Rneasy tissue mini isolation kit

Primary hepatocytes were isolated from 3- to 4-month-old C57BL/6 mice as described previously [25 (link)]. Primary hepatocytes were kept in culture medium (DMEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% FBS, 1% penicillin-streptomycin, and l-glutamine (PAN Biotech). After overnight incubation, the medium was replaced by William’s E medium (PAN Biotech), and cells were treated for 6 h with IL-1β (20 ng/ml; Peprotech, Hamburg, Germany), IL-6 (40 ng/ml; Sigma, St. Louis, MO, USA), TNFα (30 ng/ml; Peprotech), and the HNF4α antagonist BI6015 (50 μM; Millipore, Burlington, MA, USA) or for 24 h with TGFβ1 (10 ng/ml; R&D Systems, Minneapolis, MI, USA). Alternatively, the cells were transfected with the siRNA against HNF4α (60 pmol; Thermo Fisher Scientific ID: 158081 and 158082, Germany) according to the manufacturer’s protocol (Lipofectamine® RNAiMAX™ Transfection Reagent, Thermo Fisher Scientific, Germany) and cultured for 48 h. RNA was isolated via RNeasy tissue mini isolation kit (Qiagen, Hilden, Germany). The RNA samples were translated to cDNA with the M-MLV reverse transcriptase kit (Promega, Madison, WI, USA) and random hexamers (Thermo Scientific, Waltham, MA, USA). The relative expression of genes of interest was determined using specific primers (Additional file 1: Table S1). The mouse ribosomal gene L7 was used as an internal loading control.

Liver tissues from nine patients who underwent liver surgery at the University of Aachen between the years 2006 and 2018 were analyzed. Unaffected surrounding portions of liver tissue from nine patients collected during oncological surgery for exclusion of liver malignancy were used as controls (Supplementary Table S1). RNA was isolated using the RNeasy tissue mini isolation kit (Qiagen, Hilden, Germany). RNA was translated to cDNA using the M-MLV reverse transcriptase kit (Promega, Madison, WI, USA) with random hexamer primers (Thermo Scientific, Waltham, MA, USA). The relative expression of genes of interest was determined using qPCR using specific primers (Supplementary Table S2). The human ribosomal gene RPLPO was used as an internal loading control.
Further experimental procedures (e.g., proteomics, bioinformatics, and statistical analysis) are given in the supplementary materials.