Statistical analyses Results are expressed as mean 6 SEM. Student’s t test and 1- way ANOVA (Tukey’s post-hoc test) were used where convenient. p,0.05 was considered statistically significant. Supporting Information Figure S1 Lactate does not support EC survival through increased oxygen consumption and CHC is devoid of intrinsic toxicity. (A) HUVEC death was determined with a NucleoCounter. Confluent cells were assayed after 4-h of culture in serum-free DMEM containing the indicated substrates. *p,0.05 compared to glucose; n = 3. (B) EPR oximetry was used to determine the rate of oxygen consumption by HUVECs in DMEM containing 4500 mg/L glucose 610 mM lactate. Slopes calculated from linear regressions are not statistically different (21.97360.174 for glucose, 21.90260.275 for glucose+lactate, p = 0.84, n = 3). (A and B) Error bars reflect mean 6 SEM. (PDF) Figure S2 Lactate induces the nuclear translocation of HIF-1a in HUVECs. HIF-1a protein expression was detected by Western blot in the cytosol (laminin A/C-negative fraction, 8 mg of total protein per well) and nucleus (laminin A/C-positive fraction, 10 mg of total protein per well) of HUVECs after a 24-h treatment with hypoxia or lactate as indicated. Densitometric analyses of HIF-1a protein expression (% of control) are provided below the correspond- ing bands; the experiment was repeated twice with similar results. (PDF) Figure S3 Membrane expression of VEGFR2 and MCT1 in ECs. (A) HUVECs were treated during 24 h with 10 mM lactate or not, after which the cells were immunostained with an antibody raised against an epitope comprising an extracellular domain of VEGFR2. Nuclei were stained with DAPI. Shown are representative pictures indicating that lactate stimulates the membrane expression of VEGFR2 in normoxic ECs. Omission of primary antibodies was used for control. (bar = 20 mm). (B) HUVECs were immunostained using antibodies against MCT1, its chaperon protein CD147, and CD31, and nuclei were stained with DAPI. Shown are representa- tive pictures, including a merged picture, indicating that all 3 proteins colocalize at the cell membrane. Omission of primary antibodies was used for control. (bar = 20 mm). (PDF) Figure S4 Inhibition of lactate-induced HIF-1a expres- sion by 2-oxoglutarate, oxamate and LDH-B silencing. Representative western blots are shown. (A and B) HIF-1a and b- actin protein expressions were detected in BAECs treated during 24-h as indicated with (A) 10 mM lactate and increasing concentrations of the PDH substrate 2-oxoglutarate (2-OG), (B) 10 mM lactate and/or 10 mM oxamate. (C) HUVECs were transfected with a specific siRNA against LDH-B or with a References 1. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. 2. Wu R, Racker E (1959) Regulatory mechanisms in carbohydrate metabolism. IV. Pasteur effect and Crabtree effect in ascites tumor cells. J Biol Chem 234: 1036–1041. 3. Semenza GL (1998) Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Dev 8: 588–594. 4. Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, et al. (2004) Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 64: 3892–3899. 5. Gordan JD, Thompson CB, Simon MC (2007) HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation. Cancer Cell 12: 108–113. 6. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407: 249–257. Targeting MCT1 Blocks Tumor Angiogenesis scrambled siRNA, and treated during 24-h with 20 mM lactate where indicated. The representative western blot shows HIF-1a, LDH-B and b-actin protein expressions. (PDF) Figure S5 Protein extinctions in HUVECs after siRNA/ shRNA delivery. Representative western blots and quantifica- tion graphs are shown for HUVECs transfected with (A) a specific siRNA against LDH-B or a control siRNA (n = 2), or (B) a specific shRNA against MCT1 or a control shRNA. n = 3. (A and B) Error bars reflect mean 6 SEM. (PDF) Figure S6 CHC is devoid of intrinsic toxicity for ECs. HUVEC death was determined with a NucleoCounter. Confluent cells were assayed after 24-h culture in EBM2-MV containing glucose and serum and supplemented with lactate or CHC as indicated. ns, non-significant; n = 4. Error bars reflect mean 6 SEM. (PDF) Figure S7 Various MCT1 inhibitors block lactate-induced HIF-1a protein expression in normoxic ECs. BAECs were treated during 24-h with 10 mM lactate and the MCT1 inhibitors indicated. *p,0.05, **p,0.01 compared to control; ##p,0.01, ###p,0.005 compared to lactate alone; n = 4. Error bars reflect mean 6 SEM. NPPB, 5-nitro-2-[3-phenylpropylamino]-benzoate. (PDF) Figure S8 MCT1 modulates basal and lactate-induced angiogenesis. HUVECs transfected with a shRNA against MCT1 (target extinction = 86.763.3%) or with a scrambled (Scr) shRNA were seeded on undiluted growth factor-reduced Matrigel containing 10 mM lactate or not and monitored for their ability to generate vascular tubes. Shown are representative pictures (n = 3) captured after 12-h of culture. Bar = 1 mm. (PDF) Table S1 Genes activated by lactate in normoxic ECs (RT2 Profiler PCR Array). (PDF) Acknowledgments The authors thank Prof. Chuan-Yuan Li (University of Colorado, Denver) for the kind gift of the ODD-Luc construct, Dr Paolo Porporato (UCL, Brussels) for scientific and technical advices, and Elise Beneteau for excellent technical assistance. Author Contributions Conceived and designed the experiments: PS BG CM MWD OF. Performed the experiments: PS TC CJDS FV JV KMK EJM SD PD FF AR. Analyzed the data: PS TC CJDS FV JV KMK EJM SD PD FF BG AR CM MWD OF. Contributed reagents/materials/analysis tools: PS BG AR CM MWD OF. Wrote the paper: PS MWD OF. 7. Pugh CW, Ratcliffe PJ (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9: 677–684. 8. Berra E, Benizri E, Ginouves A, Volmat V, Roux D, et al. (2003) HIF prolyl- hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF- 1alpha in normoxia. EMBO J 22: 4082–4090. 9. Hirsila M, Koivunen P, Gunzler V, Kivirikko KI, Myllyharju J (2003) Characterization of the human prolyl 4-hydroxylases that modify the hypoxia- inducible factor. J Biol Chem 278: 30772–30780. 10. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, et al. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399: 271–275. 11. Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721–732. PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e33418