Hypoxic Cell Cycle Arrest by PHD3 Inhibition Figure 4. siPHD3 does not activate marked apoptosis. (A) SCC2 cells transfected with the indicated siRNAs were exposed to hypoxia for 6 to 30 hours. Cells were subsequently stained for caspase-3 (red) and Hoechst nuclear stain (blue). In siPHD3-transfected cells increased activation of caspase-3 was detected after 24 hour hypoxia. (B) Quantification of caspase-3 activation at the indicated hypoxic time points. Means and SD for five visual fields are shown. (C) Flow cytometric analysis (FACS) of the sub-G (apoptotic) SCC2 cells. SCC2 cells were transfected with the indicated siRNA and exposed to 24–48 hours of hypoxia followed by staining with propidium iodide (PI). The amount of cells in sub-G1 was determined. There were no changes in the size of apoptotic sub-G1 phase population of siPHD3 samples relative to siScr control. (D) Quantification of the apoptotic sub G1 population in SCC2 cells. Three independent experiments are shown as folds vs. control (siScr). doi:10.1371/journal.pone.0027112.g004 tantly, PHD3 depletion strongly enhanced the hypoxic decrease already at 6 hours of hypoxia. At 48 hours the pRb level was further suppressed in siPHD3 transfected cells (Fig. 6A). We next analyzed the expression of cyclins B and D. Cyclin D1 forms a complex with cyclin dependent kinases 4 and 6 (CDK4\6) which phosphorylate Rb and drive the cell cycle. Cyclin D1 expression is elevated during G1 to S transition [41]. Cyclin B1 is a regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and is needed for G2 to M phase transition of cell cycle. Cyclin B1 expression serves as a switch for cells to proceed to mitosis. In accordance with the reduced G1 to S transition, we detected a decrease in cyclin D1 levels in hypoxic siPHD3-treated cells (Fig. 6A). Also a clear reduction in cyclin B1 expression was detected in cells exposed to hypoxia. The reduction was futher enhanced by PHD3 depletion (Fig. 6A). PHD3 depletion did not affect the expression of other cyclins such as cyclin A or cyclin E (not shown). PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27112