following: GLUTI (NM_006516) F-AAGGTGATCGAG- GAGTTCTACA, R-ATGCCCCCAACAGAAAAGATG; hexoki- nase-2 (HK-2) (NM_000189), F-GAGCCACCACTCACCC- TACT, R-ACCCAAAGCACACGGAAGTT; PFKL (X16930) F-CTGGGA GAACTTCATGTGTGAG, R-TAGCTGGAC- GAGATGGGCT; LDHA1 (NM_005566) F- CTCCAAGCTGGTCATTATCACG, R-AGTTCGGGCTG- TATTTTACAACA; PDK1 (NM_002610) F- TCCTGGACTTCGGATCAGTGA, R-CGGATGGTGTCCT- GAGA AGATT; PGK1 (NM_000291) F-TTAAAGG- GAAGCGGGTCGTTA, R-TCCATTGTCC AAGCA- GAATTTGA; MCT4 (NM_004207) F- TGTGTGCGTGAACCGCTTT, R-AAAC CCAACCCCGT- GATGAC; PKLR (D13243) F-CTTCGGTCATGGGTCTC- TAAGT; GAPDH (NM_002046) F-ATGGGGAAGGT- GAAGGTCG, R-GGGGTCATTGATGGC AACAATA. Primers for the HIF1A gene were F-ATCCATGTGACCAT- GAGGAAATG and R-CTCGGCTAGTTAGGGTACACTT. As an internal control for standardization, a gene encoding TATA-binding protein (TBP, NM_003194) was used with the following primers: F-TTTCTTGCCAGTCTGG AC, R-CAC- GAACCACGGCACTGATT. The PCR cycling conditions were the following: 10 min at 95uC, 40 cycles of 10 s at 95uC and 1 min at 60uC. Applied Biosystems 7900 systems software was used for analysis. Ct values were determined for the internal control (TBP) and for the test genes at the same threshold level in the exponential phase of the PCR curves. Relative quantification (comparative Ct (DDCt) method) was used to compare the expression level of the test genes with the internal control. Dissociation curve analysis was performed after every run to check the specificity of the reaction. Three or four reactions (each in triplicate) were run for each gene and the standard deviation was calculated. Biochemical assays Activated B-cells, EBV-infected B-cells and LCLs were assayed for L-lactate, pyruvate and lactate dehydrogenase. Peripheral blood B-cells (56106) were activated with anti-CD40 and IL4, or infected with EBV and grown for 5 days. LCLs (56106) were also grown for 5 days. Colorimetric assays were as described by Bioassay Systems (Hayward, CA) for medium lactate (L-Lactate Assay Kit, ECLC-100), pyruvate (Pyruvate Assay Kit, EPYR-100) and lactate dehydrogenase (Lactate Dehydrogenase Kit, DLDH- 100). To measure lactate concentration, the cell culture medium was collected. For pyruvate and lactate dehydrogenase assays, 36106 cells were sonicated in 300 ml of 100 mM potassium phosphate/2 mM EDTA (pH 7.0) and the absorbance of stan- dards and unknowns was measured on a microplate reader at 565 nm. Results HIF1A protein is stabilized in LCLs HIF1A levels in lymphoblastoid cells were determined by Western blotting of whole cell lysates for HIF1A and its hydroxylated form (HIF1A-OH). Because HIF1A is usually hydroxylated and degraded very fast under normal conditions, HIF1A was also assayed after blocking proteasomal degradation with MG132. A strong HIF1A signal was obtained with LCL lysates, prepared with or without MG132 (Figure 1A, left panel). This suggests that HIF1A protein is stabilized in LCLs. To answer the question whether EBV plays a role in HIF1A stabilization, HIF1A levels were measured in freshly infected B-cells and in CD40+IL4-activated B-cells by Western blotting. Cells were EBV-Encoded Proteins Stabilize HIF1 Alpha collected after 5 days of activation/infection, and the levels of HIF1A and prolylhydroxylases 1 and 2 were compared in NiCl2- treated and -untreated cells (Figure 1B). Ni+2 and Co2+ ions can replace iron cations in the active catalytic center of HIF1A