Supporting Information

Hong et al. 10.1073/pnas.1315319110 SI Methods glucose. The microsomal proteins were quantified using the Isolation of Primary Hepatocytes and Kupffer Cells. Hepatocytes and Micro bicinchoninic acid (BCA) Protein Assay kit (PI23235; Kupffer cells were isolated from mice by collagenase digestion Thermo Scientific). Liver microsomes (0.05 mg) were incubated in and two-step Percoll Gradient method with slight modifications. the dark at 37 °C in PBS containing 10 μM2’,7’-dichlorodihydro- fl Briefly, mice were anesthetized, and the peritoneal cavity was uoresceine diacetate (H2DCFDA; D399; Life Technologies) for + + opened. Livers were perfused with Ca2 and Mg2 free-HBSS 1 h in a 96-well black plate. The levels of reactive oxygen species containing EDTA (1 mM) and then digested with a collagenase were detected by measurement of the fluorescent product using solution containing liberase thermolysin medium research grade a fluorescence microplate reader (excitation, 485 nm; emission, (0.1 mg/mL; 01 401 119 001; Roche), glucose (1%), CaCl2 (5 528 nm). mM), and DNase I (10 μg/mL) in HBSS. Digested livers were removed and rinsed twice with HBSS and then gently teased with Western Blotting. Cell and liver samples were homogenized in lysis forceps until they were in solution. The cell suspensions were buffer [10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, fl filtered through a sterile 100-μm nylon cell strainer (BD Bio- 1 mM phenylmethylsulfonyl uoride, 0.2 mM sodium orthova- sciences) to remove undigested tissue and connective tissue. The nadate, 0.5% Nonidet P-40 containing protease and phosphatase cells were centrifuged for 5 min at 650 × g and resuspended with inhibitor mixture tablets (Roche)] at 4 °C for 30 min. Protein fi HBSS. After the cell suspensions were centrifuged for 3 min at was quanti ed using the Micro BCA Protein Assay kit (Thermo fi – 35 × g, the supernatants (nonparenchymal cells such as Kupffer Scienti c kit), and proteins were resolved on 7.5 10% SDS/ cells) were transferred to a new tube, and the pellets (paren- PAGE gels. The membranes were blocked for 1 h in tris-buffered chymal cells such as hepatocytes) were resuspended with HBSS. saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% skim For Kupffer cell isolation, the supernatants obtained as de- milk and then incubated overnight at 4 °C with a primary anti- scribed above were centrifuged using a two-step Percoll gradient body (see below) in the same buffer. The blots were then washed [25%/50% (vol/vol)] for 15 min at 1,800 × g with the brake option three times in TBS-T for 15 min to remove excess antibody, and off. Subsequently, the upper layers were removed, and the then the membranes were incubated for 1 h with secondary an- middle layers between 25% and 50% Percoll gradient were tibodies in TBS-T + 5% (g/vol) skim milk: anti-rabbit (NA934V; collected without contamination from the pellets. The collected GE Healthcare) or anti-mouse (NA931V; GE Healthcare). middle layers were washed twice with DMEM supplemented Following three washes in TBS-T, proteins were detected using with 10% (vol/vol) FBS then plated into six-well tissue culture Lumi-Light Western Blotting substrate (Roche). plates. One hour following seeding, nonadherent cells (cell de- Primary antibodies used were estrogen-related receptor α (ERRα) bris or blood cells) were removed by aspiration, and fresh media (2131-1; Epitomics), RPLP0 (ab88872; Abcam), p53 (sc-53394; was added. After 24 h, the cells were treated with 0.5% trypsin– Santa Cruz), caspase 3 (9662; Cell Signaling), caspase 