Small interfering rna

Primary mouse hepatocytes were isolated and maintained as previously described (Fan et al., 2017 (link)). HepG2 cells were maintained in DMEM supplemented with 10% FBS. FLAG-tagged BRG1 (Li et al., 2018e (link)) and LGALS3 promoter-luciferase constructs (Kadrofske et al., 1998 (link)) have been described previously. LPS (1 μg/ml) and palmitate (0.4 mM) were purchased from Sigma. Small interfering RNAs were purchased from Dharmacon. Transient transfection was performed with Lipofectamine 2000. Briefly, cells were plated in 12-well culture dishes (∼60,000 cells/well). The next day, 0.1 μg of reporter construct and 0.1–0.3 μg of effector construct were transfected into each well. DNA content was normalized by the addition of an empty vector (pcDNA3). For monitoring transfection efficiency and for normalizing luciferase activity, 0.02 μg of GFP construct was transfected into each well. Cells were harvested 48 hours after transfection and reporter activity was measured using a luciferase reporter assay system (Promega) as previously described (Liu et al., 2018 (link)). All experiments were repeated three times.

Human embryonic kidney cells (HEK293) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, HyClone) as previously described^{27 (link),28 (link)}. Primary murine renal fibroblasts were isolated and cultured as previously described^{29 (link)}. TheZeb1 promoter–luciferase construct was generated by amplifying genomic DNA spanning the proximal promoter and the first exon of the Zeb1 gene (-1000/+100) and ligating it into a pGL3-basic vector (Promega). The MRTF-A^{30 (link)} and Zeb1^{31 (link)} expression vectors have been previously described. Truncation mutants were made using a QuikChange kit (Thermo Fisher Scientific, Waltham, MA, United States) and were verified by direct sequencing. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24-48 hours after transfection using a luciferase reporter assay system (Promega) as previously described^{32 (link)}.

The rat intestinal epithelial cell IEC-6 (ATCC) was maintained in DMEM supplemented with 10% FBS. TGF-β was purchased from R&D. EX-527 and CP2 were purchased from Selleck. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000 as previously described (Chen et al., 2021 (link); Kong et al., 2021b (link); Liu et al., 2021a (link),b (link); Zhang et al., 2021 (link)).

Human immortalized vascular endothelial cells (EAhy926) were maintained in DMEM supplemented with 10% FBS. Primary mouse LSECs were isolated and characterized as previously described (Meyer et al., 2016 (link)). Briefly, mice were anesthetized with isoflurane. Following perfusion and digestion, the liver suspension was passed through a 70 μm cell strainer. The non-parenchymal cells were isolated by density gradient centrifugation. LSECs were further purified by selective adherence for exactly 8 min. BRG1 expression constructs, NOX4 expression construct, and NOX4 promoter-luciferase constructs have been previously described (Ago et al., 2010 (link); Bai et al., 2014 (link); Li et al., 2018b (link)). Small interfering RNAs were purchased from Dharmacon. Transient transfection was performed with Lipofectamine 2000. Cells were harvested 48 h after transfection and reporter activity was measured using a luciferase reporter assay system (Promega) as previously described (Yang et al., 2018 (link)).

Primary hepatocytes were isolated from C57B6/L mice and cultured in DMEM with 10% FBS as previously described [48 (link), 50 (link)–52 (link)]. FLAG-tagged Hes5 [53 ], LIGHT promoter-luciferase constructs [54 (link)], and MYC-tagged SIRT1 [55 (link)] have been described previously. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine LTX (for DNA plasmid) or Lipofectamine RNAiMax (For siRNA) per vendor recommendation. Luciferase activities were assayed 24-48 hours after transfection using a luciferase reporter assay system (Promega) as previously described [56 (link)–58 (link)].

Human hepatoma cells (HepG2) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone). Human T-cell leukemia cells (Jurkat) were maintained in RPMI-1640 supplemented with 10% FBS. Primary hepatocytes were isolated and cultured as previously described (Fan et al., 2020 (link)). Mouse Npnt promoter-luciferase constructs (Lanthier et al., 2011 (link)) and BRG1 expression constructs have been previously described (Chen et al., 2020c (link); Li et al., 2020a,b (link), c (link)). Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described (Wu et al., 2020 (link); Yang et al., 2020a,b (link)). For conditioned media (CM) collection, the cells were switched to and incubated with serum-free media overnight. The next day, the media were collected, centrifuged at 4000 × g for 30 min at 4°C using 3-kDa MW cut-off filter units (Millipore) and sterilized through a 0.4-μm filter.

Human hepatoma cells (HepG2) were maintained in DMEM supplemented with 10% fetal bovine serum (Hyclone). Primary murine hepatocytes were isolated and cultured as previously described.^[52 (link)
^] Primary human hepatocytes were purchased from Sigma. Human DDIT4 promoter–luciferase constructs^[53 (link)
^] and BRG1 expression constructs^[54 (link)
^] have been previously described. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega).