Rotorgene analysis software 6000

RNA was extracted from liver MTs and the cell suspensions obtained following the MT dissociation protocol at day 3 following standard TRIzol procedure with the addition of glycogen (LT-02241; ThermoFisher, Waltham, MA, USA). Extracted RNA was reverse transcribed using a M-MLV Reverse transcriptase (M1705; Promega,), M-MLV RT buffer (M531A; Promega), dNTP Mix (02-31-00100; Solis BioDyne), and Oligo dT-Primer (79237; Qiagen). The quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using TaqMan probes (Table 2) for selected oxidative stress markers (HMOX1 and NQO1) and hepatic markers characteristic of HepaRG surrogates for hepatocytes (ALB), hTERT-HSC (CD44) and THP-1 surrogates for KCs (CD68). FastStart TaqMan Polymerase (04673433001; Roche) was used to perform the qRT-PCR. Program settings: 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The Ct values were generated using the Corbett Rotorgene Analysis Software 6000 and processed on GraphPad Prism. Gene expression changes were calculated using the ΔCt method with GAPDH as a house keeping gene. Fold changes were calculated as $2$ -(Δ(ΔCt) and expressed as mean and SD of 2 biological repeats with 3 replicates each.

For detection of SLC19A1, HepaRG were treated with MTX for 3 days; for ACTA2 and DDIT3 detection, mono- and co-cultures were exposed to MTX or TGF-β1 for 7 days. mRNA was isolated following standard TRIzol extraction procedure with Glycogen (ThermoFisherScientific, Reinach, Switzerland, LT-02241) from cells lysed using QIAzol Lysis Reagent (Qiagen, Basel, Switzerland, 79306). Reverse transcription was carried out using M-MLV Reverse transcriptase (Promega, Dübendorf, Switzerland, M1705) and oligo dT (Qiagen, Basel, Switzerland, 79237). Real-time PCR was performed using either FastStart TaqMan^® Probe Master (Sigma, Taufkirchen, Germany, 4673417001; for SLC19A1) or GoTaq^® Probe qPCR and RT-qPCR Systems (Promega, Dübendorf, Switzerland, A6102; for DDIT3 and ACTA2) and TaqMan probes of selected genes (see Table 2). The q-RT-PCR Program used: 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The Ct values were generated using the Corbett Rotorgene Analysis Software 6000 (SLC19A1) or the LightCycler^® 480 Systems (DDIT3 and ACTA2) and processed on GraphPad Prism using B2M as the internal standard for normalization. Data are expressed as fold change.