A, miR-210 qPCR expression (DDCT) plotted as a function of ISCU mRNA expression (Illumina arrays) in the Oxford breast cancer series (left), and, relapse free survival for samples with high ISCU and low ISCU split by median in the Oxford breast cancer series (right). B, mRNA expression of the transcript hosting the miR-210 precursor plotted as a function of ISCU mRNA expression measure by Affymetrix U133b and U133a respectively in the Uppsala breast cancer series (left), and relapse free survival for samples with high ISCU and low ISCU split by median in the Uppsala breast cancer series (right). C, miR-210 qPCR expression (DDCT) plotted as a function of ISCU mRNA expression (Affymetrix arrays) in the Oxford-Manchester HNSCC cancer series where ISCU was initially found to be associated to hypoxia (left), and relapse free survival for samples with high ISCU and low ISCU split by median value in the Chung et al. HNSCC series (right).

A, in normoxia, transfection of mimic-210 significantly increases superoxide production at 48 hrs as measured by MitoSox staining. Representative images from mimic-210 transfected cells are shown on the top panels and the mean ± s.e.m. of approximately 300 cells are shown on the bottom panels (***, p<0.001). B, exposure of cells to 0.1% oxygen for 48 hrs enhances the production of superoxide; this effect is almost completely reversed upon transfection of anti-210. Representative images from miR-210 transfected are shown on the left panels and the mean ± s.e.m. of approximately 300 cells are shown on the right panels (***, p<0.001). C, ROS production induced by mimic-210 is inhibited in MCF7 cells by cotrasfection with the ISCU2 expressing plasmid. Mean ± s.e.m. of approximately 300 cells is shown (*** p<0.001). Bars  = 10 µm.

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 ± 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 ± 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 ± s.e.m. of three biological replicates is shown (* p<0.05, ** p<0.01, *** p<0.001).

A, miR-210 increases under hypoxia, with the most robust induction seen at 48 hrs with 0.1% oxygen. B, under the same conditions, the strongest downregulation of ISCU mRNA is observed at 48 hrs with 0.1% oxygen. The expression levels of miR-210 and ISCU mRNA under hypoxia are relative to their normoxic expression levels at 24 hrs (N). Mean ± s.e.m. of three independent experiments is shown (* p<0.05, ** p<0.01, *** p<0.001). C, transfection of anti-210 partially rescues the hypoxic suppression of ISCU mRNA and mimic-210 overexpression decreases ISCU mRNA in normoxia at 48 hrs. Expression of ISCU mRNA is relative to the anti-ctrl in normoxia (N). Mean ± s.e.m. of three independent experiments is shown (** p<0.01, *** p<0.001). D, (top panel) ISCU protein is downregulated in MCF7 lysates at 0.1% oxygen compared to normoxic lysates (N) when the anti-ctrl is transfected for 48 hrs. This decrease in protein level under hypoxic conditions is partially rescued when the anti-210 is transfected. Under normoxic conditions, mimic-210 suppresses ISCU protein levels compared to mimic-ctrl transfected cells. D, (bottom panel), quantification of the rescue of ISCU protein level upon transfection of anti-210. Transfection of anti-210 rescues the ISCU protein levels in MCF7 by approximately 40%. Mean ± S.E.M of two independent experiments is shown. (* p<0.05).

A, apoptosis (Annexin V+ cells) was measured in MCF7 (left) and HCT116 (right) cells treated with miR-210 inhibitor or mimic, after 48 hours exposure to 0.1% oxygen or normoxia. Mean ± s.e.m. is representative of 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.001). B, after transfecting MCF7 cells with scrambled LNA or mir-210 LNA and exposed to 0.1% oxygen or normoxia (48 hrs), the cells were re-plated (100 and 500 cells/well). After a 12-day incubation, colonies were fixed, stained, and the surviving fractions were calculated based upon the plating efficiency. C, surviving fractions were calculated as in (B) in HeLa cells, which were transfected with anti-ctrl or anti-210 (Applied Biosystems/Ambion, Austin, TX, USA) and exposed to 0.01% oxygen or normoxia. In all colony assays, mean ± s.e.m. is representative of 2 independent experiments carried out in triplicate employing 2 different plating densities. (** p<0.01, *** p<0.001). D, RT-PCR for ISCU and miR-210 in U87 xenografts treated with anti-angiogenic therapy (bevacizumab). Relative expression of ISCU normalised to β-actin, miR-210 normalised to three small nucleolar controls, RNU43, RNU44 and RNU48. Mean expression ± s.e.m. in 4 animals/group is shown (** p<0.01, ***p<0.001).