prolylhydroxylases, and thereby block their activity [14]. This leads to HIF1A protein stabilization, because only the hydroxyl- ated form is recognized by VHL, the E3 ubiquitin ligase for HIF1A [15]. Importantly, the levels of HIF1A and prolylhydroxylases 1 and 2 in LCLs did not change under conditions mimicking hypoxia, indicating that HIF1A is stabilized in LCLs. Q-PCR was performed to check whether the high HIF1A levels were simply due to enhanced transcription. This was not the case because HIF1A mRNA expression was similar in primary peripheral blood B-cells, mitogen-activated B-cells and B-cells freshly infected with EBV (Figure 1C). Together with the absence of hydroxylated HIF1A, our findings indicate that the HIF1A protein is stabilized in lymphoblastoid cells. EBNA-3 forms a complex with HIF1A. To test whether the EBV-encoded transforming proteins, EBNA-3, -5, and -6, are involved in stabilization of HIF1A, a GST pulldown assay was performed, using glutathione S-transferase (GST) alone and GST fusion proteins with HIF1A or ARNT immobilized on the surface. Proteins from whole lymphoblastoid cell lysates were captured by the support and then analyzed by Western blotting. The membranes were probed with mouse anti-EBNA-3, anti-EBNA- 6 and anti-EBNA-5. This showed that EBNA-3 was bound specifically by GST-HIF1A, whereas EBNA-5 and EBNA-6 did not bind to GST-HIF1A or GST-ARNT (Figure 2A). Immunoprecipitates were prepared from LCL lysates using either anti-EBNA-3 or anti-EBNA-5 coupled to CNBr Sepharose beads and normal mouse serum (NMS) as a control for the lysates. Western blotting analysis showed coprecipitation of HIF1A and EBNA-3 but not with EBNA-5 in this preparation (Figure 2B). HIF1A colocalizes with EBNA-3 and EBNA-5 in the nucleus. To monitor the cellular distribution of HIF1A in the presence of EBNA-3 and EBNA-5, cells of the MCF7 breast carcinoma line were transfected with plasmids encoding GFP- HIF1A, RedDs-EBNA-5 and FLAG-tagged EBNA-3. After fixation and rehydration with PBS, GFP-HIF1A+RedDs-EBNA- 5-transfected cells were stained with rabbit sera against PHD1 and PHD2, followed by goat anti-rabbit biotinylated antibody and AMKA streptavidin. Cells expressing GFP-HIF1A and RedDs- EBNA-5 showed a high degree of nuclear colocalization of these proteins (Figure 2C). In the doubly transfected cells, PHD1 (white), which is normally found in the cytoplasm, was located in the nucleus colocalized with both HIF1A and EBNA-5. This suggests that EBNA-5 may form a complex with PHD1 and so impair its enzymatic activity. Immunostaining showed much more limited colocalization of PHD2 with HIF1A and EBNA-5 in the nucleus. MCF7 cells were also cotransfected with GFP-HIF1A and FLAG-EBNA-3 plasmids and stained for PHD1 and PHD2 (in blue, Figure 2D). Subsequently, the cells were stained with mouse monoclonal anti-FLAG and horse anti-mouse Texas Red conju- gated antibodies (red signal) to visualize FLAG-tagged EBNA-3. The staining pattern for PHD1 and PHD2 was similar to that described above in MCF7 cells transfected with EBNA-5 and HIF1A. The normally cytoplasmic PHD2 (blue signal) was found in the nucleus, where it colocalized with GFP-HIF1A (green signal) and EBNA-3 (in white; Figure 2D). PHD1 was also colocalized with GFP-HIF1A in the nucleus, but to a lesser extent than PHD2. PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e42072