Animals
All animal procedures were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23 revised in 1996). TRAF1-KO mice (C57BL/6J background) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; Stock No. 008076), and the knockout was confirmed via PCR analysis using the primers 5′-GCCAGAGGCCACTTGTGTAG-3′, 5′-CAGAACCCCTTGCCTAATCC-3′, and 5′-TCCTAGAGGCCTGCTGCTAA-3′. To generate hepatocyte-specific TRAF1 TG mice, full-length murine TRAF1 cDNA amplified with the forward primer 5′-GAACTCGAGCCACCATGGCCTCCAGCTCAGCCCC-3′ and reverse primer 5′-GAAGGATCCTAAGCACTAGTGTCCACAA-3′ was cloned downstream of the albumin promoter. This construct was microinjected into fertilized embryos (C57BL/6J background) to produce hepatocyte-specific TG mice, and four independent TG lines were established. Genomic DNA was extracted from the tail for genoty** using PCR analysis with the primers 5′-AAGGGTGGCAACTTCTCCTC-3′ (forward) and 5′-ATAAGGAATGGACAGCAGGG-3′ (reverse). Only 8- to 10-week-old (24–27 g) males were used. All mice were housed in an environment with controlled light (12 h light/dark), humidity, and temperature, with food and water available ad libitum.
Mouse hepatic I/R injury model
A non-lethal model of segmental (70%) warm hepatic I/R injury was performed after the mice were anesthetized by pentobarbital sodium (50 mg/kg, i.p., Sigma-Aldrich, P3761, St. Louis, MO, USA). First, a midline laparotomy was performed after the abdominal wall of the mice was shaved and cleaned with Betadine. An atraumatic microvascular clamp (Fine Science Tools, North Vancouver, BC, Canada) was used to occlude the vasculature supplying the left and median lobes (ischemic lobes) of the liver for 60 min. Evidence of ischemia was confirmed by visualizing the pale blanching of the ischemic lobes. The mice in whom no ischemic color changes were observed or who exhibited a lack of response to reperfusion were immediately killed. The abdomen was covered during the ischemic period, and the body temperature of the mice was maintained at 36.5–37.5 °C through the use of a homeothermic blanket. After the removal of the clamp, reperfusion was confirmed by the color change of the ischemic lobes. Finally, the abdomen was closed with a continuous 4-0 polypropylene suture. Sham mice underwent the same surgical procedure without vasculature occlusion.
Liver damage assessment
The serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels were determined by using a spectrophotometer (Chemix 180i, Sysmex, Shanghai, China). Liver MPO activity was measured in frozen tissues according to the published methods,43 with slight modifications. In brief, 100 mg of liver tissue was homogenized in 2 ml of buffer containing 3.4 mmol/l of KH2HPO4 and 16 mmol/l of Na2HPO4 (PH 7.4). After the mixture was centrifuged for 20 min at 10 000 × g, the precipitation was resuspended in 10 volumes of the buffer, which contains 43.2 mmol/l of KH2HPO4, 10 mmol/l of EDTA, 6.5 mmol/l of Na2HPO4, and 0.5% hexadecyltrimethylammonium (pH 6.0), and the sample was sonicated for 10 s. The supernatant was reacted with 3,3′3,5′-tetramethylbenzidine after being heated at 60 °C for 2 h. Finally, the change in absorbance was read by a spectrophotometer at 655 nm. For histopathological analysis, hematoxylin and eosin (H&E) staining was performed after the liver tissues were fixed in 10% formalin, embedded in paraffin, and sectioned (5 μm per section). The percentage of necrotic area was estimated by random evaluation of five high-power fields ( × 200) for each H&E section.
Cytokine measurement
Serum TNF-α, IL-1β, IL-2, IL-6, and IL-10 cytokine levels were measured by a commercially available enzyme-linked immunosorbent assay kit (Peprotech, Rocky Hill, NJ, USA; RayBio, Norcross, GA, USA; Invitrogen, Camarillo, CA, USA). Additional details are included in the Supplementary Table 1.
Analysis of apoptosis
Apoptosis was evaluated by a TUNEL assay with the ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (EMD Millipore Corporation, S7111, Billerica, MA, USA) according to the manufacturer’s instructions. A commercially available cell death detection enzyme-linked immunosorbent assay kit (11585045001, Roche Applied Science, Penzberg, Germany) was used to assess the DNA fragmentation in primary hepatocytes according to the manufacturer’s instructions.
