Cell culture
TNBC cell lines MDA-MB-231 and MDA-MB-231/ADR were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Yansheng Industrial Co., LTD, Shanghai, China). Adriamycin-resistant MDA-MB-231/ADR cells were screened from MDA-MB-231 cells exposed to adriamycin in different doses under optimal growth conditions (10 ng/mL of adriamycin in the beginning, and gradually increasing the dose to 2ug/mL), the survival of adriamycin-resistant cell line MDA-MB-231/ADR cells after half a year of screening). MDA-MB-231 cells were cultured in DMEM, while MDA-MB-231/ADR cells were cultured in L15 medium supplemented with 10% FBS and 1% Penicillin–streptomycin. The cells were incubated at 37 °C in 5% CO2 environment. MDA-MB-231/ADR cells were specifically cultured in a medium supplemented with adriamycin (500 ng/ml) to sustain the drug-resistant phenotype.All cells were cultured in a drug-free medium for 36 h before in vitro assay.We regularly used mycoplasma reagents to monitor mycoplasma, and there was no mycoplasma contamination.
Western blotting assay
Cells were processed in RIPA buffer (Beyotime, China) at 4 °C for 30 min. The proteins were separated using SDS-PAGE and then proteins were transferred to Polyvinylidene Fluoride(PVDF) membranes. The membrane is cut to size according to the location of the gene protein.and then the nitrocellulose membranes were blocked with 5% BSA (Beyotime, China) for 2 h at room temperature on a rocker. The membranes were then incubated overnight at 4 °C with the primary antibodies (1:1000 dilution), including anti IKKα (CST#61294), p-IKKα (CST#2697S), Bax (Bioss, #bs-0127R), Bcl-2 (Bioss, #bs-0032R), caspase3 (Bioss, #bs-20364R), and GAPDH (CST #5174). Then, incubated with HRP-conjugated anti-IgG secondary antibodies (Zhongshanjinqiao, China) at 1:2500 dilution for 2 h at room temperature on a rocker. The membranes were scanned using an enhanced chemiluminescence detection system (Vision Works).