All B-cell lines, LNCAP and PC3 cell lines were propagated in RPMI supplemented with 10% fetal bovine serum albumin, penicillin, streptomycin, and glutamine. Hela, 293, MDA-MB-231, MDA-MD-361, CaoV3, and BG1 cells were propagated in Dulbecco’s Modified Eagles Medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum albumin, penicillin, streptomycin, and glutamine. Cells were cultured at 37°C in a humidified 5% CO2-containing atmosphere.
Transient transfection experiments were performed by using a modified version of the calcium phosphate precipitation procedure (a detailed protocol is available at flemingtonlab.com). Briefly, 106 cells were plated onto 100-mm-diameter tissue culture dishes. The following day, the medium was replaced with 8 ml of fresh supplemented DMEM; 4 h later, DNA precipitates were generated by mixing 0.5 ml of 1x HEPES-buffered saline (0.5% HEPES, 0.8% NaCl, 0.1% dextrose, 0.01% anhydrous Na2HPO4, 0.37% KCl [pH 7.10]) with a total of 30 μg of plasmid DNA (10 μg retroviral vector plus 10 μg VSV-G expression vector, plus 10 μg pVPACK dGI packaging vector). A total of 30 μl of 2.5 M CaCl2 was added, and samples were mixed immediately. Precipitates were allowed to form at room temperature for 20 min before being added drop wise to cells. Cells were incubated at 37°C with 5% CO2 for 16 h before the medium was replaced with 10 ml of fresh DMEM (plus 10% FBS).
24 hrs later, viral supernatants were collected and subjected to two rounds of centrifugation to get rid of floating 293 cells. Infections were carried out in 6 well plates with 1 ml virus plus 106 MutuI or DG75 cells suspended in 1 ml DME (+10% fetal bovine serum). Polybrene was added to a final concentration of 8 ug/ml and the mixture was mixed by gently rocking. Cells were spun in 6 well plates at 1000 g for 1 hr followed by 4 hr incubation in a tissue culture incubator. Cells were then collected, spun down and resuspended in 2 ml RPMI (+10% fetal bovine serum, + penicillin, streptomycin, and glutamine) per well. Cells were cultured for 2 days prior to antibiotic selection.
Western blot analysis
After a single 1× PBS wash, cells were immediately suspended in 300 μl of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis loading buffer (125 mM Tris [pH 6.80], 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromphenol blue) and boiled for 20 min to shear the genomic DNA. Whole-cell extracts were measured with the Bio-Rad protein assay kit according to the manufacturer's instructions. An equal weight of cell lysates was subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blots were blocked for 30 min in Tris-buffered saline containing 5% low-fat powdered milk and 1% FBS and then incubated with the primary antibody (in blocking buffer) overnight at 4°C. The blots were washed three times with 1× TBST (140 mM NaCl, 3 mM KCl, 25 mM Tris–HCl [pH 7.4], 0.1% Tween 20) (each wash was carried out for approximately 10 min). The blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad) in blocking buffer for 1 h at room temperature. Blots were washed as described above and analyzed with an enhanced chemiluminescence detection system (Perkin-Elmer) according to the manufacturer's recommendations, and filters were exposed to Fuji Super RX film. The following antibodies were used for Western blot analysis: EBNA1 (Advanced Biotechonologies Incorporated, CAT# 13-156-100, 1:1000 dilution), HA.11 (Boehringer Mannheim, Cat# 1 867 431, 1:1000 dilution), Actin (Sigma, Cat# A 4700, 1:1000 dilution).
Real time RT-PCR
RNAs from cells were prepared using a RNeasy® Mini kit from Qiagen (Cat# 74104) according to vendor’s protocol. Normal tissue RNAs were purchased from BD Biosciences (Cat# 636643). Individual RNAs from primary lung (Cat#64013-1), colon (Cat#64014-1), and breast (Cat#64015-1) tumors were purchases from BD Biosciences. qPCR human matched pair total RNA panel was purchased from BD Biosciences (Cat# 636691).
cDNA synthesis was carried out using Superscript™ III first-strand synthesis system for RT-PCR (Invitrogen, Cat#. 18080–051). 2 ug of RNA were used for first strand synthesis and then diluted to a final volume of 200 ul.