Immunofluorescence staining
Immunofluorescence staining was performed as described previously.44,45 In brief, paraffin-embedded liver tissues were cut into 5-μm sections and processed for immunofluorescence staining. After a 5-minute high-pressure antigen retrieval process in sodium citrate buffer (100 × , pH 6.0), liver sections were washed in PBS containing 10% goat serum for 1 h and incubated with the indicated primary antibodies at 4 °C overnight: rabbit anti-Mac1 (CD11b, 1 : 100 dilution, ab75476, Abcam, Cambridge, MA, USA), anti-CD3 (1 : 100 dilution, ab16669, Abcam); rat anti-Ly6G (1 : 100 dilution, 551459, BD Biosciences, San Jose, CA, USA) and mouse anti-PCNA (1 : 100 dilution, 2586, Cell Signaling Technology, Beverly, MA, USA). After washing with PBS, the sections were incubated with the appropriate secondary antibodies for 1 h at 37 °C. The secondary antibodies used included anti-mouse immunoglobulin G (IgG) Alexa Fluor 568 conjugate (Invitrogen), anti-rat IgG Alexa Fluor 568 conjugate, and goat anti-rabbit IgG Alexa Fluor 568 conjugate. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained using a fluorescence microscope (OLYMPUS DX51, Tokyo, Japan) and DP2-BSW software (version 2.2, Tokyo, Japan), and the images were analyzed with Image Pro Plus (version 6.0, Media Cybernetics, Rockville, MD, USA).
Primary hepatocyte isolation and culture
Primary hepatocytes were cultured according to the published methods,43 with a slight modification. Liver perfusion medium (Invitrogen, 17701-038) was used to perfuse the livers in situ via the portal vein and was followed by liver digest medium (Invitrogen, 17703-034) after anesthetizing the mice with pentobarbital sodium. The liver was excised and minced in William’s E medium (Life technologies, A12176-01, Grand Island, NY, USA). The cell suspension was mixed gently several times with a pipette and strained through a steel mesh sieve after removing the liver capsule. The dispersed hepatocytes were collected via centrifugation at 50 × g for 5 min at 4 °C and washed twice with William’s E medium. Hepatocytes were isolated by Percoll separation and washed twice with William’s E medium. The final pellet was resuspended in William’s E medium. The hepatocytes were counted, and their viability was determined by trypan blue exclusion. The hepatocytes were cultured under normoxic conditions (air/5% CO2) for further experiments.
Adenoviral infection and hypoxia-reoxygenation model in vitro
Adenoviruses harboring sequences encoding mouse TRAF1 and a short hairpin RNA targeting TRAF1 (shTRAF1) were generated. The open reading frame (ORF) of mouse TRAF1 was purchased from OriGene (MG206422, Rockville, MD, USA) and subcloned into a pCMV6-AC-GFP shuttle vector. Recombinant adenoviruses were generated using an AdEasy vector kit (Stratagene, La Jolla, CA, USA). Inserts were cloned into the pShuttle-CMV vector. Plasmids were recombined with the pAdEasy backbone vector according to the manufacturer’s instructions, and HEK293 cells were transfected with the vector using FuGENE transfection reagent (E2312, Roche, Indianapolis, IN, USA). Recombinant adenoviruses were plaque-purified, titrated to 109 PFU/ml, and verified by restriction digestion. To generate shTRAF1, the hairpin-forming oligonucleotides TRAF1-f, 5′-GATCCGCGTGTGTTTGAGAACATTGTTCTCGAGAACAATGTTCTCAAACACACGTTTTTTACGCGTG-3′ and TRAF1-r, 5′-AATTCACGCGTAAAAAACGTGTGTTTGAGAACATTGTTCTCGAGAACAATGTTCTCAAACACACGG-3′ were synthesized, annealed, and subcloned into the shuttle vector distal to the U6 promoter. Recombinant adenovirus was generated using a similar process. Cultured hepatocytes were transfected with adenovirus at a multiplicity of infection (MOI) of 100 for 48 h. To establish an I/R model in vitro, cultured cells were washed twice with PBS before the addition of serum-free DMEM/F12 medium saturated with 95% N2/5% CO2 at 37 °C. The culture plates were then transferred to an experimental hypoxia chamber in a saturated atmosphere of 95% N2 and 5% CO2 for 60 min. The medium was then replaced with fresh normal maintenance medium that had been warmed, and oxygenated, and the plates were returned to a 37 °C humidified incubator under normoxic conditions (air/5% CO2) for the indicated times. Cells exposed to normoxic medium for the same periods were used as controls.