6 (9762; 0.02% EDTA to remove endothelial cells, and the attached Cell Signaling), cleaved caspase 6 (9761; Cell Signaling), caspase Kupffer cells were cultured for another 24 h, at which time they 9 (9504; Cell Signaling), cleaved caspase 9 (9509; Cell Signaling), were ready for experimental use. HMGB1 (3935; Cell Signaling), nuclear factor κB (NF-κB)-p65 For hepatocyte isolation, the pellet suspensions obtained as (4764; Cell Signaling), NF-κB-p50 (1559-1; Epitomics), Lamin described above were centrifuged using 50% Percoll for 15 min at B1 (sc-56145; Santa Cruz), Tubulin (CLT9002; Cedarlane), and 1,800 × g with the brake option off. After centrifugation, the IκBα (4814; Cell Signaling). healthy hepatocytes were pelleted as damaged hepatocytes, or α nonparenchymal cells could not penetrate into 50% Percoll so- ELISA. Circulating IL-1 and HGF levels were detected in serum α lution. The pellets were washed twice with DMEM supplemented from WT and ERR -null mice acutely treated with DEN for the with 10% FBS, and then 5 × 105 hepatocytes were seeded into six- indicated time points. ELISA kits were obtained from R&D α well tissue culture plates. After 24 h, nonadherent cells were re- Systems (MLA00 for IL-1 and MHG00 for HGF). moved by aspiration, and fresh media was added. Histological and Immunohistological Analyses. For histological fi ALT Measurements. Mouse blood samples were transferred into 1.1 analyses, livers were xed in 10% buffered formalin for 48 h and fi mL Z-Gel microcentrifuge tubes containing a serum-gel clotting subsequently paraf n-embedded. Serial sections were stained activator (41.1378.005; Sarstedt). The samples were allowed to with Hematoxylin and Eosin, and stained slides were examined clot for 30 min at room temperature before centrifugation for using an Aperio XT Slide Scanner (Aperio). For BrdU detection 30 min at 2,348 × g. Serum samples were removed without dis- (550803; BD Bioscience) the tissue sections were incubated with turbing the gel layers. Alanine aminotransferase (ALT) levels 0.1 M citrate buffer (pH 6.0) at 95–100 °C for 1 h. After blocking were determined using the International Federation of Clinical with 10% normal donkey serum, slides were incubated with Chemistry comparative method at the diagnostic laboratory of primary antibodies [cleaved caspase 3 (9664; Cell Signaling) and the Comparative Medicine Animal Resource Centre at McGill Tubulin] at 4 °C overnight. Following this, slides were washed University. and incubated simultaneously with the corresponding Alexa- Fluor conjugated secondary antibodies from Life Technologies Microsomal Reactive Oxygen Species Measurements. Whole livers diluted in TBS with 1% donkey serum at room temperature for from diethylnitrosamine (DEN)-induced acute liver injury were 1 h. After washing, slides were mounted in ProLong Gold anti- homogenized with lysis buffer (0.25 M sucrose, 5 mM Hepes, fade reagent (P36931; Invitrogen) and examined using an Axi- 1 mM EDTA pH 7.2). The homogenates were centrifuged at oskop microscope (Zeiss). For immune-reactive Ki-67 (ab15580; 500 × g for 10 min at 4 °C, and the supernatants were transferred Abcam) and cell death (11 684 812 910; Roche) in situ, paraffin- into new tubes and centrifuged for 10 min at 9,500 × g.Aftercen- embedded tissue sections were dewaxed and rehydrated. Cell trifugation, the supernatants were removed by aspiration, and the death analysis was done using the In Situ Cell Death Detection pellets were resuspended in ice-cold PBS containing 20 mM Kit as per the manufacturer’sspecifications. For immune-reactive

