Supporting Information

Gruhne et al. 10.1073/pnas.0810619106 SI Materials and Methods detected by immunofluorescence. Cells (3 ϫ 104)in100␮L PBS Cell Lines. Details of the cell lines used in this study are shown in were deposited on glass slides by cytospin centrifugation and fixed Table S3. All cell lines were maintained in RPMI 1640 supple- in fresh 4% formaldehyde (Merck), pH 8, for 10 min. The slides mented with penicillin (100 U/mL), streptomycin (0.1 mg/mL), were then stained overnight at 4°C with anti-pH2AX (Upstate) or FCS (10%), and glutamine (2 mM) (all from Sigma–Aldrich) with anti-ATM (Novus Biologicals) and developed with goat anti- (complete medium). The stable EBNA-1 transfected BJAB-E1 rabbit or goat antimouse IgG Alexa Fluor 488 (Molecular Probes, cell line was kept in complete medium supplemented with 2 Invitrogen). Digital images were captured using a LEITZ-BMRB mg/mL Geneticin (Gibco). A BJAB subline that stably expresses fluorescence microscope (Leica) equipped with a CCD camera a tetracycline-regulated EBNA-1 (BJAB-tTAE1) was pro- (Hamamatsu Photonics) and analyzed with Photoshop software duced by transfection of the pTRE2pur-FlagEBNA-1 plasmid (Adobe Systems). into the BJAB-tTA cell line that carries a tet-off–regulated transactivator (kind gift of Martin Rowe, University of Birming- Detection of Reactive Species. ham, United Kingdom) followed by selection in 1 mg/mL were detected by staining with DCFDA (Invitrogen), a cell- puromycin and 500 mg/mL hygromycin B (Calbiochem). The permeable indicator that becomes fluorescent when its acetate pTRE2pur-FlagEBNA-1 plasmid was produced by cloning the group is removed by intracellular oxidases. Cells (75 ϫ 104) were FlagEBNA-1 ORF excised as a HindIII-NotI fragment from labeled for 45 min with 2 ␮M DCFDA in PBS and then incubated pCDNA3-FlagEBNA-1 vector into the same sites of pTRE2 overnight at 4°C. Fluorescence was analyzed using a FACScali- shuttle vector. An EcoRV-NotI fragment was then cloned into bur cytometer (Becton-Dickinson) with excitation at 488 nm and the PvuII-NotI sites of pTRE2pur vector (Clontech) to produce emission at 533 nm and recorded in FL-1. Ten thousand events the puromycin-selectable pTRE2pur-FlagEBNA-1 plasmid. In- were scored for each sample. The values of fluorescence inten- ducible expression of EBNA-1 was determined by culture in the sity were converted to the molecule equivalent of fluorescence presence or absence of 2 ␮g/mL tetracycline (Sigma-Aldrich). (MEFL) using Shero rainbow calibration particles (BD Bio- EBNA-1 was detected by SDS-PAGE and Western blots using sciences). The results were expressed as MEFL. Where indicated the specific mouse monoclonal antibody OT1X (gift of Jaap the cells were pretreated overnight with 3.5 mM ebselen (Alexis Middeldorp, Vrije Universiteit, Amsterdam, The Netherlands). Biochemicals), a glutathione mimetic that scavenges TWO3, HeLa, and HEK293 cell lines were transfected with ROS, or with 1 mM citric acid (Sigma-Aldrich). pCDNA3-FlagEBNA1 using the jetPEI (Polyplus-transfection) reagent according to the manufacturer’s instructions. The EBV- Analysis of EBV-Regulated . Twenty-four microarray gene negative BL cell line DG75 was transfected using the nucleo- expression datasets from 18 EBV-negative and -carrying Burkitt’s fection protocol according to the manufacturer’s instructions lymphoma lines and EBV-transformed lymphoblastoid cells lines (program K25; Amaxa Biosystems). The expression of NOX2 was (Table S2) were extracted from the profiles of 336 examined 48 h later by Western