Human insulin

MCF-10A (non-tumorigenic epithelial cell line), MCF7, and A594 cells were purchased from ATCC. MCF-10A cells were cultured in Mammary Epithelial Cell Growth Medium (Takara) supplemented with 0.1 μg/ml cholera toxin (Sigma). MCF7 cells were cultured in MEM (Gibco) supplemented with 10% FBS (Gibco), 1% sodium pyruvate (Gibco), 1% glutamine (Gibco), and 0.1% insulin (Cell Science Technology). A594 cells were cultured in DMEM supplemented with 10% FBS. MCF-10A and TP53 (^−/−) cells were purchased from horizon. MCF-10A and TP53 (^−/−) cells were cultured in DMEM/F-12 medium (Gibco) containing 2.5 mM L-glutamine and 15 mM HEPES (Gibco), supplemented with 5% horse serum (Gibco), 0.5 μg/ml hydrocortisone (Sigma), 10 μg/ml human insulin (R&D System), 20 ng/ml hEGF (Sigma), and 0.1 μg/ml cholera toxin (Sigma). MDA-MB-231, T47D, U2OS, and 293T cells were generously provided by the Japanese Foundation for Cancer Research. MDA-MB-231 cells were cultured in DMEM/F-12 (Sigma). T47D cells were cultured in RPMI 1640 (Sigma) supplemented with 10% FBS and 0.2 unit/ml insulin. U2OS and 293T cells were cultured in DMEM supplemented with 10% FBS. Cells were cultured at 37 °C with 5% CO₂. All cell lines were examined to be mycoplasma negative before experiments.