Hong et al. www.pnas.org/cgi/content/short/1315319110 1of6 Ki-67 assay, the tissue sections were incubated with blocking buffer primer sets amplifying non-ERRα and NF-κB (p65) bound ge- (1% BSA in PBS) at 37 °C for 1 h. Slides were then incubated nomic regions (Tables S2 and S3). with primary antibodies (1:200) at room temperature for 1 h, washed in PBS, and incubated with the corresponding Alexa- RNA Isolation, Reverse Transcription, and qRT-PCR. Total RNA ex- Fluor conjugated secondary antibody. After washing, slides were tracts from mouse liver or cells were prepared using the RNeasy mounted in ProLong Gold antifade. Proliferation was assessed Mini Kit (Qiagen). Reverse transcription (RT) was performed at μ by the percentage of Ki-67–positive cells. Histology experiments 42 °C for 50 min using 1 g of total RNA and 200 units of Su- were performed in collaboration with the Goodman Cancer perScript II together with oligo(dT) primers and reagents pro- Research Centre Histology Core Facility. vided by Life Technologies. Quantitative RT-PCR was carried Primary hepatocytes were grown in standard culture medium as out using specific primers (Table S4), a Quantitect SYBR Green described above and cultured in eight-well chambers on tissue PCR Kit (Qiagen), and a Roche LightCycler 480 instrument culture glass slides (BD Bioscience). The cells were washed in (Roche) equipped with a 96-well optical reaction plate. All PBS and fixed in 100% methanol at −20 °C for 20 min. Slides experiments were run in triplicate, and mRNA values were cal- were air-dried and then rehydrated in PBS. The fixed cells were culated based on the cycle threshold and monitored for an am- blocked with 10% goat serum at room temperature for 30 min plification curve. and incubated overnight with primary antibodies at 4 °C. Fol- lowing this, slides were washed and incubated with Alexa-Fluor Mitochondrial and Nuclear DNA Quantification. The relative number conjugated secondary antibodies (Life Technologies) diluted of mitochondria in primary hepatocytes obtained from WT and 1:1,000 in PBS containing 1% normal goat serum at room tem- ERRα-null livers was determined by measuring the ratio of mi- perature for 1 h. After washing, slides were mounted in ProLong tochondrial DNA (mtDNA) to nuclear DNA. DNA was isolated Gold antifade reagent and examined using an Axioskop micro- using a Qiagen DNeasy Blood and Tissue Kit (Qiagen), and the scope (Zeiss). relative levels of mtDNA and nuclear DNA were quantified using primers specific for mitochondrial cytochrome b (mt-Cytb) ChIP. Mouse liver ChIP samples were prepared from 2- to 3-mo- (forward, 5′-CTCCTCTTCCTCCACGAAACAG-3′,andreverse, old male mice treated with or without DEN (100 mg/kg) for 48 h. 5′-GTTTATTGGGGATTGAGCGTAG-3′) and the nuclear gene fl Brie y, livers were isolated, weighed, and homogenized for a few Pgk2 (forward, 5′-GAAGACAAGGTCAGCCATGTGAGC-3′, seconds (until no tissue clumps) in cold PBS using a Brinkmann and reverse 5′-GGTTGAGTTGGTTCTGGTCCTGTG-3′). polytron homogenizer. The samples were centrifuged at 1,721 × g for 2 min at 4 °C, and the cell pellets were resuspended in cold LC-MS/MS Detection of Energy Metabolites. Primary hepatocytes PBS. Following centrifugation, the pellets were resuspended in were grown in standard culture medium with or without cytokines fi – PBS containing formaldehyde (1% nal), and DNA protein as described above. The cells were washed with cold ammonium cross-linking was allowed to occur at room temperature for 10 formate and resuspended in lysis buffer [50% (vol/vol) acetoni- × min with rotation. Samples were then centrifuged at 1,721 g for trile]. Quantification of energy metabolites