shape and ppm position. VNMRJ software by Varian (Cary, NC) was used to acquire and analyze spectra. All samples were tuned and shimmed before data acquisition. For enzymatic measure- ments, confluent HUVECs were incubated with 10 mM L-lactate, washed twice with PBS containing 10 mM D-Lactate (to saturate MCTs), and lysed in 200 ml ice-cold RIPA Buffer (50 mM Tris HCL pH 7.4, 150 mM NaCl, 1% Triton-x 100, 0.05% sodium deoxycolate, 0.1% SDS,1 mM EDTA, 1 mM Na3VO4 and protease inhibitors). Samples were filtered through centrifugation columns with a 10 KDa cutoff. L-Lactate and pyruvate concentra- tions were quantified in clear cell lysates using specific enzymatic assays on an ISCUSflex analyzer (CMA Microdialysis AB). Oximetry Determination of cell oxygen consumption rates was carried out using electron paramagnetic resonance (EPR) in DMEM contain- ing 4500 mg/liter glucose, dextran 10% [57]. Briefly, 26107 viable cells/ml were sealed in glass capillary tubes in the presence of 0.08 mM of the O2 sensor 4-oxo-2,2,6,6-tetramethylpiperidine- d-15N-1-oxyl (CDN isotopes) and with 10 mM lactate where indicated. Cells were maintained at 37uC during recording on a Bruker EMX EPR spectrometer operating at 9 GHz. Dual luciferase reporter assays Dual luciferase reporter assays were performed with the dual luciferase kit (DLR) from Promega using pGL3-(PGK-HRE6)- TK-Luc as reporter of HIF-1 activity [10] and pODD-Luc as reporter of PHD activity [30]. Cells were transfected using electroporation (Amaxa Nucleofector, Lonza). Western blotting, immunohistochemistry and antibodies We used previously disclosed protocols for immunocytochemistry and immunohistochemistry [58] except that HUVECs were permeabilized with 0.05% saponin for MCT1 detection. Western blotting (WB) was done as previously shown [59] with the slight modification that proteins were not heat-denaturated for HIF-1a protein detection. This allowed the detection, in whole cell lysates, of basal HIF-1a that would otherwise not be detected [60]. For Figure S2, nuclear extraction was performed according to manufacturer’s recommendations using the nuclear extraction kit from Active Motif. Primary antibodies were: a mouse monoclonal against HIF-1a (BD), a rabbit polyclonal raised against an intracellular epitope of MCT1 (AB3538P, Chemicon), a rabbit polyclonal against caveolin-1 (BD), a rabbit polyclonal against laminin A/C (Santa Cruz), a goat polyclonal raised against an epitope comprising an an extracellular domain of VEGFR2 (R&D) for immunohistochemistry, a mouse monoclonal against VEGFR2 (Santa Cruz) for WB, a mouse monoclonal against CD147 (BD), a mouse monoclonal against b- actin (Sigma), a mouse monoclonal against CD31 (Dako) for Figure S3, and a rat monoclonal against CD31 (BD) for Figure 8. For immunohistochemistry, areas of positive staining were quantified using the Framework for Image Dataset Analysis (FRIDA) software developed by Johns Hopkins University. ELISA VEGF-A and bFGF protein levels were measured in the supernatant of confluent HUVECs using the Human VEGF Quantikine ELISA Kit (R&D) and the Human bFGF Quantikine ELISA kit (R&D) according to manufacturer’s instructions. PCR and RNA interference The effects of a 24-h exposure of HUVECs to lactate and saline were compared using the Human Angiogenesis RT2 Profiler PCR Targeting MCT1 Blocks Tumor Angiogenesis Array from SABiosciences, according to manufacturer’s instruc- tions. In each condition, total mRNA extracted from 3 independent dishes was pooled. To silence MCT1 and LDH-B mRNAs, HUVECs were transfected with specific siRNAs as reported previously [61]. siRNAs (Qiagen) targeted the following sequences: hMCT1 AAGAGGCUGACUUUUCCAAAU, hLDHB AAGA- UUGUAGUGGUAACUGCA. Allstar siRNA (Qiagen) was used as negative control. MCT1 shRNA was purchased from Open Biosystems (clone TRCN0000038340); scrambled control shRNA was Addgene plasmid 1864. The following primers were used for SYBR green quantitative PCR [61]: hMCT1 sense, 59-GTGG- CTCAGCTCCGTATTGT-39, antisense, 59-GAGCCGACCTA- AAAGTGGTG-39; hMCT2 sense, 59-CAACACCATTCCAAG- ACAGC-39, antisense 59-TGGCTGTTATGTACGCAGGA-39; hMCT3 sense, 59- GGATGTGTT-GAAGAACTATGAGATC- 39, antisense 59-CCGGGTTCCTCTGCAACA-39; hMCT4 sense, 59-CAGTTCGAGGTGCTCATGG-39, antisense, 59- ATGTA- GACGTGGGTCGCAT-39. Housekeeping human ribosomal pro- tein L19 (hRPL19) primers were sense, 59-CAAGCGGATTCT- CATGGAACA-39, antisense, 59-TGGTCAGCCAGGAGCTT- CTT-39. Endothelial cell migration assays To image EC migration, we used an assay analogous to a scratch injury test [62]. BAECs were cultured to confluence on gelatin- coated circular coverslips, which were then transferred to adhere onto 100 mm gelatin-coated dishes containing EBM-EGM without serum and growth factors, and supplemented with lactate (10 mM) or not. Images of migrating cells were captured at the migration front around the coverslip margin. For quantification, BAECs were prestained with calcein and 50,000 cells were seeded in the upper chamber of the Matrigel-coated insert plate of a Biocoat Tumor Invasion System (BD). Bottom wells contained EGM-MV without serum and growth factors, supplemented with lactate (10 mM), VEGF (10 ng/ml, Sanvertech), CHC (5 mM) and/or saline. Twenty-four hours later, cells on the underside of the Fluoroblock membranes were quantified by measuring fluorescence at 535 nm after an excitation at 485 nm. Detached membranes were also fixed in 4% paraformaldehyde for 10 minutes and processed for immunohistochemistry using anti-caveolin-1 antibodies. Endothelial tube formation and aortic ring angiogenesis assays Capillary-like tube formation [63] and mouse aorta ring assays [35] were performed as previously described. Where indicated, inhibitors were present both in the gels and in the supernatants. In vivo experiments All mice were from Elevage Janvier. Where indicated, vehicle or CHC (25 mmol in 200 ml, [64]) was daily injected i.p. We used RJ:NMRI mice and an existing protocol for Matrigel plug implantation [58]. The protocols used for intramuscular tumor growth and intravital microscopy have also been reported previously [65,66]. LLc cells were injected into syngeneic male 6–8 week-old C57/Bl6 J mice and TLT cells into syngeneic male RJ:NMRI mice. Surgery and imaging were performed on anesthetized (ketamine/xylazine) animals. All in vivo experiments were performed with approval of Duke University Institutional Animal Care and Use Committee (specific approval ID for this study was A069-03-02) and by Universit´e catholique de Louvain (UCL) authorities (Comit´e d’Ethique Facultaire pour l’Experimentation Animale, specific approval ID for this study was TUMETABO) according to national animal care regulations. PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33418