Flow cytometry
Flow cytometry was performed to assess kupffer cell, inflammatory monocyte, and neutrophil infiltration into the liver. In brief, ischemia liver lobes were aseptically collected from mice after 1 h of ischemia and 12 h of reperfusion and prepared as a single-cell suspension. Nonparenchymal cells (NPCs) were isolated from ischemic lobes (I/R) after 12 h of reperfusion. Groups were pooled and analyzed by flow cytometry as described previously.46 We defined neutrophils as CD11bhiLy6Ghi, inflammatory monocytes as CD11bintLy6Chi, and Kupffer cells as CD11b+F4/80+. Antibodies were purchased from Biolegend (San Diego, CA, USA): CD16/32 (clone 93), F4/80 (APC; clone BM8), Ly6G (PE; clone 1A8), Ly6C (FITC; clone HK1.4), CD11b (PerCP/Cy5.5; clone M1/70). Data were acquired with CyAn Advanced Digital Processing (ADP) analyzer (Beckman Coulter, Miami, FL, USA) and analyzed with Summit v4.2. Each experiment was repeated a minimum of three times.
Quantitative real-time PCR and western blotting
TRIzol Reagent (15596-026, Invitrogen) was used to isolate total RNA from the samples of liver tissue and primary hepatocytes. Two micrograms of total RNA was used for cDNA synthesis with the Transcriptor First Stand cDNA Synthesis Kit (04896866001, Roche). The differentially expressed genes were determined by quantitative real-time PCR using SYBR green (Roche, 04887352001), and the results were normalized using GAPDH expression. The primers used are presented in Supplementary Information (Supplementary Table 2). A western blot analysis was performed as previously described.44,45,47,48 RIPA lysis buffer (720 μl RIPA, 20 μl PMSF, 100 μl Complete, 100 μl Phos-stop, 50 μl NaF, and 10 μl Na3VO4 in 1 ml lysis buffer) was used to extract total protein from the liver tissue or cultured cells. The protein concentration was determined by the Pierce BCA Protein Assay Kit (Pierce, 23225, Rockford, IL, USA). Twenty micrograms of protein was resolved via 10% SDS-PAGE (Invitrogen, NP0301BOX) and then electrically transferred to a PVDF membrane (Millipore, IPVH00010, Billerica, MA, USA), which was blocked with TBS containing 5% skim milk powder for 90 min at room temperature. After incubation with the indicated primary antibody overnight at 4 °C, the membrane was incubated with a secondary peroxidase-conjugated antibody (Peroxidase-Affinipure Goat Anti-Mouse IgG (H+L) (115-035-003), Peroxidase-Affinipure Goat Anti-Rabbit IgG (H+L) (111-035-003), Jackson ImmunoResearch Laboratories, Lincoln, NE, USA). Finally, a FluorChem E (Cell Biosciences, Santa Clara, CA, USA) imaging system was used for signal detection. Protein expression levels were quantified and normalized to the loading control GAPDH. All of the antibodies used in this study are listed in Supplementary Table 3.
Assay of cell viability
The MTT Cell Viability Assay Kit (11465007001, Roche) was used to determine cell viability, and lactate dehydrogenase (LDH) release was assessed by a colorimetric LDH cytotoxicity assay (G1782, Promega, Madison, WI, USA) according to the manufacturer’s protocol.
