Ab41902

Synthetic LXR ligand 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl) amino] propyloxy] phenylacetic acid hydrochloride (GW3965) was kindly donated by Jon Collins (GlaxoSmithKline, Research Triangle Park, NC). Dihydroethidium (DHE) and TRIzol Reagent were from Life Technologies (Carlsbad, CA). Mouse monoclonal antibody against LXRα (ab41902) and rabbit polyclonal antibody against LXRβ (ab28479) were from Abcam (Cambridge, UK); rabbit anti-mouse nitrotyrosine antibody (06-284) was from Millipore (Billerica, MA); rabbit anti-cleaved caspase-3 (5A1E, #9664), rabbit anti-nuclear factor kappa-light-chain-enhancer of activated B cell p65 (NF-κB p65, C22B4; #4764), rabbit anti-Akt (#9272), rabbit anti-phospho-Akt (D9E, Ser473, #4060), rabbit anti-p38 mitogen-activated protein kinase (p38 MAPK, #9212), rabbit anti-phospho-p38 MAPK (D3F9, Thr180/Tyr182, #4511), rabbit anti-c-Jun N-terminal kinase (JNK, 56G8, #9258), rabbit anti-phospho-JNK (81E11, Thr183/Tyr185, #4668), rabbit anti-Histone H3 (#9715) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 14C10, #2118) were from Cell Signaling Technology (Beverly, MA). IRDye 800CW goat anti-mouse (926-32210) and anti-rabbit IgG (926-32211) secondary antibodies were from LI-COR Biosciences (Lincoln, NE).

CAFs (2 × 10⁴ cells per 12-well dish), Hep3b and SNU423 (5 × 10⁵ cells per 60 mm dish) and SNU398, SNU449, HLF, Huh7 and PLC/PRF5 (4 × 10⁵ cells per 60 mm dish), after the specified treatments, were washed in ice-cold phosphate buffer saline (PBS), pH 7.4, lysed and analyzed by immunoblot as described^{20 (link)}, with antibodies at the following dilutions: fibronectin, 1:10,000 (Sigma-Aldrich, Stockholm, Sweden, F3648); fatty acid synthase (FASN), 1:1,000 (ab22759); calponin, 1:2,000 (EP798Y, ab46794); LXRα, 1:1,000 (ab41902); Smad3, 1:1,500 (ab40854), all from Abcam, Cambridge, United Kingdom; α-smooth muscle actin (αSMA), 1:500 (Santa Cruz Biotech Inc., Santa Cruz, CA, USA, sc1a4); GAPDH, 1:50,000 (Ambion, ThermoFisher Scientific, Fyrislund, Sweden, AM4300). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (ThermoFisher Scientific, Fyrislund, Sweden) were used at 1:20,000 dilution. Triplicate (n_b = 3) biological experiments were performed in 2 technical replicates (n_t = 2) per condition. Densitometric quantification of protein bands was performed using the Fujifilm Intelligent Dark Box II program of a Fuji Aida digital scanner (Fujifilm Nordic AB, Stockholm, Sweden).

Isolated hepatocytes were fixed in 1% formalin in suspension for 10 min at room temperature and then quenched in 125 mM glycine. Cells were pelleted and washed twice with cold PBS, and subsequent steps of sonication and immunoprecipitation were performed as described (Shang et al., 2002 (link)). Two µL of anti-FOXO1 (#2880, Cell Signaling) or anti-LXRα antibody (ab41902, Abcam) was used per ChIP sample. Primers used for ChIP-qPCR are listed in Supplementary file 9.

Paired human breast cancer and adjacent non-tumor paraffin tissue microarrays were purchased from Shanghai Zuocheng Biotech (Shanghai, China). The sections were subjected to antigen retrieval and incubated with primary antibodies against NR1H3 (ab41902, abcam) and CD68 (ab955, abcam) at 4°C in a humid chamber overnight. The next day, the sections were incubated with biotinylated secondary antibody for 60 min. Protein levels of NR1H3 and CD68 were evaluated as follows: the slides were appraised for the intensity of the staining (0–3) and the percentage of positively stained cells (0–4). Index of protein levels was calculated as the intensity of the staining × the percentage of positively stained cells. Therefore, slices were divided into 4 groups: negative (score 0), low expression (score 1–4), medium expression (score 5–8) and high expression group (score 9–12).

The reagent strips for ALT/AST and TG were purchased from Changchun Huili Biotech Co., Ltd. (Changchun, China). Primary antibodies against GAPDH (ab8245), MPO (ab45977), lipin 1 (ab70138), LXRα (ab41902), lipin 2 (ab176347), CYP2E1 (ab28146), NLRP3 (ab4207), Sirt1 (ab110304), PPARγ (ab19481), SREBP1 (ab3259), F4/80 (ab6640), and Opti-MEM® were obtained from Abcam (Cambridge, MA, United States). Primary antibodies against ASC (sc514414), PPARα (sc9000), LXRβ (sc34341), IL6 (sc28343), IL1R1 (sc393998), and caspase 1 (sc622) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States). Primary antibodies against FXR (cs4173), p-AMPKα (cs2531), AMPKα (cs2532), p-AMPKβ1/2 (cs4181), AMPKβ1/2 (cs4150), LKB1 (cs3047), p-LKB1 (cs3482), p-ACC (cs11818), and ACC (cs3676) were purchased from Cell Signaling Technology (Beverly, MA, United States). Lipofectamine® 2000 Transfection Reagent was purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). The BCA Protein Assay Kit was obtained from Beyotime (Jiangsu, China). All other chemicals and reagents were obtained from Sigma-Aldrich (Shanghai, China).