Geneticin

Cells were seeded at 5 × 10⁴ per well (approximately at 70% confluence) of the 24-well plate in culture medium and cultured overnight. Next day, culture medium was replaced by the fresh complete culture medium (500 μL) including 50 μL of DNA–lipid complex (0.5 μg of plasmid pcDNA 3.1–CYP3A4 or empty plasmid pcDNA 3.1 as negative control; GenScript, Piscataway, NJ). Cells were transfected by mixing with Lipofectamine 3000 reagent according to the instructions of manufacturer (Life Technologies). After 48 hours of transfection, cells were washed and supplemented with the fresh culture medium. Next day, cells were harvested and seeded at approximately 25% confluence onto 24-well plate in culture medium with various concentrations (100, 500, and 1000 μg/mL) of Geneticin (Santa Cruz Biotechnology, Dallas, TX) for selection of Geneticin-resistant cells. Selective media were replenished every 3 days and percentage of surviving cells was monitored. After 9 days, 500 μg/mL of Geneticin was selected to maintain cell line expressing CYP3A4. CYP3A4 expression was monitored by qPCR and immunoblotting.

Strains used in this study (Supplementary Table 6) are derivatives of the S288c strain RDKY5964^{20 (link)}, with exception of strain AH109 (Clontech Laboratories) that was used for Y2H analysis. Strains were cultivated at 30 °C in yeast extract-peptone-dextrose media (YPD) or appropriate dextrose-containing synthetic dropout (SD) medium for selection of plasmids markers, lacking lysine (Lys⁻) or threonine (Thr⁻) (to select for lys2-10A or hom3-10 frameshift revertants, respectively), or SD medium lacking arginine (Arg⁻) supplemented with 60 mg/L canavanine, to select canavanine-resistant (Can^R) mutants. 5-fluoroorotic acid (5-FOA, US Biological) plates were done in SD medium supplemented with 1 g/L 5-FOA. Antibiotics were used at the following final concentrations: 200 μg/mL geneticin (Santa Cruz Biotechnology), 300 μg/mL hygromycin B (Thermo Fisher Scientific), and 100 μg/mL nourseothricin (clonNAT, Werner BioAgents). Gene deletions and gene tagging were performed using standard PCR-based recombination methods^{51 (link),52 (link)}, followed by confirmation by PCR. Tags and junctions were confirmed by PCR and sequencing. Yeast strains carrying mutations in MLH1, MLH2, or PMS1 genes, were generated by pop-in/pop-out strategy using pRS306-based integrative vectors^{51 (link)}, and were confirmed by sequencing.

Luciferase-bearing PC-3M and PC-3S cells were clonally derived from the human cell line PC-3 [11 (link)]. Du-145, CWR22v1 and LNCaP cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown at 37°C in a 5% CO₂ atmosphere in RPMI 1640 medium supplemented with 10% fetal bovine serum, L-glutamine, non-essential aminoacids, sodium pyruvate, penicillin/streptomycin (PAA Laboratories, Coelbe, Germany) and geneticin (Santa Cruz Biotechnologies, Santa Cruz, CA).