blotting. human B cell representative of a wide selection of normal, transformed, and experimentally manipulated B cells Scoring of Chromosomal Abnormalities. Rapidly growing cells were (PubMed ID: 15778709; National Center for Biotechnology Infor- treated with 30 ng/mL colcemide (KaryoMAX; Invitrogen) for mation Gene Expression Omnibus: GSE2350). The Affymetrix 90 min to induce metaphase arrest, washed in hypotonic buffer GeneChip HG-U95Av2 array including Ϸ10,000 human genes was containing 75 mM KCl (Sigma-Aldrich), fixed, dropped onto used in this analysis. One hundred thirty-four genes involved in cold glass slides, and mounted in DAPI containing Vectashield ROS metabolism (Table S1) were identified according to their (Vector Laboratories). Digital images were captured using a annotation in the database. One hundred twenty- LEITZ-BMRB fluorescence microscope (Leica) equipped with one probe sets corresponding to 103 of the 134 genes were present a CCD camera (Hamamatsu Photonics) and analyzed using in the HG-U95Av2 array. Significance analysis of microarray Photoshop software (Adobe Systems). Fifty metaphases for each (SAM) was used to identify genes that are differentially regulated cell lines were examined for the presence of dicentric chromo- in EBV-positive compared with EBV-negative cells. SAM calcu- somes, fragments, rings, chromosome gaps, and lates a ␦ value for each probe set that represents the significance of double minutes. the observed differential gene expression. The differential regula- tion of 4 genes showing a cut-off ␦ value Ն2 was validated by Detection of DNA Double-Strand Breaks and DNA Damage Response. RT-PCR, and NOX2 expression was detected by Western Double-strand DNA breaks were detected by the comet assays as blots. Expression of regulated genes was investigated by RT-PCR. described by Blasiak et al. (1) with minor modifications. Briefly, 105 RNA was isolated from 4 ϫ 106 cells by lysis in 0.5 ml TRIzol cells were resuspended in 37°C warm 1% low-melting agarose and (Invitrogen), followed by incubation on ice for 5 min. After adding then dropped onto a slide coated with 1% agarose. The drops were 0.1 mL chloroform (Merck), cell debris were spun down for 5 min covered with a small coverslip and incubated at 4°C until solidified. at 14,200 ϫ g, and the water phase was mixed with 0.25 mL The slides were then incubated in lysis buffer (2.5 M NaCl, 100 mM isopropanol (Sigma-Aldrich) and spun down again for 10 min at EDTA, 10 mM Tris⅐HCl, 0.5% Triton-X-100, and 10% DMSO; pH 14,200 ϫ g. The pellet was washed with 70% ethanol and resus- 9.5)foratleast1hat4°Candthensoaked in electrophoresis buffer pended in ddH2O. To produce cDNA, 3 ␮g RNA was mixed with (300 mM sodium acetate and 100 mM Tris⅐HCl; pH 8.5). Electro- ddH2O and oligo-dT (0.05 mg/mL; Invitrogen), boiled for 10 min phoresis was carried out for 25 min at 25 V in a horizontal chamber at 70°C, and cooled on ice for 5 min, and then 5ϫ First Strand buffer kept at 4°C. Thereafter slides were washed 3 times in neutralization (250mM Tris-HCi pH 8.3, 375mM KCi, 15mM MgCl2), 200 nM buffer (0.4 M Tris, pH 7.5) and stained with DAPI (Vectashield; DTT, 1 mM dNTPs, 100 U MNLV-RT, and 10 U RNase (all from Vector Laboratories). Images were taken using a Nikon Eclipse Invitrogen) was added. The mixture was incubated for1hat37°C. TE300, and comet length was analyzed using Comet Score 1.5 Two microliters of DNA was mixed with the primers (GAPDH: software (Tritec). Activated ATM and phosphorylated H2AX were CAT CAC CAT CTT CCA GGA GC, GAG TCC TTC CAC GAT

