miR-210 Regulates ISCU Figure 2. MiR-210 negatively regulates aconitase and complex I activity in MCF7 and HCT116, and upregulates glycolysis. Cells transfected with siRNA against ISCU or with mimic-210 show a significant decrease in the activity of the Fe-S protein aconitase (A) and complex I (B) at 48 hrs. Activity is expressed relative to scr for siISCU3 or to mimic-ctrl for mimic-210. Mean 6 s.e.m. of three independent experiments is shown (* p,0.05, ** p,0.01, *** p,0.001). C, hypoxia decreases aconitase and complex I activity in MCF7 and HCT116. Activity is expressed relative to normoxia (N). Transfection of MCF7 with anti-210 increases the level of aconitase activity, compared to anti-ctrl. Mean 6 s.e.m. of three independent experiments is shown (* p,0.05, ** p,0.01, *** p,0.001). D, in normoxia, MCF7 transfected with mimic-210 have an increase in the secreted lactate concentration and a reduction in the extracellular pyruvate after 48 hours. Lactate: Pyruvate ratio shows a significant increase under the same conditions. Transfection with anti-210 in hypoxia reduced lactate production compared to anti-ctrl transfected cells. Mean 6 s.e.m. of three biological replicates is shown (* p,0.05, ** p,0.01, *** p,0.001). doi:10.1371/journal.pone.0010345.g002 nucleic acid antagonist (LNA) miR-210 molecule (Figure 4B). Although most of the reduction in clonogenicity in hypoxia is clearly via other mechanisms, the effects of the anti-210 resulted in decreased clonogenic survival in hypoxia, which we also found in a more hypoxia-resistant cell line, HeLa cells (Figure 4C). In vivo suppression of ISCU by hypoxia and clinical significance of ISCU expression We investigated whether expression of ISCU gene expression was regulated in vivo, by studying human tumour xenografts. Xenografts of the glioblastoma cell line U87 were treated with the VEGF inhibitor Avastin (Bevacizumab), or with vehicle PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10345