Human liver tissues
Liver biopsy FFPE samples were obtained from the UC Davis Cancer Center Biorepository funded by the National Cancer Institute. Samples from 4 different patients with alcoholic liver disease and 4 normal livers were used in the studies. The tissues were processed for immunohistochemistry and real-time qPCR assays.
Animals and cell culture
All animal experiments were conducted according to the experimental procedures approved by the Institutional Animal Care and Use Committee at the University of California Davis. Conditional HSC NOX4 knockout mice (NOX4HSCKO) were generated by crossing the fl/fl NOX4 mice with C57B6 background (from R. Brandes, Goethe University, Frankfurt, Germany) and GFAP-cre mice (B6.Cg-Tg(Gfap-cre)73.12Mvs/J, Stock #012886, Jackson Laboratory, Bar Harbor, ME) through several generations. The cell specific knockout of NOX4 was verified with cell isolation and FACS sorting, followed by RT qPCR. In brief, mice were subjected to sequential liver perfusion and enzyme digestion, and hepatocytes were isolated first as described previously45. Then the non-parenchymal fraction was labeled with PE-conjugated anti-F4/80 (Biolegend, San Diego, CA) and FACS sorting was performed to purify F4/80+ macrophages and auto-fluorescent HSC. The cells were processed for PCR to analyze NOX4 mRNA. NOX4HSCKO or fl/fl NOX4 control littermates were pair-fed with the Lieber-DeCarli alcohol liquid diet (4% vol/vol, BioServe Inc., Flemington, NJ) and isocaloric control for 5 weeks following the manufacturer’s instructions.
Primary stellate cells were isolated from mice as described previously45. The cells were cultured in medium 199 (Millipore Sigma, St. Louis, MO) with 20% fetal bovine serum (FBS) and antibiotics. Human immortalized HSC, LX-2 cells were maintained in DMEM supplemented with 5% FBS and antibiotics.
Immunohistochemistry and immunofluorescence
The FFPE sections from ALD patients and normal controls were deparaffinized and boiled in citric buffer (pH = 6.0) for antigen retrieval. After blocking, the tissues were incubated with anti-NOX4 (1:1000, kindly provided by Dr. A. Shah, King’s College London, UK) at 4 °C overnight. The tissues were reacted with biotinylated secondary antibody followed by diaminobenzidine staining. For immunofluorescence, the slides were incubated with antibodies to NOX4 and αSMA (1:1000; Epitomics, Burlingame, CA) then probed with appropriate fluorescence conjugated secondary antibodies (Life Technologies, Carlsbad, CA). In vitro, after 100 μM acetaldehyde treatment for 24 hours, primary HSC were fixed with 4% paraformaldehyde, and then permeabilized with 0.2% TritonX-100 and 0.1% tween 20 in PBS. After blocking, the cells were probed with anti-HuR antibody (1:50, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), followed by appropriate fluorescence secondary antibody (Life Technologies) and mounted with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained by confocal microscopy.
ROS production was studied using lucigenin assay. Fresh liver tissues were homogenized on ice in sucrose buffer (0.3 M sucrose, 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM Spermidine, 1 mM DTT pH 7.4, with protease inhibitors (PI, Roche, Basel, Switzerland). The homogenate was centrifuged at 1000 × g for 5 minutes. The supernatant was further centrifuged at 100,000 × g for 1 hour at 4 °C to obtain the membrane enriched fraction. The pellet was suspended in Kreb’s buffer (100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 1.0 mM K2HPO4, 25 mM NaHCO3, 20 mM Na-HEPES, 0.2% glucose, pH 7.4) with PI. The membrane fraction was incubated with lucigenin (5 μM, Invitrogen) at room temperature for 15 min. Then 100 μM of NADPH (Sigma-Aldrich) was added and the chemiluminescence intensity was read with luminometer every 1 minute, up to 10 counts. The data were adjusted to the protein concentration.