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 1of13 ACC; NOX2: forward: ATC CAT GGA GCT GAA CGA TTG, Mission shRNA NOX2 transduction particles; Invitrogen) and reverse: CTC TGT CCA GTC CCC AAC GAT; GPX1: forward: nontarget shRNA (Mission nontarget shRNA control transduction GAA CGC CAA GAA CGA AGA GAT T, reverse: GCA TGA particles; Invitrogen) were used for transduction of 2.5 ϫ 105 AGT TGG GCT CGA A; ACOX1, forward: CTC ACT CGC AGC BJAB-E1 cells at 2.7, 2.9, and 9.6 transfection units per milliliter, CAG CGT TA, reverse: ATT GAG GCC CAC AGG TTC CCA), respectively, and NOX2 expression was assayed after 48 h by 10ϫ PCR RXA buffer, 50 nM MgCl, 1 mM dNTPs (all from Western blots. Invitrogen), and 1 U Taq Polymerase (New England BioLabs). The GAPDH PCR was run for 21 cycles at 55°C annealing temperature, Western Blotting. Total cell lysates were prepared in lithium do- NOX2 for 31 cycles at 65°C annealing temperature, GPX1 for 28 decyl (LDS) sample buffer, fractionated in precast 4%–12% cycles at 61°C annealing temperature, and ACOX1 for 29 cycles at SDS-PAGE gradient gels (Invitrogen), and transferred to PVDF 57°C annealing temperature. The products were resolved on a 1% membranes (Millipore). The blots were probed with anti-EBNA1 agarose gel for 15 min at 80 V. (OT1X, given by Y. Middeldorp, Vrije Universiteit, Amsterdam, The Netherlands), ␤-actin (AC-15, 1:2,000; Sigma-Aldrich), rabbit phox NOX2 Activity. A reporter plasmid was serum anti-Nox2 (1:200; White Label), rabbit serum anti p22 (FL-195; Santa Cruz Biotechnology), rabbit serum anti-p47phox constructed by cloning a PCR fragment corresponding to nucleo- phox tide Ϫ533 to ϩ6 of the human NOX2 gene (forward primer: (Millipore), anti-p67 (cl.29; BD Biosciences), and anti-Rac1 5Ј-GATGGTAGATCTTTTTCAGCACA CACACACAAG- (BD Biosciences), followed by the appropriate HRP-conjugated TATA-3Ј, reverse: 5Ј-GTGGCAGATCTTGAATGTGTTGTGT secondary antibody (Zymed), developed by enhanced chemilumi- T TGCCTTTCTTC-3Ј, BAC-RPCI-11, clone RP11–715D15 gene nescence. CYBB, from RZPD Deutsches Ressourcenzentrum fur Genomfor- Immunostaining. Cells (3 ϫ 104) in 0.1 mL PBS were deposited on schung) into the pGL3-Enhancer vector (Promega). HL60 cells glass slides by cytospin centrifugation and fixed in fresh 4% were transfected with 1 ␮g of each NOX2-Luc reporter plasmid formaldehyde (Merck), pH 8, for 10 min. The slides were then (Amaxa electroporator solution V and program T-19), either alone stained overnight at 4°C with anti-pH2AX (Upstate) or with or with increasing amounts of the EBNA-1-expressing plasmid anti-ATM (Novus Biologicals) and developed with goat antirab- pCDNA3-FlagEBNA-1. Empty pCDNA3 was used to equalize the ␮ bit or goat antimouse IgG Alexa Fluor 488 (Molecular Probes, amount of transfected DNA, and 0.1 g Renilla luciferase plasmid Invitrogen). Digital images were captured using a LEITZ- was cotransfected as a normalization control. Maximal and back- BMRB fluorescence microscope (Leica) equipped with a CCD ground activities were recorded in cells transfected with the pGL3- camera (Hamamatsu Photonics) and analyzed with Photoshop SV40 promoter plasmid and empty pGL3-enhancer plasmid, re- software (Adobe Systems). spectively. Cells were lysed with passive lysis buffer (Promega) 48 h after transfection, and luciferase activities were determined by Methyl Thiazolyl Tetrazolium Assay. Yellow methyl thiazolyl tetra- consecutive addition of Luciferase Assay Buffer and Stop and Glow zolium (MTT) is reduced to purple formazan in the mitochon- Buffer (both from Promega). Luminescence was detected with a dria of living cells. BJAB and BJAB-E1 cells (2 ϫ 105) were luminometer reader (Novostar; BMG LABTECH) and, after resuspended in color-free MEM medium (Gibco) and MTT normalization for Renilla activity, relative luciferase activity was buffer (Roche). After incubation for4hat37°C, the cells were calculated as (NOX2-Luc Ϫ pGL3)/(pGL3-SV40 Ϫ pGL3) ϫ 100. spun down 4 min at 14,200 ϫ g and resuspended in 0.1 M HCl The expression of EBNA-1 was confirmed in Western blots probed in isopropanol (Sigma-Aldrich) to dissolve the insoluble purple with the OT1X antibody. formazan into a colored solution. The suspension was incubated at room temperature with constant shaking for at least 1 h. The Inhibition of NOX2. For chemical inhibition of NADPH oxidase absorption at 570 nm was measured with a spectrophotometer activity the cells were treated overnight with 0.1 mM Apo, which and normalized to the absorption at 750 nm. specifically blocks the assembly of the NOX2 oxidase, or 40 ␮M DPI, which inhibits all NADPH oxidases by suppressing flavopro- Statistical Analysis. Statistical analysis was performed using Stu- teins (both from Sigma-Aldrich). Lentiviruses expressing NOX2- dent’s t test. All data are shown as mean Ϯ SD; probabilities of specific shRNAs (TRC0000064588, cl.1, TRC0000064590, cl.2; P Յ 0.05 were considered significant.

