Hs00187842 m1

Total RNA is isolated using RNeasy Micro Kit (Qiagen, Cat# 74004) or RNAqueous Phenol-free total RNA isolation (Invitrogen, Cat# AM1912) followed by the recommended protocol provided from the manufacturers. cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as previously described^{51 (link)}. Level of human MCJ (DnaJC15) mRNA was determined by real-time RT-PCR using the designated primer/probes previously described^{111 (link)}: probe, 5′-CCTTGCCAGCAGATGGGCTTACACCTAAA-3′; sense primer, 5′-CAGAAAATGAGTAGGCGAGAAGC-3′; and antisense primer, 5′-TGAC TCTCCTATGAGCTGTTCTAATC-3′. Values of gene of interest were normalized to β2-microglobulin (Thermo Fisher, Hs00187842_m1) or HPRT (Thermo Fisher; Hs02800695_m1) by the comparative delta CT method.

Cryopreserved differentiated HepaRG™ cells (HPRGC10, ThermoFisher Scientific, Waltham, MA, USA) were seeded according to the manufacturer’s protocol and cells were treated by studied compounds after 72 h of stabilization (control (DMSO 0.1%), CITCO 10 µM, diazepam 30 and 50 µM, nordazepam, temazepam, and oxazepam 50 µM, respectively) for 48 h. Total RNA was isolated using TRIZOL^® and reverse transcription was performed with the RevertAid RT Reverse Transcription Kit. qPCR was performed using TaqMan™ Fast Advanced Master Mix with TaqMan probes for CYP2B6 (Hs04183483_g1), GAPDH (Hs02786624_g1), and B2M (Hs00187842_m1) genes (all reagents for qRT-PCR were obtained from Thermo Fischer Scientific Catalog number: 4331182, Waltham, MA, USA). The delta-delta method was used for gene expression quantification normalized to GAPDH and B2M gene expression average. Three technical replicates were used for each reaction.

Quantitative real-time PCR (qRT-PCR) was performed with 100 ng of total RNA isolated from cell lines transfected with siCLDN1 using the TaqMan RNA-to-CT One Step Kit (Thermo Fisher Scientific; Waltham, MA; #4392938). Relative expression levels of CLDN1 (Hs00221623_m1), CD133 (Hs01009241_m1) and CD44 (Hs01075862_m1; Thermo Fisher Scientific) were measured using beta-2-microglobulin as an internal loading control (Hs00187842_m1; Thermo Fisher Scientific). qRT-PCR was performed using the 7500 Fast Real-Time PCR System (ThermoFisher Scientific, Grand Island, NY. Cat# 4351106) and data was analyzed using Microsoft Excel (Microsoft Corporation, Redmond, WA).