4 nitroquinoline

Hoxb8 immortalized [23 (link)] R26-CreER^T2Recql4^fl/+ (control) and R26-CreER^T2Recql4^fl/K525A cells were maintained in IMDM, 10% FBS (non-heat inactivated) and 1% GM-CSF containing media (BHK-HM5 cell conditioned media). The cells were treated for 4 days with 400nM 4-hydroxy tamoxifen (Merck Millipore) then genotyped to confirm complete recombination. Cells were then plated at 10,000 cells/well in 96 well plates (Corning, CLS3610) and incubated for 48 hours with the indicated concentration of drugs in triplicates per dose (dose range Doxorubicin: 0–0.5μM, Hydroxyurea: 0–0.5mM, 4-Nitroquinoline: 0–2μM and Topotecan: 0–0.5mM). Doxorubicin was obtained from St. Vincent’s Hospital Pharmacy. Hydroxyurea was purchased from Selleck. 4-Nitroquinoline and Topotecan were purchased from Sigma-Aldrich. Cell viability was measured using ATP-Lite (Perkin Elmer) as directed by the manufacturer and measured on an EnSpire plate reader (Perkin Elmer). Data were plotted and the IC₅₀ value calculated using Prism 7 software. The dose-response curve was plotted as mean±SEM.

2-acetylaminofluorene (2-AAF), 2,4-diaminotoluene (2,4-DAT), 4-nitroquinoline (4-NQO), 7,12-dimethylbenzaanthracene (DMBA), acrylamide (AA), cyclophosphamide (CPA), dimethylsulfoxide (DMSO), etoposide, methylmethanesulfonate (MMS), omeprazole (OME) phenobarbital (PB), rifampicin (RIF), phenacetin, diclofenac, bupropion, midazolam, acetaminophen, 4′-OH-diclofenac, 1′-OH-midazolam, OH-bupropion, diclofenac-d₄ (internal standard), and acetaminophen-d₄ (internal standard) were purchased from Sigma (Saint-Quentin-Fallavier, France). 2-amino-1-methyl-6-phenylimidazo[4-5b]pyridine (PhIP) and 2-amino-3-methylimidazo[4-5f]quinolone (IQ) were obtained from Toronto Chemical Research (Toronto, ON, Canada).