1. Blasiak J, Kowalik J, Malecka-Panas E, Drzewoski J, Wojewodzka M (2000) DNA damage 9. Pizzo PA, et al. (1981) Examination of Epstein-Barr virus and C-type proviral sequences and repair in human exposed to three anticancer platinum drugs. Teratog in American and African lymphomas and derivative cell lines. Cancer Res 41:3165–3171. Carcinog Mutagen 20:119–131. 10. Klein G, Dombos L, Gothoskar B (1972) Sensitivity of Epstein-Barr virus (EBV) producer 2. Klein G, Giovanella B, Westman A, Stehlin JS, Mumford D (1975) An EBV-genome- and non-producer human lymphoblastoid cell lines to superinfection with EB-virus. Int negative cell line established from an American Burkitt lymphoma; receptor charac- J Cancer 10:44–57. teristics. EBV infectibility and permanent conversion into EBV-positive sublines by in 11. Cohen JH, et al. (1987) B-cell maturation stages of Burkitt’s lymphoma cell lines vitro infection. Intervirology 5:319–334. according to Epstein-Barr virus status and type of chromosome translocation. J Natl 3. Benjamin D, et al. (1982) Immunoglobulin by cell lines derived from African Cancer Inst 78:235–242. and American undifferentiated lymphomas of Burkitt’s and non-Burkitt’s type. JIm- 12. Lombardi L, Newcomb EW, Dalla-Favera R (1987) Pathogenesis of Burkitt lymphoma: munol 129:1336–1342. Expression of an activated c-myc oncogene causes the tumorigenic conversion of 4. Menezes J, Leibold W, Klein G, Clements G (1975) Establishment and characterization EBV-infected human B lymphoblasts. Cell 49:161–170. of an Epstein-Barr virus (EBC)-negative lymphoblastoid B cell line (BJA-B) from an 13. Steinitz M, Klein G (1980) EBV-transformation of surface IgA-positive human lympho- exceptional, EBV-genome-negative African Burkitt’s lymphoma. Biomedicine 22:276– cytes. J Immunol 125:194–196. 284. 14. Lenoir GM, Vuillaume M, Bonnardel C (1985) The use of lymphomatous and lympho- 5. Rowe M, et al. (1995) Restoration of endogenous antigen processing in Burkitt’s blastoid cell lines in the study of Burkitt’s lymphoma. IARC Sci Publ (60):309–318. lymphoma cells by Epstein-Barr virus latent membrane protein-1: Coordinate up- 15. Basso K, et al. (2005) Reverse engineering of regulatory networks in human B cells. Nat regulation of peptide transporters and HLA-class I antigen expression. Eur J Immunol Genet 37:382–390. 25:1374–1384. 16. Gotch FM, et al. (1985) Characterization of the HLA-A2.2 subtype: T cell evidence for 6. Gregory CD, Rowe M, Rickinson AB (1990) Different Epstein-Barr virus-B cell interac- further heterogeneity. Immunogenetics 21:11–23. tions in phenotypically distinct clones of a Burkitt’s lymphoma cell line. G Gen Virol 17. Ben-Bassat H, et al. (1977) Establishment in continuous culture of a new type of 71(Pt 7):1481–1495. from a ‘‘Burkitt like’’ malignant lymphoma (line D.G.-75). Int J Cancer 7. Rowe M, et al. (1985) Distinctions between endemic and sporadic forms of Epstein-Barr 19:27–33. virus-positive Burkitt’s lymphoma. Int J Cancer 35:435–441. 18. Klein G, et al. (1976) Inducibility of the Epstein-Barr virus (EBV) cycle and surface marker 8. Epstein MA, Barr YM (1964) Cultivation in vitro of human lymphoblasts from Burkitt’s properties of EBV-negative lymphoma lines and their in vitro EBV-converted sublines. malignant lymphoma. Lancet 1:252–253. Int J Cancer 18:639–652.

