Abcg2

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).

Total protein was isolated from MMTV-PyMT tumor-derived cells after 24 h treatment with the same chemotherapeutic agents as above using lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 % Triton-X, 0.5 % NP-40, 50, mM NaF, 1 mM Na₃VO₄, 5 mM Sodium pyrophosphate, 0.2 mM PMSF, 1x protease inhibitor cocktail (Fisher) and 1x phosphatase inhibitor cocktail 2 (Sigma). 30 μg of protein were separated by SDS-PAGE (8 % gel), and transferred onto Immobilon-P membrane (Millipore). After transfer, membranes were blocked in 5 % non-fat dry milk in 1 % TBS with 0.1 % Tween (TBS-T) for 1 h at room temperature. Blots were incubated with the primary antibody (MDR1, 1:1000 in 1 % bovine serum albumin in TBS-T, Cell Signaling, or ABCG2, 1:100 in 5 % non-fat dry milk in TBS-T, Abcam) for 2 nights at 4 °C or β-actin (1:25000, 1 % BSA in TBS-T, Sigma) for 1 h at room temperature. Secondary antibody (anti-rabbit, anti-rat or anti-mouse IgG-HRP, 1:1000) was diluted in the same diluent as the corresponding primary antibody and incubated at room temperature for 1 h. Blots were developed with Clarity ECL regent (Bio-rad) and a ChemiDoc MP Imaging System (Bio-rad). Densitometry quantification was performed using ImageJ software (NIH). Blots are representative of three runs.

Cells were prepared in lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 25 mM beta-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 0.1 mM PMSF, and a Protease Inhibitor cocktail (leupeptin, pepstatin, aprotinin, and antipain; each 5 μg/ml). For secretory protein preparation, the culture medium was centrifuged, and cellular components and debris were discarded. The culture medium was concentrated using 10 K cut-off microcon (Amicon), or by adding ice-cold acetone, the precipitated protein was resuspended in lysis buffer. The proteins were separated on SDS-PAGE and transferred to a 0.45-μm Immobilon P-transfer membrane (Millipore). The membrane was blocked in 5% (w/v) non-fat milk and then probed with a primary antibody; anti-human GLRX3 antibody, beta-catenin, E-cadherin, GAPDH (Santacruz, Dallas, Texas, US), c-MET, PI3K, pAKT (Cell signaling, Danvers, Massachusetts, US), AKT, Wnt1, 3, 5a, 7b,11, 16, RhoA, RhoB, pJNK, RAC1, Dvl2 (Santacruz, Dallas, Texas, US), and ABCG2 (Abcam, Cambridge, UK). The immunoreactive material was then visualized using SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, Illinois, US) according to the manufacturer’s instructions.

Cell suspensions were seeded on the central concavity of a special 35 mm glass bottom plate (Nest, China) at ~ 50% confluent density. The cells were cultured overnight with or without PLA. After three washes with ice-cold PBS, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, cells were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with an appropriate concentration of primary antibodies against Oct4, ABCG2, ALDH1, SOX2, or CD49f (Abcam, London, UK, 1:100 dilution) overnight at 4 °C. Then, the cells were incubated with Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (Abcam, London, UK, 1:200 dilution) or goat anti-mouse IgG (Abcam, London, UK, 1:200 dilution) secondary antibodies for 1 h. The stained cells were observed under a fluorescence confocal microscope.