Hypoxic Cell Cycle Arrest by PHD3 Inhibition Figure 1. PHD3 is overexpressed in human HNSCC and induced by hypoxia. (A) Five different primary human head and neck squamous cell carcinoma-derived cell lines (SCC) were cultured in normoxia or in hypoxia for 6 and 48 hours. The PHD3 mRNA expression was detected and quantified by Q-RT-PCR. (B) PHD3 expression in three different SCC cell lines detected by western blot analysis. (C) PHD3 mRNA expression in 28 HNSCC patient tumour samples and in 11 noncancerous anatomically matching samples was determined by Q-RT-PCR and normalized to EF-1a levels in each sample. Each dot represents the expression in one sample. The median values are indicated by dashed line and numerical value. (D) The statistical significance of PHD3 mRNA expression in patient and control samples was determined using Spearman correlation and two-tailed Student’s t-test. The upregulation of PHD3 gene shows statistical significance (p-value,0,0001) and correlates with Glut-1 expression (p- value,0,0001). (E) Four of five independent head and neck cancer studies extracted from Oncomine database show overexpression of PHD3 in cancer tissue relative to normal tissue. Three of the studies show statistically significant overexpression of PHD3 in cancer samples compared to normal tissue. (F) Detailed data analysis extracted from one Oncomine database study [37] for PHD3 expression. The expression is shown in five anatomically different HNSCC sites relative to control samples. doi:10.1371/journal.pone.0027112.g001 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27112