Mda mb 231

Breast cancer cell lines BT-474, MCF7, Hs-578-T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 (Cobioer, Nanjing, China) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sunncell, Wuhan, China) with 10% fetal bovine serum (FBS) and certain cell growth factor. BT-549, HCC1937, and T47D cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sunncell, Wuhan, China) with 10% or 20% FBS.
For the following function experiments, T47D, MCF7, MDA-MB-453, HCC1937, MDA-MB-231, and MDA-MB-468 cells were treated with 100 or 500 nM GDC-0941 (PI3K selective inhibitor, MedChemExpress, New Jersey, USA) or dimethyl sulfoxide (DMSO) for 24 h. In addition, MDA-MB-231 and HCC1937 cells were treated with 100 nM OSU-T315 (ILK inhibitor, SolelyBio, Beijing, China), tumor necrosis factor (TNF)-α, IgG, or TNF-α antibody (ab6671, Abcam, Cambridge, UK).
In addition, MCF7 and MDA-MB-453 cells were transfected with ILK, and MDA-MB-231 and HCC1937 cells were transfected with shILK.

The human breast cancer cell lines MCF-7, T47D, SKBR-3, HCC1954, HCC38, HCC1187, MDA-MB-231, BT549, MDA-MB-453, and HCC1937, and the non-tumorigenic human mammary gland cell line MCF-10A were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Ten cell lines of our included were subjected to westernblot assays to validate the expression profile of TMEM158 in various types of breast cancer cell lines. MDA-MB-231 cells were cultured in DMEM, and MCF-7, T47D, SKBR-3, HCC1954, HCC38, HCC1187, BT549, MDA-MB-453, and HCC1937 cells were cultured in RPMI1640, each containing fetal bovine serum (FBS, 10%), streptomycin (100 μg/ml), and penicillin (100 U/ml). MCF-10A cells were cultured in DMEM-F12 containing FBS (10%), penicillin (100 U/ml), streptomycin (100 μg/ml), insulin (10 µg/ml), hydrocortisone (0.5 µg/ml) and epidermal growth factor (EGF, 20 ng/ml). All cells were cultured at 37°C in a humidified incubator with 5% CO₂, with medium changed every 1-3 days, based on the number of cells and their growth rates. Where indicated, MDA-MB-231 cells were pretreated overnight in the presence or absence of 20 μM PD98059 (MedChem Express, USA), an inhibitor of ERK1/2 signaling. Cells were harvested and assayed by western blotting.

The human breast cancer cells (BT-549, Hs578T, MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7, and T47D) were obtained from the American Type Culture Collection (ATCC, USA). All cells were cultured in the recommended medium (Gibco, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 1% streptomycin/ penicillin (Beyotime, Shanghai, China) at 37 °C with 5% CO₂. To establish cisplatin (DDP)-resistant MDA-MB-231 cell line (MDA-MB-231/DDP), doxorubicin-resistant BT549 cell line (BT-549/DOX) and tamoxifen-resistant MCF-7 cell line (MCF-7/TAM), MDA-MB-231, BT-549 and MCF-7 cells were exposed to repetitive and incremental concentrations of drugs over a period of 6 months. To maintain the resistance phenotype, MDA-MB-231/DDP, BT-549/DOX, and MCF-7R cells were, respectively, cultured in the presence of 2 μM cisplatin (Med-ChemExpress, NJ, USA), 2 μM doxorubicin (Med-ChemExpress, NJ, USA), and 1 μM tamoxifen (Med-ChemExpress, NJ, USA). Arachidonic acid was obtained from Sigma-Aldrich (St. Louis, MO, USA). Giripladib, an inhibitor of cytoplasm phospholipase A2, was purchased from USBiological Life Sciences (Swampscott, MA, USA).