from the cellular 2 min at 4 °C, and the pellets were washed twice with cold PBS · extracts of primary hepatocytes was achieved using a liquid and resuspended in nuclei lysis buffer (50 mM Tris HCl, pH 8.1, chromatography-tandem mass spectrometry (LC-MS/MS) ap- 10 mM EDTA, 1% SDS) supplemented with protease inhibitors proach at the Metabolomic Core Facility of McGill University. (Roche). Samples were sonicated on ice at power 10 for 30-s Ultra high performance liquid chromatography-mass spectrom- pulses using a VirSonic 100 (Virtis) sonicator at 30-s intervals to etry (UHPLC-MS) analysis was performed on an Agilent 1290 prevent the samples from heating. Sonicated material was Infinity LC system coupled to an Agilent 6430 triple quadrupole centrifuged at 15,871 × g for 15 min at 4 °C to remove cellular (QQQ)-LC-MS/MS mass spectrometer (Agilent). Chromato- debris and chromatin (100 μg) was diluted in 2.5× ChIP dilution graphic separations were performed on a Pursuit 3 penta- buffer (0.5% Triton X-100, 2 mM EDTA, 100 mM NaCl, 20 mM fl μ × · uorophenyl (PFP) column (3.0 m, 2.0 150 mm; Agilent) at Tris HCl, pH 8.1) and incubated overnight with either anti- fl α – κ a ow rate of 0.2 mL/min and then were followed on a Pursuit 3 ERR monoclonal antibody (Epitomics), anti NF- B(p65) μ × polyclonal antibody (Santa Cruz), or a rabbit IgG antibody as PFP MetaGuard column (3.0 m, 2.0 2.0 mm; Agilent). The a control (Santa Cruz) and 60 μL of Dynabeads Protein A (In- following gradient program was used: 100% A [water/0.2% for- vitrogen 10008D). Beads were washed six times with LiCl buffer mic acid (FA), pH 3.25] with a 7-min gradient to 30% B [0.2% (1% Nonidet P-40, 500 mM LiCl, 1% Na-deoxycholate, pH 8.0, FA in methanol (MeOH)] followed by a 3-min step using 100%. 100 mM Tris·HCl, pH 8.1). The beads were next washed briefly A subsequent reequilibration time for 6 min should be performed with TE buffer (10 mM Tris·HCl, pH 7.5, 1 mM EDTA, pH 8.0) before next injection. Mass spectrometer is equipped with an and de–cross-linked (1% SDS, 0.1 M NaHCO3) at 65 °C over- electrospray ionization source, and samples were analyzed in night. De–cross-linked samples were purified using the QIA- positive mode. Data were quantitated by integrating the area quick Spin Kit (Qiagen). under the curve of each optimized multiple reaction monitoring ChIP samples from murine macrophage cells (Raw 264.7 cells) transition on standards for each metabolite (ATP, ADP, AMP, were prepared with cell culture medium containing formaldehyde creatine, phosphocreatine, and creatinine; Sigma). Absolute (1% final), and DNA–protein cross-linking was allowed to occur quantitation was performed using calibration curves processed at room temperature for 10 min with rotation. The cross-linked with Agilent Mass Hunter Quantitative Analysis software. Tran- cells were washed twice with cold PBS and resuspended in nuclei sitions for quantifier and qualifier ions were, respectively, 508.0 → lysis buffer (50 mM Tris·HCl, pH 8.1, 10 mM EDTA, 1% SDS) 136.0 and 508.0 → 410.0 for ATP, 428.0 → 136 and 428.0 → 348.1 supplemented with protease inhibitors (Roche) and sub- for ADP, 348.0 → 119 and 348.0 → 136 for AMP, 212.0 → 44.1 sequently processed as described above for liver ChIP samples. and 212.0 → 114.1 for phosphocreatine, and 132.0 → 44.1 and ERRα and NF-κB (p65) standard ChIP enrichments were 132.0 → 90.0 for creatine. Gas temperature and flow were set at quantified by qPCR analysis using specific primers and normal- 350 °C and 10 L/min, respectively; nebulizer pressure was set at ized to the average enrichments obtained using two control 50 psi, and capillary voltage was set at +4,000 V.

