 2 × 10⁴ cells from the 1st were suspended in 2 mL sphere formation medium with or without PQR309 and seeded into a well of an ultra-low-attachment 6-well plate. A 500 μL fresh sphere formation medium was added to the well every 3 days. The cells were incubated for 14 days, and the 2nd spheres were observed via inverted light microscopy. The area and diameter of spheres were analyzed by ImageJ software (version 1.4.3.67).