Targeting MCT1 Blocks Tumor Angiogenesis Figure 2. Lactate activates HIF-1 in normoxic endothelial cells. HUVECs were incubated during 24-h with 10 mM lactate or not. (A) The transcriptional activity of HIF-1 was determined using a dual luciferase reporter assay in HUVECs. *p = 0.0207; n = 4. (B) VEGF protein level was measured using ELISA in the supernatant of cells. ns, non-significant; n = 6. (C) bFGF protein level was measured using ELISA in the supernatant of cells. ***p,0.0001; n = 5. (D) VEGFR2 protein expression was detected by western blot in cell lysates. **p = 0.0084; n = 4–5. (A–D) Error bars reflect mean 6 SEM. doi:10.1371/journal.pone.0033418.g002 consequence of MCT1 inhibition was addressed by measuring changes in VEGFR2 expression. CHC blocked lactate-induced upregulation of VEGFR2 protein expression in HUVECs (Figure 5D) and in BAECs (Figure 5E). It also reduced basal HIF-1a and VEGFR2 protein expressions in HUVECs (Figure 5D). We ruled out a major contribution of two known Figure 3. Lactate induces an increase in HIF-1a protein expression in normoxic endothelial cells. HIF-1a protein expression was detected by western blot in normoxic ECs. (A) HUVECs were incubated during 24-h with 10 mM lactate or not. **p = 0.0014; n = 8. (B) as in (A) but with BAECs. ***p,0.0001; n = 16–17. (C) BAECs were incubated during 24-h with the indicated concentrations of lactate. **p,0.01; n = 4. (D) BAECs were incubated during the indicated times with 10 mM of lactate. *p,0.05; n = 5–12. (A–D) Error bars reflect mean 6 SEM. doi:10.1371/journal.pone.0033418.g003 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33418