Hypoxic Cell Cycle Arrest by PHD3 Inhibition Figure 2. PHD3 is required for SCC cell survival in prolonged hypoxia. (A) PHD3 is induced in hypoxia. Knock-down of PHD3 expression with siRNA (siPHD3) is specific and does not affect PHD2 or HIF-1a expression. siScr = scrambled control siRNA. (B) SCC2 cells were plated on glass slides, transfected with siRNAs and exposed to hypoxia for 48 h followed by Hoescht nuclear staining. The samples with PHD3 siRNA showed marked reduction in cell number in hypoxia. No effects were seen in the normoxic counterparts. (C) Quantification of the cell number in three optical fields from each sample in three independent experiments. The change in cell number is shown as folds vs. control (siScr). The reduction in cell number of siPHD3-transfected samples was statistically significant (**; p-value,0,01). (D) The proliferation rate of cells transfected with control (siScr), PHD2 or PHD3 siRNA was determined using BrdU incorporation. Results from absorbance changes in three independent experiments are shown as folds vs. control (siScr). (E) To validate the specificity of the effect of PHD3 inhibition on cell survival a point-mutated PHD3 siRNA (siPHD3mut) we used as a control. siPHD3mut did not affect the viability of SCC cells. (F) Quantification of the cell number in three optical fields. (G) Western blot analysis shows the effect of the indicated siRNAs on PHD3 expression. doi:10.1371/journal.pone.0027112.g002 PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27112