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Gruhne et al. www.pnas.org/cgi/content/short/0810619106 3of13 Fig. S1. Expression of EBNA-1 induces a slowdown of cell proliferation that is reversed by treatment with antioxidants and inhibitors of the leukocyte NADPH oxidase. Slow growth rate in E1-positive cells due to high levels of ROS. Cells were seeded at a concentration of 0.2 ϫ 106/mL, and proliferation was monitored daily by counting the number of live cells by trypan blue dye exclusion. After 4 days, cells were reseeded at original cell density. (a) Growth curves of BJAB/BJAB-E1 and BJAB-tTAE1 ϩTet/ BJAB-tTAE1 –Tet. Mean cell count recorded in 4 independent experiments are shown. (b) Representative growth curves of BJAB-E1, BJAB-tTAE1 -Tet treated with 1 mM citric acid (CA), 3.5 ␮M ebselen (Ebs), or (c) 0.1 mM Apo. Three independent experiments were performed. Antioxidants did not affect the proliferation of EBNA-1 negative cells (not shown).

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 4of13 Fig. S2. The increase of ROS is independent of respiratory chain activity. Cells were treated with different concentrations of the C inhibitor natrium fluoride (NaF) for 4 h. (a) Respiratory chain activity was measured after NaF treatment using the MTT assay. Error bars represent SD from 3 independent experiments. (b) Relative ROS levels after NaF treatment were measured by FACS sorting. Error bars express SEM of 2 independent experiments.

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 5of13 Fig. S3. (a) SAM plot analysis illustrating the differential expression of 4 of 124 genes involved in ROS metabolism in EBV-positive compared with EBV-negative B cell lines. Three genes were upregulated (red dots), and 1 gene was downregulated (green dot) in EBV-positive cells. (b) Normalized average expression levels in EBV-positive and EBV-negative cells and fold change of the 4 regulated genes. (c) NOX2 upregulation is induced in EBV-carrying cells independent of their latency type. The normalized expression levels of NOX2 were plotted against the reported patterns of EBV latent gene expression. Only EBNA-1 is detected in cells expressing latency I; latency II cells express EBNA-1 together with LMP1 and LMP2, and latency III cells express EBNA-1–6 and the LMPs.

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 6of13 Fig. S4. Schematic representation of the NOX2 promoter fragment cloned in the NOX2-Luc reporter. The known regulatory sequences are indicated, and the transcription factors binding to these regions are shown in text with arrows pointing to the corresponding sequence. PU.1 is a leukocyte-specific transcription factor that binds to the hematopoietic-associated factor (HAF)-1 sequence region. PU.i is a key regulator of NOX2. IFN regulatory factors (IRF)-1, -2, and signal transducers and activators of transcription (STAT)-1 activate the NOX2 promoter through 2 IFN-stimulated response elements (ISRE) and a ␥-activated sequence (GAS) mediating IFN-␥ response. The GATA-1 and GATA-2 transcription factors regulate NOX2 expression through the GATA . The GLI Kru¨ ppel-related zinc finger transcription factor family member YY1 binds to the 5 sequence elements on NOX2 promoter denoted as binding increased during differentiation (BID). CCAAT box-binding protein CP1 and nuclear transcription factor (NF)-Y bind to the 2 CCAAT box elements present in the proximal promoter region; CCAAT displacement protein (CDP) binds to 5 sites in the promoter and functions as a NOX2 repressor. A region of homology to the EBNA-1 binding sites in the EBV Qp promoter and oriP Dyad symmetry is indicted. Strictly conserved bases are highlighted with white text on black background.