Malondialdehyde (MDA) assay
Lipid peroxidation was measured by MDA assay (Abcam, Cambridge, MA) following the manufacturer’s instructions. Briefly, liver tissues were homogenized on ice in MDA lysis buffer followed by centrifugation at 13,000 × g for 10 minutes. The supernatant was incubated with TBA solution at 95 °C. The light absorbance was measured at 532 nm. The values were normalized to the tissue weight.
RNA extraction and reverse transcription and quantitative real-time polymerase chain reaction
Total RNA was extracted from liver tissues or cells using the RNeasy kit (Qiagen, Germantown, MD). The RNA from human FFPE livers was extracted with a kit from Life Technologies following the instruction. One μg of RNA was reverse-transcribed (iScriptTM cDNA synthesis kit, Bio-Rad, Hercules, CA) and RT-qPCR was performed on the 7900 HT system (Applied Biosystems, Foster City, CA) using Power SYBR Green PCR Master Mix (Applied Biosystems). The absolute amount of target genes was calculated based on the standard curve generated from the templates, and adjusted to the housekeeping gene. The primer sequences used are listed in Suppl. Table 1.
Luciferase reporter assay
Using the GeneJuice® transfection reagent (MilliporeSigma, Temecula, CA) and following the manufacturer’s protocol, the human stellate cells LX-2 were transfected with the human NOX4 promoter-luciferase reporter construct. The cells were then treated with 100 μM acetaldehyde (MilliporeSigma). After 24 hours, the cells were collected and the luciferase reporter assay was performed (Promega, Madison, WI) following the manufacturer’s instructions. Chemiluminescence for each sample was normalized to the protein content.
Messenger RNA half-life measurement
LX-2 cells were transduced with either adeno-NOX4 or control virus (adeno-CMV) (Applied Biological Materials Inc. Richmond, BC). To knock down HuR, LX-2 cells were transfected with either siHuR or control siRNA (Thermo Fisher, Waltham, MA) using RiboJuice transfection reagent (MilliporeSigma) following the instruction. The cells then were treated with 2.0 μg/ml actinomycin D (MP Biomedicals, Solon, OH) at the indicated time points. Total RNA was extracted with the TRIzol reagent (Life Technologies) following the manufacturer’s instructions. The amount of CCR2 and CCL2 mRNA was measured by RT-qPCR. mRNA half-life was calculated as T1/2 = Ln(2)/λ.
LX-2 cells transduced with adeno-NOX4 or adeno-CMV were serum starved for 16 hr. Cytoplasmic proteins were collected using an ER Isolation kit (MilliporeSigma). Fifty micrograms of cytoplasmic proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidine difluoride (PVDF) membranes. The membranes were blocked and incubated with anti- HuR (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phospho-HuR (Ser221) (MilliporeSigma), followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Detection was performed by chemiluminescence method.
Macrophage migration assay
WT and NOX4KO HSC were isolated and plated in a 24-well plate from a cell migration assay kit (Cell Biolabs, Inc. San Diego, CA). The cells were transfected with a CCR2 expression plasmid DNA (GeneCopoeia, Rockville, MD) using GeneJuice® transfection reagent, followed by transduction with adeno-CCL2 (Vector Biosystems Inc. Malvern, PA). Control plasmid and virus were used in parallel. Twenty four hours later, HSC were washed and macrophages from WT mice were seeded on the upper chambers and co-cultured with HSC. After 12 hours of co-culture, migration was assessed following manufacturer’s instruction. In brief, the non-migrating cells from the upper side of chambers were removed and the cells migrated to the other side were labeled with a fluorescent dye. The cells were then lysed and the fluorescence intensity was measured at 480 nm/520 nm.
All data represent at least three independent experiments and express as the mean ± standard error of the mean (SEM). ANOVA and two-tailed student’s t-test were used to analyze the significance of the data. A value of p < 0.05 was considered significant.