Plasmid constructs
The mouse TRAF1 CDS was PCR amplified with TRAF1-5′ and TRAF1-3′ primers, digested with BamHI and XhoI, and ligated into pSicoR-EGFP-myc digested with BamHI and XhoI to create the EGFP-myc-TRAF1 recombinant. To obtain TRAF1 fragments consisting of residues 1 to 187 and 188 to 409, EGFP-myc-TRAF1 was PCR-amplified with TRAF1-5′ and TRAF1-N-3′ primers and the TRAF1-C-5′ and TRAF1-3′ primers, respectively. The products were digested with BamHI and XhoI and ligated into pSicoR-EGFP-myc to create an in-frame fusion with EGFP-myc. Human ASK1 CDS was amplified with the ASK1-5′ and ASK1-3′ primers, digested with SalI, and ligated into pSicoR-Flag-Cherry digested with XhoI to construct the Flag-Cherry-ASK1 recombinant. To obtain ASK1 fragments consisting of residues 1 to 320, 321 to 649, 650 to 940 and 940 to 1375, Flag-Cherry-ASK1 was PCR-amplified with the ASK1-5′ and ASK1-N1-3′ primers, the ASK1-N2-5′ and ASK1-N2-3′ primers, the ASK1-K-5′ and ASK1-K-3′ primers, and the ASK1-C-5′ and ASK1-3′ primers, respectively. The products were digested with BamHI and SalI, BamHI and SalI, BamHI and SalI or BglII and SalI, respectively, and ligated into the pSicoR-Flag-Cherry digested with BamHI and XhoI to create an in-frame fusion with Flag-cherry. The primers for making these constructs are presented in Supplementary Table S2. All plasmids were verified by sequencing.
Immunoprecipitation
Transient transfections of HepG2 cells with pSicoR-myc-TRAF1 and pSicoR-Flag-ASK1 into HepG2 cells were performed using FuGENE transfection reagent according to the manufacturer’s instructions (Roche). After 48 h of incubation, the cells were lysed in ice-cold IP buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40) containing a protease inhibitor cocktail (Roche). For co-immunoprecipitation experiments, 500–1000 μg of cell lysates was precleared with 10 μl of protein A/G-agarose beads (11719394001, 11719386001, Roche) and immunoprecipitated with the indicated antibody with protein A/G-agarose beads. After 4 h of incubation at 4 °C, the beads were washed 5–6 times with 800 μl of cold IP buffer, resuspended in 1 × loading buffer, and heated at 95 °C for 5 min. The cell lysates and immunoprecipitates were analyzed by western blotting as described above.
Confocal microscopy
HepG2 cells were seeded onto coverslips in 24-well plates. After co-transfection of pCherry-ASK1 and pEGFP-TRAF1 for 48 h, the cells were fixed with 4% fresh paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with 1 g/ml DAPI for 15 min. Between each step, the coverslips were washed three times with 1 × PBS. The coverslips were removed from the wells and placed on a slide with mounting solution (D2522, Sigma). Images were acquired with an Olympus FV1000 confocal microscope.
FRET
For FRET, the TRAF1 (1–187 aa/1–409 aa) and ASK1 genes were cloned into pEYFP-N1 and pECFP-N1 as EcoRI-BamHI fragments. The following PCR primers were used: for TRAF1 (1–187 aa), sense: 5′-ATGGCCTCCAGCTCAGCCCCT-3′; anti-sense: 5′-CTAAGCACTAGTGTCCACAA-3′; for TRAF1 (1-409aa), sense: 5′-ATGGCCTCCAGCTCAGCCCCT-3′; anti-sense: 5′-CTAAGCACTAGTGTCCACAATGC -3′; and for ASK1, sense: 5′-ATGAGCACGGAGGCGGACGA-3′; anti-sense: 5′-TCAAGTCTGTTTGTTTCGAAA-3′. For FRET analysis, 293T cells (4 × 106 cells/100 mm dish) were co-transfected with ECFP-ASK1 and EYFP-TRAF1 (1–187 aa/1–409 aa) expression plasmids. CFP-ASK1 and YFP-TRAF1 were used as the donor–acceptor FRET pair. FRET was detected using the donor dequenching approach in which CFP fluorescence was measured before and after photobleaching of the YFP. The percentage dequenching FRET efficiency was calculated as E=(DQ−Q/DQ) × 100, where DQ and Q are the dequenched and quenched donor fluorescence, respectively. Images were captured using a Nikon AIRsi.
Statistical analysis
Data are presented as the mean±S.D. Differences between the groups were assessed by an ANOVA followed by Tukey’s post-hoc test. Comparisons between two groups were performed by Student’s t-test. P<0.05 was considered significant.
