Bcrp abcg2

Human melanoma A375 cells (ATCC, Manassas, VA, USA) were maintained in DMEM medium supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin-streptomycin, 1% v/vl-glutamine. Cells were maintained in a humidified atmosphere at 37 °C, 5% CO₂. To measure the expression of ABC transporters, 1 × 10⁶ cells were rinsed and fixed with 2% w/v paraformaldehyde (PFA) for 2 min, washed three times with PBS and stained with anti-P-glycoprotein (Pgp/ABCB1) (Kamiya, Hamamatsu City, Japan), anti-MDR-related protein 1 (MRP1/ABCC1) (Abcam, Cambridge, UK) and anti-breast cancer resistance protein (BCRP/ABCG2) (SantaCruz Biotechnology Inc., Santa Cruz, CA, USA) antibodies for 1 h on ice, followed by an AlexaFluor 488-conjugated secondary antibody (Millipore, Billerica, MA, USA) for 30 min. One-hundred-thousand cells were analyzed with EasyCyte Guava™ flow cytometer (Millipore), equipped with the InCyte software (Millipore). Control experiments included incubation with non-immune isotype antibody.

Cells were rinsed in lysis buffer (125 mM Tris-HCl, 750 mM NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mM MgCl₂, 5 mM EDTA, 25 mM NaF, 1 mM NaVO₄, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, pH 7.5), sonicated and centrifuged at 13,000 x g for 10 min at 4°C. 20 μg cell lysates were subjected to Western blotting and probed with the following antibodies: phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2 (Millipore); ERK1/2 (Millipore); HIF-1α (BD Bioscience, San Jose, CA); FPPS (Abcam, Cambridge, UK); Pgp/ABCB1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); MRP1/ABCC1 (Abcam); MRP2/ABCC2 (Abcam); MRP3/ABCC3 (Santa Cruz Biotechnology Inc.); MRP4/ABCC4 (Abcam); MRP5/ABCC5 (Santa Cruz Biotechnology Inc.); BCRP/ABCG2 (Santa Cruz Biotechnology Inc.); β-tubulin (Santa Cruz Biotechnology Inc.), followed by the secondary peroxidase-conjugated antibodies (Bio-Rad Laboratories). Proteins were detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA). To assess HIF-1α phosphorylation, the whole cell lysate was immunoprecipitated with a polyclonal anti-HIF-1α antibody (Santa Cruz Biotechnology Inc.), then resolved by SDS-PAGE and probed with a biotin-conjugated anti-phosphoserine antibody (Sigma-Aldrich), followed by polymeric streptavidin-horseradish peroxidase-conjugates (Sigma-Aldrich).

Cultured cells were washed twice in PBS and lysed in RIPA buffer (Beyotime, China) with complete protease inhibitors (Roche, Germany). Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% low-fat dry milk in TBST for 2 hours at room temperature and probed with the indicated primary antibodies overnight at 4°C; Antibodies for the following targets were used: PLOD2 (Proteintech, USA), Bax (Signalway Antibody, USA), P-gp(MDR1), MRP1, BCRP(ABCG2), Bcl2 (Santa Cruz, USA), and GAPDH antibody (Abcam, USA). After washed with TBST, the membranes were incubated with HRP-conjugated anti-IgG secondary antibody at room temperature for 1 hour. Signals were analyzed using an Enhanced Chemiluminescence (ECL) Detection System (Tanon, China).