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 7of13 Fig. S5. EBNA-1 does not affect the production of ROS and the activity of the NOX2 promoter in epithelial cells. (a) Expression of EBNA-1 does not affect the endogenous levels of ROS in epithelial cells. ROS levels were detected by DCFDA staining NOX2 in EBNA-1–negative and -transfected sublines of the B lymphoma line DG75 and the epithelial cell lines HeLa, HEK293, and TWO3 that is derived from a nasopharyngeal carcinoma. DG75 was transiently transfected using the Amaxa electroporator. Cells were harvested after 48 h. (b) EBNA-1 does not upregulate NOX2 in epithelial cells. The NOX2 antibody detected a nonspecific band of Ϸ100 kDa in all epithelial cells. The intensity of this band was not affected by EBNA-1 expression. (c) The NOX2 promoter is inactive in epithelial cells and is not activated by EBNA-1. HEK293 cells were cotransfected with the NOX2-Luc reporter and increasing amounts of pCDNA3-FlagEBNA-1 plasmid. Relative luciferase activity was calculated as the ratio between that activity of NOX2-Luc and the maximal activity of an SV40-Luc reporter. All values were normalized to the activity of a cotransfected SV40-Renilla reported. Similar results were obtained with the HeLa and TWO3 cell lines (not shown)

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 8of13 Table S1. Genes involved in ROS metabolism identified by their Gene Ontology annotations Gene symbol Gene name Function in ROS metabolism

ABP1 Amiloride binding protein 1 (amine oxidase copper ROS production containing) ACOX1* Acyl- oxidase 1, palmitoyl ROS production ACOX2 Acyl-coenzyme A oxidase 2, branched chain ROS production ACOX3 Acyl-coenzyme A oxidase 3, pristanoyl ROS production ALOX12 Arachidonate 12- ROS production ALOX12B Arachidonate 12-lipoxygenase, 12R type ROS production ALOX12P2 Arachidonate 12-lipoxygenase pseudogene 2 ROS production ANGPTL7 Angiopoietin-like 7 Response to ROS AOC2 Amine oxidase, copper containing 2 (retina-specific) ROS production AOC3 Amine oxidase, copper containing 3 (vascular ROS production adhesion protein1) AOF2 Amine oxidase (flavin containing) domain 2 ROS production AOX1 Aldehyde oxidase 1 ROS production APOA4 Apolipoprotein A-IV Antioxidant APOE* Apolipoprotein E Antioxidant ATOX1 ATX1 antioxidant protein 1 homologue (yeast) Antioxidant BNIP3 BCL2/adenovirus E1B 19-kDa interacting protein 3 Response to ROS BNIP3L BCL2/adenovirus E1B 19-kDa interacting protein Response to ROS 3-like CAT Antioxidant CCL5* Chemokine (C-C motif) ligand 5 Response to ROS CCS Copper chaperone for superoxide dismutase Antioxidant CSDE1† Cold shock domain containing E1, RNA-binding Response to ROS CYBA Cytochrome b-245, ␣ polypeptide ROS production CYBB Cytochrome b-245, ␤ polypeptide (chronic ROS production granulomatous) CYGB† Cytoglobin Response to ROS CYP2C9 Cytochrome P450, family 2, subfamily C, ROS production polypeptide 9 DGKK† Diacylglycerol kinase, ␬ Response to ROS DHCR24 24-dehydrocholesterol reductase Antioxidant DUOX1† ROS production DUOX2† ROS production DUSP1 Dual specificity phosphatase 1 Response to ROS DUSP10* Dual specificity phosphatase 10 Response to ROS DUSP11 Dual specificity phosphatase 11 (RNA/RNP complex Response to ROS 1-interacting) DUSP14 Dual specificity phosphatase 14 Response to ROS EPHX1 Epoxide 1, microsomal (xenobiotic) Antioxidant EPHX2 Epoxide hydrolase 2, cytoplasmic Antioxidant EPX peroxidase Antioxidant FOXM1 Forkhead box M1 Response to ROS GCLC Glutamate- , catalytic subunit Antioxidant GCLM Glutamate-cysteine ligase, modifier subunit Antioxidant GLRX2† Glutaredoxin 2 Antioxidant GPR156† G protein-coupled receptor 156 Response to ROS GPX1 1 Antioxidant GPX2 Glutathione peroxidase 2 (gastrointestinal) Antioxidant GPX3 Glutathione peroxidase 3 (plasma) Antioxidant GPX4 Glutathione peroxidase 4 (phospholipid Antioxidant hydroperoxidase) GPX5† Glutathione peroxidase 5 (epididymal Antioxidant androgen-related protein) GPX6† Glutathione peroxidase 6 (olfactory) Antioxidant GPX7 Glutathione peroxidase 7 Antioxidant GSR Antioxidant GSS* Glutathione synthetase Antioxidant GSTZ1 Glutathione zeta 1 (maleylacetoacetate Antioxidant ) GTF2I* General transcription factor II, i Response to ROS HAO1 Hydroxyacid oxidase (glycolate oxidase) 1 ROS production KRT1† Keratin 1 (epidermolytic hyperkeratosis) Response to ROS

