Specific antibodies

ChIP assays were performed using the Magna Chip G kit (Milipore) according to the manufacturer’s instructions. 100% HCV infected (generated as described above) and non-infected Huh7.5 cells were fixed with 1% formaldehyde to cross-link proteins to DNA. Cells were then lysed, and chromatin was fragmented by sonication (Covaris S220) to generate DNA fragments of 200–1000 bp. Chromatin was incubated overnight with either specific antibodies against H3K9Ac, H3K43Me, H3K93Me and H3K273Me (Millipore) or normal Rabbit IgG Ab (Milipore) as a negative control for the immunoprecipitation experiments. Cross-links were reversed, and DNA purified.
Libraries for next generation sequencing (NGS) were prepared by using NEBNext Ultra TM DNA Library Prep Kit for Illumina (NEB) and sequenced using Illumina HiSeq 2500 platform with single-end reads of 50 bp according to the manufacturer’s instructions. For each ChIP, at least three biological replicates were performed. For resected liver tissues: ChIP assays were performed using the iDeal ChIP seq kit for Histones (Diagenode) according to the manufacturer’s instructions. For each sample, 20–40 mg of liver tissue was chopped, homogenized, and subjected to ChIP using H3K9Ac or a Rabbit IgG Ab as negative control (Diagenode).

Expression of COLI, COLII, and ACAN was determined in cell culture using specific antibodies (Sigma-Aldrich, Madrid, Spain). The cells were cultured on poly-L-lysine-coated cover slides in chondrogenic culture medium for up to 6 wk as described above. Then, they were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 min. Once washed, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, and after three washes, they were incubated for 30 min with blocking solution (1% bovine serum albumin (BSA) and 1.1% Tween-20 in PBS). The cells were then incubated with the appropriate primary antibody (diluted in antibody diluent at 1 : 100 for COLI and ACAN and 1 : 500 for COLII) overnight at 4°C. After three washes, cells were incubated with 1 : 200 secondary anti-mouse (COLI and ACAN) or anti-rabbit (COLII) FITC-conjugated antibody (Sigma-Aldrich, Madrid, Spain). After the final washes, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the samples were analyzed using the Leica DM2500 fluorescence microscope (Leica, Wetzlar, Germany).

The expression of type I collagen (COLI), type II collagen (COLII), and aggrecan (ACAN) was determined in cell culture using specific antibodies (Sigma-Aldrich, Madrid, Spain). Cells were cultured with proliferation or differentiation culture media for up to 6 weeks as described above. Then, cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min. Once washed with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, and after three washes, they were incubated for 30 min with blocking solution (1% bovine serum albumin [BSA] and 1.1% Tween-20 in PBS). The cells were then incubated with the appropriate primary antibody (diluted in antibody diluent solution at 1:100 for COLI and ACAN and 1:500 for COLII antibody) overnight at 4 °C. After three washes, cells were incubated with the secondary anti-mouse (COLI and COLII) or anti-rabbit (ACAN) FITC-conjugated antibody (Sigma-Aldrich, Madrid, Spain) diluted 1:200. After the final washes, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and the samples were analyzed with a Leica DM2500 fluorescence microscope (Leica, Wetzlar, Germany).

The expression levels of type II collagen (COLII) and aggrecan (ACAN) were assessed in a cell culture using specific antibodies obtained from Sigma-Aldrich, Madrid, Spain, as previously documented [31 (link)]. Scaffolds containing cells were transferred onto 8-well cell culture slides (Millicel, Sigma-Aldrich, Madrid, Spain). The cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min, then washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Following three washes, they were incubated for 30 min with a blocking solution (1% bovine serum albumin [BSA] and 1.1% Tween-20 in PBS). Subsequently, the scaffolds were incubated overnight at 4 °C with the appropriate primary antibodies (diluted in antibody diluent solution at 1:100 for ACAN and 1:500 for COLII antibody). After three additional washes, the scaffolds were incubated with the following corresponding secondary antibodies: an anti-mouse (for COLI and COLII) or anti-rabbit (for ACAN) FITC-conjugated antibody (Sigma-Aldrich, Madrid, Spain), diluted at 1:200. Following the final washes, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the samples were examined using a Leica DM2500 fluorescence microscope (Leica, Wetzlar, Germany)