Adriamycin

The σ₂ receptor ligand A011 was prepared as previously reported (Feng et al., 2019 (link)). KO143 was purchased from MedChemExpress (Monmouth Junction, NJ, United States). Cisplatin (DDP), adriamycin (ADR), paclitaxel, verapamil, mitoxantrone and rhodamine 123 (Rh123) were acquired from Solarbio (Beijing, China). Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories (Japan). Pgp-Glo™ Assay Systems were purchased from Promega (Madison, USA). Anti-P Glycoprotein antibody, ABCG2, GAPDH, Goat Anti-Rabbit IgG H&L Secondary Antibody (Alexa Fluor 488), Goat Anti-Rabbit IgG H&L Secondary Antibody and Goat Anti-Mouse IgG H&L Secondary Antibody were purchased from Abcam (Cambridge, United Kingdom). SlowFade^TM Gold antifade reagent was purchased from Thermo Fisher (MA, United States).

MDA-MB-231 and MDA-MB-468 cell lines of logarithmic growth were seeded into 96-well plates at the concentration of 1×10⁴ per well. After treatment by different concentrations of cisplatin (Shandong Qilu Pharmaceutical, China), adriamycin (Solarbio, China), paclitaxel (Bristol-Myers Squibb Company, USA) and gemcitabine (Eli Lily and Company, USA) for 48 h, the TNBC cell lines were incubated by MTT solution (Beyotime bIotechnology, China) for 4 h. Optical density (OD) of the cell lines was measured on the microplate reader (model: iMark, BioRad, USA) at the wavelength of 570 nm, and proliferation inhibition rate was calculated in line with the formula of. $\left(\frac{\text{1-OD\hspace{0.17em}test\hspace{0.17em}group}}{\text{OD\hspace{0.17em}control\hspace{0.17em}group}}\right)\times 100\%.$ The half maximal inhibitory concentration (IC50) values of each drug for each cell were calculated with online Quest Graph™ IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator, AAT Bioquest, USA).

C21 steroidal glycosides were extracted and purified from E.kansui and supplied at 95% purity by the Key Laboratory of Resources and Medical Chemistry, Kunming University of Science and Technology. The structures of compounds 1–12 were identified by Dr. Jian-chun Li (Kunming University of Science and Technology). The purity (>98%) of those C21 steroidal glycosides was determined by HPLC and NMR tests. Compounds 1–12 and adriamycin (Solarbio, Beijing, China) were subsequently made up to a stock of 50 mmol∙L-1 in dimethyl sulfoxide (DMSO, Aladdin, Shanghai, China) and then stored at 4 °C. ATP1A1, p-ATP1A1, ERK, p-ERK, AKT, p-AKT, p62, LC-3 (I/II), Bcl-2 and Bax antibodies were purchased from CST (Cell Signaling Technology, Danvers, MA, USA); cleaved caspase 8 was purchased from Affinity (NanJing, China); β-actin from Servicebio (Wuhan, China); the FITC rabbit antibody from ZhongshanJinqiao Biotechnology (Beijing, China); DAPI from Beyotime (Beijing, China); the Annexin V/PI kits from BD (Franklin Lakes, NJ, USA); and the Hoechst from Sigma Aldrich (St. Louis, MO, USA).