Hypoxic Cell Cycle Arrest by PHD3 Inhibition release the cells were exposed to hypoxia for the indicated time. Population sizes of the G1 and S phase cells were determined by FACS and the G1 to S relation was calculated (arbitrary units). doi:10.1371/journal.pone.0027112.g005 The expression analysis of cell cycle regulators was further performed in synchronized HeLa cells (Fig. 6B). In line with the data from SCC cells, a marked decrease in the amount of pRb in synchronized siPHD3-treated Hela cells was detected (Fig. 6B). In HeLa cells a decrease in cyclin D1 levels in siPHD3-treated cells was evident as well, while hypoxia per se showed little effect on cyclin D1 level. The suppression of Rb hyperphosphorylation with reduced expression of cyclins D1 and B1 in cells exposed to siPHD3 validates the effect of siPHD3 on cell cycle arrest seen in flow cytometric analyses. Figure 6. PHD3 inhibition reduces the amount of hyperphosphorylated Rb and increases p27 in hypoxia. (A) SCC2 cells were transfected with the indicated siRNAs and exposed to normoxia or hypoxia for 24 to 48 hours followed by western blot analysis of PHD3, phosphorylated Rb (p-Rb) and cyclin B1. (B) HeLa cells were transfected with the indicated siRNAs, synchronized and exposed to normoxia or hypoxia for 6 to 24 hours after release. PHD3, phosphorylated Rb (p-Rb) and cyclin D1 were analyzed from samples by western blotting. (C) SCC2 cells were transfected with the indicated siRNAs and exposed to normoxia or hypoxia for 24 hours followed by western blot analysis of p21(Cip1). (D) Cells transfected with the indicated siRNAs and exposed to normoxia or hypoxia for 24 hours followed by western blot analysis of p16 and p27. Hypoxia was monitored by HIF-1a expression. (E) Cells transfected with the indicated siRNAs and exposed to normoxia or hypoxia for 24 and 48 hours followed by western blot analysis of p27. doi:10.1371/journal.pone.0027112.g006 PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27112