Hong et al. www.pnas.org/cgi/content/short/1315319110 2of6 Fig. S1. Increased hepatocellular death and DNA damage in mice lacking ERRα following acute DEN treatment. (A) Hepatocyte death and DNA damage in WT and ERRα-null mice following an acute DEN exposure were evaluated using a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. Immunofluorescence detection shows DNA strand breaks (green) and nuclei stained with DAPI (blue). (Scale bar, 50 μm.) (B) Quantification of TUNEL staining. Values represent means ± SEM. *P < 0.05.

Fig. S2. Serum levels of the proinflammatory cytokines (Left) IL-1α and (Right) HGF of WT and ERRα-null mice following acute DEN treatment are shown. Values represent means ± SEM. *P < 0.05.

Hong et al. www.pnas.org/cgi/content/short/1315319110 3of6 Fig. S3. (A) Standard ChIP qPCR analysis of p65 occupancy on the NF-κB target genes Il1a, Il6, Tnf, and Hgf in livers of WT and ERRα-null mice treated with or without DEN for 48 h. Data are expressed as percentage of input. Values represent means ± SEM. *P < 0.05. (B) Standard ChIP qPCR analysis of ERRα occupancy on the IκBα-encoding gene, Nfkbia, in mouse liver and the mouse macrophage cell line Raw 264.7. ERRα binding to the Esrra promoter is shown as a positive control. Data are expressed as percentage of input. Values represent means ± SEM. *P < 0.05.

Fig. S4. Loss of ERRα results in decreased Nfkbia expression. qRT-PCR expression analysis of Nfkbia in (A) livers and (B) primary Kupffer cells of WT and ERRα- null mice. (C–E) qRT-PCR expression analysis of Nfkbia in Raw 264.7 macrophage cells treated with or without C29, XCT790, or a vector expressing ERRα for 48 h. Data are normalized to RPLP0 levels. Values represent means ± SEM. *P < 0.05.

Hong et al. www.pnas.org/cgi/content/short/1315319110 4of6 Fig. S5. Loss of ERRα induces p53-regulated gene expression. qRT-PCR expression analysis of the p53 target genes (Left) Bcl2 and (Right) Bcl2l1 involved in the regulation of apoptosis in livers of WT and ERRα-null mice following acute DEN (100 mg/kg) treatment. Data are normalized to RPLP0 levels. Values represent means ± SEM. *P < 0.05.

Table S1. Phenotypic characterization of DEN-treated WT and ERRα-null mice Characteristic 1 m 3 m 6 m 9 m 12 m

Number of mice WT 499 9 9 KO 7 14 11 11 9 Tumor incidence, % WT 0 0 22.2 25 100 KO 0 0 36.4 100 100 No. tumors/liver WT 0 0 0.22 ± 0.15 1.50 ± 1.13 7.78 ± 2.86 KO 0 0 0.55 ± 0.28 6.90 ± 1.29 14.88 ± 2.54 Average tumor area, mm2 WT ——0.17 ± 0.11 2.69 ± 0.89 18.51 ± 3.90 KO ——0.92 ± 0.39 13.12 ± 2.48 30.73 ± 4.03 Liver weight/body weight WT 5.65 ± 0.12 5.45 ± 0.12 4.91 ± 0.07 5.24 ± 0.11 6.42 ± 1.13 KO 5.58 ± 0.09 5.26 ± 0.12 4.69 ± 0.13 6.69 ± 0.51 11.17 ± 1.98 ALT, U/L WT 36.25 ± 1.65 60.22 ± 9.14 56.00 ± 4.18 59.13 ± 3.42 86.67 ± 24.29 KO 50.57 ± 6.34 49.14 ± 3.21 64.82 ± 5.13 140.70 ± 30.65 151.33 ± 36.52

Results in bold are statistically different between WT and ERRα-null mice.

Table S2. Mouse primers used for ERRα ChIP quantitative PCR analysis Primer Sequence

Control set1 Forward 5′-TTGGCATTGATATTGGGGGTGGGAGCAACT-3′ Reverse 5′-GACTTCTTACTTTGACGCTTTCCTCCATCG-3′ Control set2 Forward 5′-CCAAGCACAAATATCTAATCACCCTTTC-3′ Reverse 5′-CTTCTTGATAGGTTTATGGGTTGGGC-3′ Esrra Forward 5′-GTGGCCCCGCCTTTCCCCGTGACCTTCATT-3′ Reverse 5′-ACCCCTGAGGACCCTCAAGTGGAGAAGCAG-3′ Nfkbia Forward 5′-AAGGCCTGCTTGGACTACCT-3′ Reverse 5′-CTCCAAGTGCTTCCGTTTTC-3′

Hong et al. www.pnas.org/cgi/content/short/1315319110 5of6 Table S3. Mouse primers used for NF-κB (p65) ChIP quantitative PCR analysis Primer Sequence

