Insulin solution

Dulbecco's modified eagle medium (DMEM) and the amino acid-deprived DMEM formulations were purchased from Welgene (Daegu, Korea). Fetal bovine serum (FBS), mitomycin C, recombinant human epithelial growth factor and insulin solution were obtained from Sigma-Aldrich (St.Louis, MO). Dialyzed FBS (dFBS), HEPES, and anti-FAK (pY397) antibodies were purchased from Life Technologies (Grand Island, NY). The anti-FAK antibody was obtained from Millipore (Billerica, MA). Penicillin-Streptomycin (pen/strep) solution was purchased from Mediatech, Inc. (Manassas, VA). Matrigel was obtained from Corning (Corning, NY) and protein assay reagent kits were obtained from Bio-Rad Laboratories (Hercules, CA). A10021B and Modified Diets were obtained from Central Lab. Animal, Inc. (Seoul, Korea; details in Supplementary Table S1).

Three millions of cells per well were seeded in 1 ml of serum-free Schneider’s media in 6-well plates and mixed with 15 μg of double-stranded RNAs against the gene of interest. After 30 minutes of incubation at 25°C, 2 ml of Schneider’s media containing 15% FBS plus penicillin/streptomycin was added. After four days of incubation at 25°C, cell media was replaced with serum-free Schneider’s media. The next day, cells were harvested, washed with cold PBS, and kept at −80°C until use. In case of no serum starvation, cells were incubated for five days prior to harvest. When cells were stimulated with insulin, insulin solution (Sigma) was added to cell media 30 minutes prior to harvesting cells at 1:1000 dilution (10 μg/ml) following the manufacturer’s instruction.
In case of reporter plasmid transfection, Effectene reagent (Qiagen) was used. Two days after RNAi treatment, 1ml of cell media was first removed to reduce the media to 2ml. Transfection of cells with a reporter construct was then performed following the Effectene manufacture’s instruction, which was followed by a direct addition (without help of Effectene) of 5 μg double-strand RNA into cell media. After two days of incubation at 25°C, cell media was replaced with serum-free Schneider’s media, followed by insulin treatment and harvest the next day.

In vitro cultures were set up in 24 well plates in Schneider’s insect medium (Gibco) supplemented with 10% (vol/vol) heat inactivated FBS (Sigma), 0.01% (vol/vol) Insulin solution (Sigma, I0516-5ML), and 1% (vol/vol) Pen/Strep (Sigma). 1 ug/ml of 20-hydroxy-ecdysone (Sigma) was added for control conditions. Dissections were performed at 48 hr and the prothoracic glands were removed prior to culture. Brains were cultured for 24 hr and the media was changed every 12 hr.

Human mammary breast cancer cells MCF-7 (ATCC, Manassas, VA, USA) were subcultured in DMEM supplemented with 10% fetal calf serum (both Gibco, Thermo Fisher Scientific, Waltham, MA, USA) 100 U/mL penicillin and streptomycin and 0.01 mg/mL insulin solution (Sigma Aldrich) and kept in a humidifier at 37 °C and 5% CO₂. Cells were passaged every 3–4 days at a confluency of 80% and regularly checked for the absence of mycoplasm contamination. For experiments, cells were seeded in culture medium for 48 h and incubation solutions were prepared using an experimental medium. The experimental medium was comprised of DMEM without phenol red (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and streptomycin and 10% dextran treated charcoal stripped fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA). Stock solutions of incubation conditions were prepared in DMSO and corresponding solvent controls were prepared accordingly applying 1% of DMSO to the experimental medium.

Preadipocytes were sub-cultured in 48-well plates at a density of 5 × 10⁴ cells/well for 24 h and treated with T863 (50 µM) and six compounds (0, 1, 50, and 100 µM) while inducing lipogenic differentiation. Dulbecco’s modification of Eagle’s medium (Corning, Manassas, VA, USA) supplemented with 10% Fetal Bovine Serum (Atlas Biologicals, Fort Collins, CO, USA), 100 units/mL penicillin, 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA), and insulin solution (Sigma-Aldrich, St. Louis, MO, USA) was used for differentiation. After 7 days of differentiation and treatments, cells were fixed and washed with DPBS. Cell lipid droplets were observed using Oil Red O staining (Sigma-Aldrich, St. Louis, MO, USA). Staining efficiency was evaluated by the OD value at 500 nm wavelength to determine the lipid accumulation level in differentiated and treated groups.

Recombinant human SCF, IL-3 and EPO were from Peprotech (Hamburg, Germany). Insulin solution, holo-transferrin, heparin, hydrocortisone, bovine albumin, crystal violet, and DEAE-Dextran were from Sigma/Aldrich (Schnelldorf, Germany). MgCl₂, CaCl₂, NaHCO₃, and formaldehyde were from Carl Roth (Karlsruhe, Germany).