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 9of13 Gene symbol Gene name Function in ROS metabolism

LOX Lysyl oxidase ROS production LOXL1 Lysyl oxidase-like 1 ROS production LOXL2 Lysyl oxidase-like 2 ROS production LPO Antioxidant MAOA* Monoamine oxidase A ROS production MAOB Monoamine oxidase B ROS production MBL2* Mannose-binding lectin (protein C) 2, soluble Response to ROS (opsonic defect) ME3 Malic 3, NADP(ϩ)-dependent, ROS production mitochondrial MGST3 Microsomal glutathione S-transferase 3 Antioxidant MPO Antioxidant MPV17 Mpv17 mitochondrial inner membrane protein Antioxidant MSRA† sulfoxide reductase A Antioxidant MT3* Metallothionein 3 [growth inhibitory factor Antioxidant (neurotrophic)] MTL5† Metallothionein-like 5, testis-specific (tesmin) Antioxidant NCF1 cytosolic factor 1, (chronic ROS production granulomatous disease, autosomal 1) NCF2 Neutrophil cytosolic factor 2 (65 kDa, chronic ROS production granulomatous disease, autosomal 2) NDUFA12† NADH dehydrogenase ͓ubiquinone͔ 1 ␣ subcomplex ROS production subunit 12 NDUFA13† NADH dehydrogenase ͓ubiquinone͔ 1 ␣ subcomplex ROS production subunit 13 NDUFA6 NADH dehydrogenase ͓ubiquinone͔ 1 ␣ subcomplex ROS production subunit 6, 14kda NDUFB4† NADH dehydrogenase ͓ubiquinone͔ 1 ␤ subcomplex ROS production subunit 4 NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, Antioxidant 75kda (NADH-coenzyme Q reductase) NDUFS2 NADH dehydrogenase (ubiquinone) Fe-S protein 2, Antioxidant 49kda (NADH-coenzyme Q reductase) NDUFS3 NADH dehydrogenase (ubiquinone) Fe-S protein 3, Antioxidant 30kda (NADH-coenzyme Q reductase) NDUFS4 NADH dehydrogenase (ubiquinone) Fe-S protein 4, Antioxidant 18kda (NADH-coenzyme Q reductase) NDUFS8 NADH dehydrogenase (ubiquinone) Fe-S protein 8, Antioxidant 23kda (NADH-coenzyme Q reductase) NME5 Nucleoside diphosphate kinase homologue 5 Response to ROS NOS1 Nitric oxide synthase 1 (neuronal) ROS production NOS1AP Nitric oxide synthase 1 (neuronal) adaptor protein ROS production NOS2A* Nitric oxide synthase 2A (inducible, hepatocytes) ROS production NOS3* Nitric oxide synthase 3 (endothelial cell) ROS production NOX1* NADPH oxidase 1 ROS production NOX4† NADPH oxidase 4 ROS production NOX5† NADPH oxidase, EF-hand binding domain 5 ROS production NUDT1 Nudix (nucleoside diphosphate linked moiety Response to ROS X)-type motif 1 NUDT13 Nudix (nucleoside diphosphate linked moiety Response to ROS X)-type motif 13 OXR1† Oxidation resistance 1 Antioxidant OXSR1 Oxidative-stress responsive 1 Response to ROS PDLIM1 PDZ and LIM domain 1 (elfin) Response to ROS PIP3-E Phosphoinositide-binding protein PIP3-E Response to ROS PNKP† Polynucleotide kinase 3'-phosphatase Response to ROS POR P450 (cytochrome) Response to ROS PRDX1 1 Antioxidant PRDX2 Antioxidant PRDX3 Peroxiredoxin 3 Antioxidant PRDX4 Peroxiredoxin 4 Antioxidant PRDX5† Peroxiredoxin 5 Antioxidant PRDX6 Peroxiredoxin 6 Antioxidant

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 10 of 13 Gene symbol Gene name Function in ROS metabolism

