single reporter assays in an ABI PRISM 7700 sequence detection system (PE Biosystems, Foster city, CA, USA). RT2 Profiler Apoptosis PCR Array (PAMM-012Z, QIAGEN) was performed according to the manufacture’s instructions. cDNAs was prepared from A20 cells infected with empty or lxn expression vectors with RT2 First Strand cDNA Kit (C-03, QIAGEN) and added to RT2 qPCR master mix (PA-012). The mixture was aliquoted across the PCR array which contains 84 apoptosis-related genes. Gene expression and quantification was performed in an ABI PRISM 7700 sequence detection system, and data was analyzed with using web-based software (http:// pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The genes with more than 2-fold change in expression and showing statistically significant (p,0.05) were chosen for candidate gene. Western Blots Cell samples were lysed at a concentration of 26107 cells/ml in a protein lysis buffer containing: 10 mM Tris pH 7.5, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 mM ZnCl2 and 1% Triton X-100, 2.8 ug/ml aprotinin (Sigma-Aldrich; St. Louis, MO), 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM sodium vanadate (Na3VO4) 1 ug/ml pepstatin, and 1 mg/ml leupeptin (Oncogene Research, MA, USA). Lysate was incubated on ice for 30 min, and then centrifuged at 15,0006g for 10 minutes to remove debris. The resulting supernatant was then aliquoted and stored at 280uC. For Western blot, protein lysates were thawed and mixed with running buffer and a reducing agent (Novex, San Diego, CA, USA, per manufacturer’s instructions) and heated at 95uC for 5 minutes. Samples were then analyzed by denaturing PAGE (Novex, 10% bis-Tris gel) using the equivalent of 46105 cells per lane. Following electrophoresis, samples were electro-transferred onto immunobi- lon-P membranes (Millipore, Bedford, MA, USA), which were subsequently blocked and probed with polyclonal rabbit anti-LXN Ig-G antibody at a 1:3000 dilution. This antibody was generated from the Lxn-specific amino acid sequence CKHNSRLPKEG- QAE at the carboxyl terminus, and was produced by Bethyl Laboratories, Inc. (Montgomery, TX). The antibody for detection of human LXN was purchased from Abcam Inc. (Cambridge, MA) and used at 1:2000 dilution. The antibodies for apoptotic proteins, Bcl-2, Bcl-xl, Bax, Bad were provide by Dr. Bondada. The antibody for Pim-2 was purchased from eBioscience (San Diego, CA). Primary antibodies were detected using alkaline phosphatase-conjugated secondary antibodies (Santa Cruz Bio- technology) and electro-chemifluorescent (ECF) reagent (Pharma- cia Biotech) according to the manufacturer’s instructions. Blots were visualized using a Molecular Dynamics STORM 860 system and Imagequant Software. Following the detection and quantifi- cation of anti-LXN antibody, immunobilon-P membrane was sequentially stripped in 40% methanol and the buffer containing 100 mm ß-mercaptoethanol, 2% sodium dodecyl sulfate and 62.4 mM Tris-HCl to remove ECF reaction product and antibodies, respectively. The stripped membrane was re-probed with anti-actin antibody (Sigma) at 1:500,000 dilution and detected as described previously. Genomic Bisulfite Sequencing To investigate the methylation pattern of Lxn promoter, CpG island analysis in the upstream sequence of Lxn open reading frame. Nucleotide sequence of Lxn in upstream region (–1000 bp) and the first 3 exons (+373 bp) was obtained from Ensembl database (www.ensembl.org) with ID number ENSG00000079257. CpG island search using CpG island searcher website (http://www.uscnorris.com/cpgislands2/cpg. Latexin: A Potential Tumor Suppressor aspx) showed a 252 bp region (–208 bp to +44 bp) in upstream of Lxn sequence enriched for CpG repeats. The criteria of 5 CpG island is: GC content .50%, ratio of CpG to GpC .0.6 and 200 bp of minimum length. Genomic DNAs were isolated using AquaPure Genomic DNA kit (Bio-Rad, Hercules, CA) and modified by sodium bisulfite using EpiTectH Bisulfite kit (QIA- GEN, Valencia, CA). For the Lxn promoter methylation study, we designed primers that could amplify a 423 bp fragment in the upstream region of Lxn containing CpG island. The forward primer sequence is 59 GTTGGTGTTTGATAAGTATGTGG 39, and the reverse primer sequence is 59 TTTAACCTTCTA- CACCTCAAACAC 39. The annealing temperatures for primers were 52uC for 2 minutes. Hot-start PCR with a total cycle number of 30 was used in all PCR amplifications. Denaturation and extension cycles were maintained for 95uC, 30 seconds and 72uC, 1 minutes respectively. The amplified fragments were cloned into the pCR2.1-TOPO vector using TOPO TA Cloning Kit (Invitrogen Carlsbad, CA) and sequenced (MWG Technology) (n$ 3 clones for cell line and n$ 8 clones for primary cells). 5-aza-29-deoxycytidine Treatment To examine the correlation of promoter hypermethylation and Lxn gene expression, the leukemia cell line, shown to have a lack or decrease of Lxn expression, was subject to 5-aza-29-deoxycytidine treatment. Cells were plated with 2 uM 5-aza-29-deoxycytidine (Sigma-Aldrich; St. Louis, MO) and incubated for 4 days. The medium and the drug were replaced every 24 hours (hrs.) and cells were harvested for RNA and DNA extraction 4 days after treatment. Infection of WEHI231 and A20 Cells with Lxn Expression Vector Cloning of the mouse Lxn gene into Sfbeta 91 retroviral vector and production of viral supernatant were performed exactly as described previously [4]. WEHI-231 and A20 cells were infected by 10 ml viral supernatant at a density of 16106 cells per 10 cm plate along with 4 mg/ml of polybrene for 48 hours. The infected cells (GFP+ cells) were sorted and expanded in culture medium. The expanded GFP+ population, if not used immediately, was cryopreserved at a concentration of 16107 cells/mL in freezing medium consisting of 80% fetal bovine serum, and 20% dimethyl sulfoxide (DMSO). Measurement of Growth of Retrovirally-transduced Tumor Cells Sorted GFP+ A20 cells over expressing Lxn or Sfbeta 91 empty vector were counted on a hemacytometer using trypan blue dye exclusion and 500,000 cells were seeded into 25 cm2 tissue culture flask in 4 mls media. Cells were incubated in a humidified atmosphere of 5% CO2 in air at 37uC and subsequently counted on days 3, 8, 12, 16 and 20. At each time point, cells were split and maintained at a concentration of 500,000 cells per 4 ml media. The cumulative cell number was calculated from the cell counts and the dilutions made at each culture split. FACS analysis was also performed at each time-point to measure the percentage of GFP+ cells. For the in vivo measurement of tumor cell growth, various numbers (5,000; 25,000; 50,000 and 100,000) of GFP+ A20 cells over expressing Lxn or Sfbeta 91 empty vector were injected in a 50 ml bolus subcutaneously in the shaved flank of BALB/cJ mice given 3.0 Gy of gamma radiation 4 hrs. prior. Lymphomas were detectable by palpation 10–12 days post- injection and all three dimensions of tumors were measured blind with calipers on days 12, 14, 16, 19, and 21. The same individual PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44979