The human hepatocarcinoma-derived cell line (HepaRG) was purchased as a differentiated confluent monolayer from Biopredic International (France). After shipment, the cells were maintained in basal medium supplemented with recovery mix for 24 h followed
by basal medium supplemented with maintenance/metabolism mix. Media and supplements were provided by the manufacturer (Biopredic, France). BEAS-2B, A549 and HepG2 cells were cultured and expanded in-house. Experiments were performed between passages 3 and 12 only. All cultures were negative for mycoplasma. Additionally, the cells were authenticated using the short tandem repeat profiling to confirm GSK269962 solubility dmso the nature of the cell cultures (LGC Standards, United Kingdom) (Nims et al., 2010). BEAS-2B, PI3K Inhibitor Library A549 and HepG2 cells were plated in 12-well tissue culture plates, at 60% confluency. A total of 6 wells per plate were treated for 48 h with 10 nM of the CYP1A1/1B1 inducer 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). HepaRG cells were not used as positive control cell line for CYP1A1/1B1,
therefore, they were not pre-induced with TCDD. After 96 h from seeding, total RNA was isolated from the cells using the RNeasy mini kit (Quiagen, United Kingdom). The RNA quantity was measured by using the NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, USA) and the quality assessed with the Agilent 2100 Bioanalyzer (Agilent, United Kingdom). The RNA was converted to cDNA using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, United Kingdom). qPCR was carried out using custom TaqMan® array 96-well plates and TaqMan® Selleckchem Venetoclax Fast Universal Master mix (Applied Biosystem, United Kingdom). Each plate contained two assays with the probes of 1 manufacturing control, 5 endogenous controls and 41 metabolism-related genes from both phase I and phase II (Table 1). qPCR amplification
mixtures (20 μL) contained 2 μL of cDNA and 18 μL of fast master mix and were amplified using the fast PCR 7500 (Applied Biosystems, United Kingdom). The cycle conditions comprised 2 min at 50 °C, 20 s at 95 °C, then 40 cycles of 3 s at 95 °C and 30 s at 60 °C. Threshold cycle (Ct) values for the genes were normalized to RPLP0, and relative expression levels were calculated using the ΔΔCt method (Livak and Schmittgen, 2001). Range finder experiments were initially carried out to select optimal concentrations for substrates, inducer and inhibitors where maximal activity, induction or inhibition were obtained without cytotoxicity. For the enzyme activity profiling, phenol free Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 0.5 mM l-Glutamine was used as a basal experimental medium. CYP1A1/1B1 activity was measured using the specific P450-Glo™ kit (Promega, United Kingdom).