Nu serum 1

NCI-H295R (RRID:CVCL_0458) and Y1 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in a humidified incubator containing 5% CO₂ at 37 °C. NCI-H295R cells were grown in DMEM/Ham’s F-12 (1:1) (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% NuSerum I (Corning, Corning, NY, USA), 1% Insulin-Transferrin-Selenium-Ethanolamine (ThermoFisher Scientific), and 1% penicillin/streptomycin (ThermoFisher Scientific). The human NCI-H2935R cell line has been authenticated using short tandem repeat profiling within the last three years. Y1 cells were cultured in High-Glucose DMEM (ThermoFisher Scientific) supplemented with 2.5% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 7.5% horse serum (ThermoFisher Scientific), and 1% penicillin/streptomycin. All experiments were performed with mycoplasma-free cells. Tegavivint was a gift from Iterion Therapeutics, INC (Houston, TX, USA), and PKF115-584 was obtained from Tocris (Minneapolis, MN, USA). All compounds were solubilized in DMSO.

Human ACC cell lines H295R and SW13 were purchased from ATTC (Rockville, MD, USA) and were grown in DMEM supplemented with 1% insulin transferrin selenium and 2.5% NuSerum I (Corning, Bedford, MA, USA). The ACC BD140A cell line [13 (link), 36 (link), 37 (link)] was kindly provided by Drs. Kimberly Bussey and Michael Demeure (TGen, Pheonix, AZ, USA), it was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin and 1% L-glutamate (Life Technologies, Grand Island, NY, USA). Human embryonic kidney cell line HEK293 was cultured in DMEM supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin. All the cancer cell lines were authenticated by short tandem repeat profiling and had testing for mycoplasma contamination. All experiments were done with cells from the original stock with no more than 15 passages.

The ACC cell lines H295R and SW13 were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in 60 cm² dishes at 37°C in a humidified incubator at 5% CO₂. The medium for H295R were consisted of DMEM/F12 (Gibco, USA), supplemented with 2.5% Nu-serum I (Corning, USA), 1% ITS+ Premix (Corning, USA), 1% L-glutamine and 1% penicillin-streptomycin (Gibco, USA). SW13 cells were grown in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.