ries, Hemel Hempstead, Herts, UK), and analysed as previously described [41]. Gene expression data-mining We previously derived a hypoxia metagene in primary head and neck cancer squamous cell carcinoma [HNSCC] [25]. Clusters of genes anti-correlated to miR-210 expression were formed as described previously [25]; Spearman correlation was used to identify genes significantly anti-correlated miR-210 and false discovery rate (FDR) was estimated using a permutation-based method [42]. Genes with FDR,0.05 were selected. We also used 5 target prediction algorithms:- TargetScanHuman (http://www. targetscan.org/); Pictar [43]; MiRanda (http://microrna.sanger. ac.uk/); DianaLab (http://diana.cslab.ece.ntua.gr/), miRDB (http://mirdb.org/miRDB/). Survival analyses and other statistical analyses Spearman correlation was used for continuous variables and Wilcox test for categorical variables. The Log-Rank test was used for univariate, and Cox survival was used for multivariate analysis. Distant-relapse free survival (DRFS) and relapse-free survival (RFS) were calculated by the STEEP criteria [44]. Statistics were carried out using non-paired t-tests and significance is represented by: *, p,0.05, **, p,0.01, and ***, p,0.001. Supporting Information Materials and Methods S1 Found at: doi:10.1371/journal.pone.0010345.s001 (0.06 DOC) MB Figure S1 Expression of miR-210 and ISCU in cancer cell lines. (A) In HCT116 cancer cells, 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 6 s.e.m. of three independent experiments is shown. (C, D) Knockdown of HIF1a but not HIF2a reverses the hypoxic suppression of ISCU mRNA at 48 hrs in MCF7 and HCT116 cell lines. Expression of ISCU mRNA is relative to scr in normoxia (N). Mean 6 s.e.m. of three independent experiments is shown. (* p,0.05, ** p,0.01, *** p,0.001). (E) A panel of breast can- cer cell lines were exposed to 1% oxygen for 24 hours (3 rep- licates/time point). miR-210 expression was measured by qPCR. Columns show mean fold difference in miR-210 expression between hypoxia and the parallel normoxia control samples using RNU48 as a reference; bars show s.e.m.. (F) ISCU was measured by qPCR comparing normoxia with cells grown in parallel under 1% oxygen; columns show downregulation under hypoxia, bars show s.e.m. Found at: doi:10.1371/journal.pone.0010345.s002 (1.01 MB TIF) Figure S2 ISCU expression is regulated by miR-210. (A) Transfection of anti-210 partially rescues the hypoxic suppression of ISCU mRNA and mimic-210 overexpression decreases ISCU mRNA in HCT116 cells in normoxia at 48 hrs. Expression of ISCU mRNA is relative to the anti-ctrl in normoxia (N). Mean 6 s.e.m. of two independent experiments for HCT116 is shown. (* p,0.05, ** p,0.01, *** p,0.001). (B) MCF7 cells were transfected with two siRNA against ISCU, siISCU1 and siISCU3. The ISCU protein normally seen under normoxic conditions is completely downregulated with both siRNAs. (C) Cellular subfractionation shows that mimic-210 downregulates both miR-210 Regulates ISCU mitochondrial and cytosolic ISCU isoforms. Immunoblotting against Tom20 (Santa Cruz Biotechnologies, CA, USA) and actin were used as loading control of the mitochondrial and cytosolic compartment respectively. Cellular subfractionation was per- formed in MCF7 cells using the Mitochondrial Isolation Kit for cells in Culture (Thermofisher, CO, USA). (D, top panel) ISCU protein is downregulated in HCT116 lysates at 0.1% oxygen compared to normoxic lysates (N) when the anti-ctrl is transfected for 48 hrs. This decrease 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 HCT116 by approximately 40%. Mean 6 S.E.M of two independent experiments is shown. (* p,0.05). (E) The 39-UTR is required for the regulation of ISCU by miR-210. A luciferase reporter construct containing the full length 39-UTR of ISCU or random sequence (Ctrl-39-UTR) was co-transfected with 20 nM of mimic-210/mimic-ctrl or 40 nM of anti-210/anti- ctrl in MCF7 cells. The cotransfection of a vector expressing Renilla luciferase was used to normalize for transfection efficiency. The luciferase activity was expressed as a ratio of ISCU-39-UTR to ctrl-39-UTR relative to expression in normoxia. All experiments were carried out in triplicate, (n = 9); error bars denote the standard deviation. (***p,0.001). (F) MCF7 and HCT116 cells were transfected with an expression plasmid for a Myc-DKK tagged ISCU2 lacking the 39-UTR. Subsequent transfection of siISCU3 reduced the protein levels of both endogenous and exogenous ISCU. Mimic-210 only affected the endogenous ISCU protein level. Found at: doi:10.1371/journal.pone.0010345.s003 (1.01 MB TIF) Figure S3 Iron uptake assessed by ferric iron staining. Knockdown of ISCU with siISCU3 led to an accumulation of ferric iron compared to control siRNA transfected HCT116 cells. This effect was reproduced when cells were transfected with mimic-210 compared to mimic-ctrl. HCT116 cells were trans- fected as described above and treated with 100 mg/L Ferric Ammonium Citrate for 16 hours. The cells were then fixed and stained with 4% Formalin and Perl’s solution (1% K4Fe(CN)6 and 1% HCl) for 30 minutes at room temperature. Cells were then incubated with 0.75 mg/ml diaminobenzidine (DAB), H2O2 in 1 M Tris pH 7.5 for 60 minutes. The reaction was then completed by washing the cells in PBS. Original magnification: 1006. Found at: doi:10.1371/journal.pone.0010345.s004 (1.19 MB TIF) Figure S4 Oncomine data. The Oncomine website (Oncomi- ne.org) was searched for microarrays containing the gene ISCU. When compared to normal tissue, ISCU was significantly downregulated in 9 tumour microarray experiments; data for relative expression units, normalised to median z-score to enable comparison across multiple studies, were downloaded and graphed. p,0.05 for all, Student’s t-test. Found at: doi:10.1371/journal.pone.0010345.s005 (0.97 MB TIF) Figure S5 Hypoxia regulates ISCU via HIF1a induction of miR-210. miR-210 represses ISCU 39-UTR and reduces ISCU protein. Reduced Fe-S assembly in the mitochondrial electron transport chain results in inhibition of major sites of electron transfer. This is a key mechanism for generating ROS, which have both positive and negative effects in hypoxia. Reduction of aconitase activity inhibits the Krebs cycle, resulting in an increase in glycolysis and lactate production. Aconitase 1 (cytosolic aconitase) when depleted of Fe-S becomes an iron regulatory protein, irp1, binding to iron response elements in the 39-UTR of the transferrin receptor. This stabilises the mRNA increasing PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10345