Real Time PCR was carried out using 5 ul of diluted cDNA for each 20 ul reaction. Analysis was carried out on a BioRad iCycler using BioRad iQ™ SYBR Green supermix (CAT#170-8882) with the following conditions: 95°C 3' then 40 cycles of 95°C 30", 60°C 30", and 72C 30”. Primer sequences for real time PCR were the following: PLAC1; PLAC1-PS1-sense, 5' - TTCACCAGTGAGCACAAAGC - 3' and PLAC1-PS1-antisense, 5' - CCAGTCTATGGAGCACAGCA - 3', PLAC1-PS2-sense 5' -GGGCAAATACAGACACAGCA -3' and PLAC1-PS2-antisense 5' -CCAGTCTATGGAGCACAGCA -3', GAPDH; GAPDH-sense, 5' - GCCAAGGTCATCCATGACAACTTTGG - 3' and GAPDH-antisense, 5' - GCCTGCTTCACCACCTTCTTGATGTC - 3', mATPsy6 ; mATGsy6-sense, 5’-GGGCGCAGTGATTATAGGCTT-3’ and mATPsy6-antisense, 5’-GGTGTAGGTGTGCCTTGTGGT-3’, RNASE T2; RNASE T2-sense, 5' -TGGGGATAAAACCATCCATC -3' and RNASE T2-antisense, 5'-AGCTGCTGGTCTTGCTTAGT-3'.
For analysis of EBV DNA in Mutu Clones, genomic DNA was isolated as describe at flemingtonlab.com. PCR analysis was carried out essentially as described above for RT-PCR analysis except that the following conditions were used: 95°C 3' then 40 cycles of 95°C 30", 55°C 30", and 72C 30”. The following primers were used to amplify different parts of the EBV genome: BamHI R primers (Rp); R-left, 5'-TAGTTAATGCCCCAGCCAGA-3', R-right, 5'-CTTTAAAAAGGCCGGCTGAC-3', BamHI M primers (BMRF1p) ; M-right, 5'-ACCTACATGACTAGCATCAAGCAA-3', and M-left, 5'-GGCCTCCATAGTTTACAGACAGAA-3', BamHI Q primers (Qp); Q-left, 5'-AAATTGGGTGACCACTGAGG-3' and Q-right, 5'-CATACACCGTGCGAAAAGAA-3', BamHI K primers (EBNA1); K-left, 5'-AAGGAGGGTGGTTTGGAAAG-3' and K-right, 5'-TGGAATAGCAAGGCCAATTCC-3'.
To generate dominant negative forms of EBNA1, full length EBNA1 was first excised from the plasmid, pCEP4, with ClaI and SacII. Since the SacII digestion cleaves the last few carboxy-terminal amino acids from the EBNA1 reading frame, two adaptors were synthesized which together encode the terminal amino acids from EBNA1 plus a BglII overhang (Adaptor 1-sense; 5’-GGAGGGTGATGACGGAGATGACGGAGATGAA-3’, Adaptor 1-antisense; 5’-CATCACCTCCTTCATCTCCGTCATCTCCGTCATCACCCTCCGC-3’, Adaptor 2-sense; 5’-GGAGGTGATGGAGATGAGGGTGAGGAAGGGCAGGAGTGA-3’, Adaptor 2-antisense; 5’-GATCTCACTCCTGCCCTTCCTCACCCTCATCTC-3’). The ClaI/SacII EBNA1 fragment was ligated to EcoRI and BglII cut pMSCV-neo vector in the presence of Adaptor 1 plus Adaptor 2, plus an EcoRI/ClaI adaptor (from eZclone Systems). Ligations were carried out in the presence of 1 ul T4 polynucleotide Kinase to phosphorylate adaptors during ligation reaction. This construction led to the generation of pMSCV-neo-EBNA1wt. pMSCV-neo-E1dn was generated from this plasmid by digesting pMSCV-neo-EBNA1wt with EcoRI and ApaI to excise the amino terminal EBNA1 sequences. The resulting vector plus carboxyl terminal EBNA1 sequences were then ligated to three adaptors which when linked together contain a translation initiation sequence, an HA epitope tag, the SV40 nuclear localization signal plus an EcoRI overhang at the 5’ end and an ApaI overhang at the 3’ end (DN adaptor 1-sense; 5’-AATTCTGCTGAAGATGATGGCCTATCCTTATG-3’, DN adaptor 1-antisense; 5’-CATCATCTTCAGCAG-3’, DN adaptor 2-sense; 5’-ATGTGCCTGACTATGCCGCCCCAAAGAAA-3’, DN adaptor 2-antisense; 5’-CATAGTCAGGCACATCATAAGGATAGGC-3’ , DN adaptor 3-sense; 5’-AAGCGAAAGGTGGCCGGCC-‘3, DN adaptor 3-antisense; 5’-GGCCACCTTTCGCTTTTTCTTTGGGGCGG-3’). Ligations were carried out in a standard ligation reaction except that 1 ul of T4 polynucleotide kinase was added to phosphorylate adaptors during ligation reaction. This strategy generated pMSCV-neo-EBNA1dn. pMSCV-puro-EBNA1dn was derived from this plasmid by simply excising the HA/nls/EBNA1dn cassette from pMSCV-neo-EBNA1dn and transferring to pMSCV-puro using appropriate standard cloning adaptors. All constructs were verified by sequence analysis.