PREX1† Phosphatidylinositol 3,4,5-trisphosphate-dependent ROS production RAC exchanger 1 PRG3† Proteoglycan 3 ROS production PRNP Prion protein (p27–30) (Creutzfeldt-Jakob disease, Response to ROS Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia) PRNPIP Prion protein interacting protein Response to ROS PTGS1 Prostaglandin-endoperoxide synthase 1 Antioxidant (prostaglandin G/H synthase and ) PTGS2 Prostaglandin-endoperoxide synthase 2 Antioxidant (prostaglandin G/H synthase and cyclooxygenase) PXDN Peroxidasin homologue (Drosophila) Antioxidant PXDNL† Peroxidasin homologue (Drosophila) Antioxidant RNF7† Ring finger protein 7 Antioxidant S100A7 S100 calcium binding protein A7 Response to ROS SCARA3† Scavenger receptor class A, member 3 Antioxidant SELS† S Antioxidant SEPP1 Selenoprotein P, plasma, 1 Antioxidant SFTPD Surfactant, pulmonary-associated protein D ROS production SGK2† Serum/glucocorticoid regulated kinase 2 ROS responsive SIRT2† Sirtuin (silent mating type information regulation 2 ROS responsive homologue) 2 SMOX* Spermine oxidase ROS production SOD1 Superoxide dismutase 1, soluble (amyotrophic Antioxidant lateral sclerosis 1) SOD2 Superoxide dismutase 2, mitochondrial Antioxidant SOD3 Superoxide dismutase 3, extracellular Antioxidant SPR Sepiapterin reductase (7,8-dihydrobiopterin:NADP Antioxidant ϩ oxidoreductase) SQLE Squalene epoxidase Antioxidant SRXN1† Sulfiredoxin-1 Antioxidant STK25* Serine/ kinase 25 (STE20 homologue, Response to ROS yeast) SUOX Sulfite oxidase ROS production TPO peroxidase Antioxidant TTN Titin Response to ROS TXNDC2† Thioredoxin domain containing 2 (spermatozoa) Antioxidant TXNRD1 1 Antioxidant TXNRD2 Thioredoxin reductase 2 Antioxidant VKORC1 Vitamin K epoxide reductase complex, subunit 1 Antioxidant WWOX* WW domain containing oxidoreductase Response to ROS

*More than 1 probe set is present in the array. †Specific probes are not present in the Affimetrix HG-U95Av2 GeneChip array.

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 11 of 13 Table S2. Cell lines included in the microarray analysis Cell line Origin EBV status Latency type Reference

Ramos* BL ϪϪ2 ST486 BL ϪϪ3 BJAB B cell lymphoma ϪϪ4 KEM type-I BL ϩ I5 Mutu type-I BL ϩ I6 ODH type-I BL ϩ I7 EB3 BL ϩ II 8 DW6 BL ϩ II/III 9 Namalwa BL ϩ III 10 KEM type-III BL ϩ III 5 Mutu type-III BL ϩ III 6 ODH type-III BL ϩ III 7 BL74 BL ϩ III 11 LCL-CB33 LCL ϩ III 12 LCL-Daikiki LCL ϩ III 13 LCL-IARC304 LCL ϩ III 14 LCL-NC6 LCL ϩ III 15 LCL-RD LCL ϩ III 16

*Data from 7 independent gene expression microarrays performed with this cell line were included in the analysis.

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 12 of 13 Table S3. Cell lines used in this study Name Origin EBV status EBV Reference

DG75 BL Negative — 6 BJAB BL Negative — 4 BJAB-B95–8 B cell lymphoma EBV converted EBNA1–6, LMP1,2 18 BJAB-E1 B cell lymphoma EBV converted EBNA1 19 Ramos BL Negative — 2 Ramos-B95–8 BL EBV converted EBNA1–6, LMP1,2 18 Ramos-P3HR1 BL EBV converted EBNA1, 3–6, LMP1–2 18 Namalwa BL Positive EBNA1–6, LMP1,2 10 Raji BL Positive EBNA1–6, LMP1,2 20 Mutu cl.148 BL Positive EBNA1 6 HL60 Promyelocytic leukemia Negative — 21 TWO3 Nasopharyngeal carcinoma Negative — 22 HeLa Cervix carcinoma Negative — 23 HEK 293 Transformed embryonal kidney Negative — 24

Gruhne et al. www.pnas.org/cgi/content/short/0810619106 13 of 13