Reversan

Murine cells were plated and treated 24 h later with each chemotherapeutic agent or a solvent control: doxorubicin (500 nM, MP Biomedicals, LLC), Valspodar (10 μM, Sigma, St. Louis, MO, USA), Reversan (5 μM, Sigma), or A69 (10 μM [33 (link),34 (link)]). Drug treatments were performed for 24 h unless otherwise indicated.

Glioblastoma cells U251, MZ-256, and MZ-327 were pre-treated with the MRP1 small molecule inhibitor MK571 (M7571 SIGMA) and Reversan (SML0173 SIGMA) at a final concentration of 25 and 15 μM; respectively, for 7 h and subsequently were seeded at 1 × 10⁴ cells/well in a 6 well plate and allowed to adhere overnight. Cells were then allowed to grow for 96 h and counted using the trypan blue exclusion method.

MDCKII-WT, MDCKII-MRP1 and MDCKII-MRP4 cells were obtained from Netherlands Cancer Institute (Dr. A. H. Schinkel), Amsterdam, Netherlands. Hep G2 cell was purchased from ATCC (Manassas, VA, USA). hCMEC/D3 cells were obtained from Dr. Pierre-Olivier Couraud (Institute Cochin, Paris, France). Cell culture medium and fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Grand Island, NY, USA). The non-selective MRP inhibitor MK 571 was from Cayman Chemical (Ann Arbor, MI, USA) and selective MRP1 inhibitor reversan[33 (link), 34 (link)] and selective MRP4 inhibitor ceefourin 1[35 (link), 36 (link)] were from Sigma (St. Louis, MO, USA) and ABCAM (Cambridge, MA, USA). [³H]Benzylpenicillin (25 Ci/mmol) and [¹⁴C]mannitol (55 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA).