Supporting Information
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Supporting Information Gruhne et al. 10.1073/pnas.0810619106 SI Materials and Methods detected by immunofluorescence. Cells (3 ϫ 104)in100L 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 gene (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 Oxygen Species. Reactive oxygen 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 peroxidase 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 Genes. 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 gene expression profiles of 336 examined 48 h later by Western blotting. human B cell phenotypes 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 Gene Ontology 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, chromosome 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 protein 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 sulfate (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 Promoter Activity. A firefly luciferase 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.