Control set1 Forward 5′-TTGGCATTGATATTGGGGGTGGGAGCAACT-3′ Reverse 5′-GACTTCTTACTTTGACGCTTTCCTCCATCG-3′ Control set2 Forward 5′-CCAAGCACAAATATCTAATCACCCTTTC-3′ Reverse 5′-CTTCTTGATAGGTTTATGGGTTGGGC-3′ Il1a Forward 5′-GTGAAGGTCACTACCGAGGCAC-3′ Reverse 5′-CTACAGCACAGGAATGAAGATGGAG-3′ Il6 Forward 5′-TCCCATCAAGACATGCTCAA-3′ Reverse 5′-AGGAAGGGGAAAGTGTGCTT-3′ Tnf Forward 5′-CTCATGTGGAGGAAGCGGTAGTG-3′ Reverse 5′-GATTCCTTGATGCCTGGGTGTC-3′ Hgf Forward 5′-CTTCCTCCCTCCCTGAAGAC-3′ Reverse 5′-CACTTGAGACCCGTTGGACT-3′

Table S4. Mouse qRT-PCR primers Primer Sequence

Bax Forward 5′-TGAAGACAGGGGCCTTTTTG-3′ Reverse 5′-AATTCGCCGGAGACACTCG-3′ Bcl2 Forward 5′-ATGCCTTTGTGGAACTATATGGC-3′ Reverse 5′-GGTATGCACCCAGAGTGATGC-3′ Bcl2l1 Forward 5′-AGGCGATGAGTTTGAACTGC-3′ Reverse 5′-TGAAGCTGGGATGTTAGATCACT-3′ Cdkn1a (p21) Forward 5′-CCTGGTGATGTCCGACCTG-3′ Reverse 5′-CCATGAGCGCATCGCAATC-3′ Cox5b Forward 5′-CTGGACCCATACAATATGCTACCTCC-3′ Reverse 5′-ATCGCTGACTGCTCGCCTTTGTG-3′ Cycs Forward 5′-CCAAATCTCCACGGTCTGTTCG-3′ Reverse 5′-GTATCCTCTCCCCAGGTGATGC-3′ Hgf Forward 5′-TTCATGTCGCCATCCCCTATG-3′ Reverse 5′-CCCCTGTTCCTGATACACCT-3′ Il1a Forward 5′-AGTATCAGCAACGTCAAGCAA-3′ Reverse 5′-TCCAGATCATGGGTTATGGACTG-3′ Il1b Forward 5′-GAAATGCCACCTTTTGACAGTG-3′ Reverse 5′-CTGGATGCTCTCATCAGGACA-3′ Il6 Forward 5′-CTGCAAGAGACTTCCATCCAG-3′ Reverse 5′-AGTGGTATAGACAGGTCTGTTGG-3′ Mdh2 Forward 5′-CTTATGCTGGAGCCCGCTTTGTC-3′ Reverse 5′-CTCAGGGATAGCCTCGGCAATC-3′ Nfkbia Forward 5′-GCAATCATCCACGAAGAGAAGCC-3′ Reverse 5′-CAGGATCACAGCCAGCTTTCAGAAG-3′ Pfkl Forward 5′-CGGTCACAGAACTCAAGAAAGAGACTG-3′ Reverse 5′-GACACATAGTCTGCCATGCTGATGC-3′ Pklr Forward 5′-GAGTCTTCCCCTTGCTCTACCGTGAG-3′ Reverse 5′-GTATAGCCAGAGCCAGGTCGCCAG-3′ Pmaip1 (Noxa) Forward 5′-GCAGAGCTACCACCTGAGTTC-3′ Reverse 5′-CTTTTGCGACTTCCCAGGCA-3′ Rplp0 Forward 5′-GCAGCAGATCCGCATGTCGCTCCG-3′ Reverse 5′-GAGCTGGCACAGTGACCTCACACGG-3′ Sdhd Forward 5′-GGTACTTGAATCCCTGCTCTGTGGTG-3′ Reverse 5′-GTCCCCATGAACGTAGTCGGTAACC-3′ Tnf Forward 5′-CCTGTAGCCCACGTCGTAG-3′ Reverse 5′-GGGAGTAGACAAGGTACAACCC-3′

Hong et al. www.pnas.org/cgi/content/short/1315319110 6of6