S crizotinib

TH588 hydrochloride (Axon Medchem), (S)-crizotinib (Sigma-Aldrich), and (R)-crizotinib (Selleckchem) were dissolved to 10 mM in DMSO for cell experiments. Nocodazole, menadione, BSO, AUR, and NAC were purchased from Sigma-Aldrich. Oxaliplatin was purchased from Selleckchem.

Chemical reagents and the standard compounds (Raclopride and (S)-crizotinib) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Tocris (Ellisville, MO, USA). N, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or ethanol was used to dissolve the various compounds. Different concentrations were then achieved by diluting stock solutions with assay buffer for the enzymatic assay or RPMI 1640 medium for cell-binding assays. [³H]TH287 was customer synthesized by ViTrax (Placentia, CA, USA) via direct catalytic tritium gas exchange, a radiochemical purity of >99% was determined by the high performance liquid chromatography (HPLC). Product identity was confirmed by Mass spectrometry and HPLC co-elution with Authentic Standard (Retention time: 8.39 min; Column: Zorbax SB-Phenyl, 4.6 × 150mm, 3.55 µm; Gradient elution: mobile phase A (0.05% TFA/water) and mobile phase B (acetonitrile). From 10% B to 90% B over 10 min at flow rate 1 mL/min.).Specific Activity was determined to be 31.2 Ci/mmol by Mass Spec. The location of the tritium label has not been determined, however, can be assumed to be located on the aromatic ring.

Clones of isogenic pol β^+/+ and pol β^−/− MEF cell lines (36.3 and 38.4, respectively)56 (link) were used for this study. Cells were grown at 34 °C in a 10% CO₂ incubator in DMEM supplemented with GlutaMAX-1 (Gibco), 10% fetal bovine serum (FBS, HyClone), hygromycin (80 μg ml⁻¹), and were routinely tested and found to be free of mycoplasma contamination. Cytotoxicity was determined by growth inhibition assays as follows57 (link): briefly, cells were seeded in six-well dishes at a density of 40,000 cells per well, and treated for 1 h with a range of concentrations of KBrO₃ in the absence or presence of 6 μM (S)-crizotinib (Sigma-Aldrich). After washing, cells were further incubated as appropriate with (S)-crizotinib until counting of nuclei collected from the plates (triplicate wells for each drug concentration) following cell lysis using hypotonic solution then detergent58 (link). Results were expressed as the number of cells in KBrO₃-treated wells relative to untreated cells (% control growth). Clones of both pol β^+/+ and pol β^−/− vector-expressing and MTH1 knockout were similarly plated and treated at 37 °C for 1 h with a range of concentrations of KBrO₃. Sensitivity was determined by growth inhibition assays as described above.