Hypoxic Cell Cycle Arrest by PHD3 Inhibition Figure 3. PHD3 hydroxylase activity is not required for hypoxic cell survival. (A) Cells were exposed to siPHD3, followed by transfection of the indicated control (EGFP) and PHD3 expressing plasmids (PHD3-EGFP and PHD3R206K-EGFP) and 48 hour incubation in hypoxia. The phase contrast images show representative examples of cell amount. (A) Quantification of cell number with transfection of indicated plasmids under siPHD3 expression and hypoxia. Means and SD of three visual fields. doi:10.1371/journal.pone.0027112.g003 G phase. The SCC2 cells were transfected with control or PHD3 siRNA and cultured in normoxia and hypoxia for 24 to 72 hours. As well established, the hypoxic exposure induced apoptosis in both control and siPHD3 cells. In control cells a 35% induction in apoptosis rate after 24-hour hypoxia was detected and after 72 hours the apoptosis rate had increased 2-fold (Fig. 4C, Fig. S3). However, when compared to the normoxic siScr control, the siPHD3 transfected cells demonstrated similar or even slightly reduced amount of cell in the sub-G phase (Fig. 4C, Fig. S3). When compared to siSCr controls at each time point, quantifi- cation of the apoptotic rate demonstrated small effect of siPHD3 on apoptosis that was insignificant (Fig. 4D). We concluded that although the exposure of SCC cells to PHD3 inhibition in hypoxia may activate caspase-3 in a subpopulation of cells this does not translate to major increase in apoptosis. PHD3 inhibition enhances hypoxia-induced block in G1 to S transition As the reduction in cell number could not be explained with increased apoptosis, we looked for possible defects in the cell cycle progression. SCC2 cells were transfected with either control or PHD3-targeted siRNA and incubated in normoxia and in hypoxia for 24 to 48 hours. Cell cycle was determined using flow cytometer with propidium iodide staining. As expected, hypoxic exposure led to an increase in the number of cells in G1 phase and a decrease in the cell population in S and M phases that peaked after 48-hour hypoxia indicating a block in G1 to S transition under hypoxia (Fig. 5A and Fig. S4). Noticeably, siPHD3 transfection led to further increase in the accumulation of cells in G1 (Fig. 5A and B). Accordingly, this was accompanied by a decrease in the amount of cells in the synthesis (S) phase. Quantification of the data from at least three independent experiments demonstrated constant increase in the G1 population under hypoxic PHD3 inhibition (Fig. 5B). Similarly, when compared to normoxic control, an approximately 10% increase in G1 was seen with PHD3 inhibition under hypoxia (Fig. S4A). In line with an increase in the G1, a marked decrease in the S phase with siPHD3 as compared to the siScr control at each time point was demonstrated under hypoxia (Fig. 5C, Fig. S4B). The percentual decrease in the S phase by siPHD3 transfection was approximately 35% and 25% at 24 and 48-hour hypoxia, respectively. Supporting reduced S-phase entry, a clear reduction of hypoxia-exposed cells in G2/M phase was also detected (Fig. 5A). The smaller increase in G1 population as compared to the reduction of S-phase reflects the fact that most cells at any given time reside in G1 phase. Noticeably, when the ratio between G1 and S phases was calculated, the difference between control and PHD3 inhibition was most evident illustrating a 2-fold increase in hypoxic siPHD3 cells as compared to control at 24- and 48-hour hypoxia (Fig. 5D). In line with previous reports on the effect of hypoxia on G1 to S transition, the difference in G1/S ratio showed 30% and 100% increase by hypoxia alone at 12- and 24-hours, respectively. This was further increased to 70% and 170% by siPHD3 exposure (Fig. S4C). To study whether the siPHD3-induced block in G1 to S transition was specific for SCC cells we used HeLa cells that were synchronized and subsequently analyzed by flow cytometry at shorter timepoints. Hela cells were transfected with PHD3 and control siRNAs, synchronized with aphidicolin followed by exposure to hypoxia for 2 to 12 hours (Fig. S5) and calculation of G1 to S phase ratio. A marked increase in the G1 to S ratio in siPHD3 cells as compared to either siScr control or siPHD2- transfected cells was seen (Fig. 5E). The most notable difference was seen after 12-hour hypoxia when the G1 to S ratio in siPHD3 cells showed a 3-fold increase compared to controls (Fig. 5E). Together the data demonstrated that in hypoxic carcinoma cells PHD3 inhibition causes cell cycle arrest at the G1-S border. PHD3 depletion reduces the amount of hyperphosphorylated retinoblastoma protein in hypoxia Hyperphosphorylation of retinoblastoma protein Rb (pRb) is required for the cell cycle to proceed from G1 to S phase [7,8]. As we detected a block in the S-phase progression, we next asked whether the PHD3 inhibition would have an effect on the phosphorylation status of Rb. SCC2 cells transfected with control or PHD3 siRNAs followed by hypoxic exposure were analyzed for phosphorylated Rb at 6 and 48-hour timepoints (Fig. 6A). As expected, a marked decrease in the amount of pRb in SCC2 cells was detected under hypoxia as compared to normoxia. Impor- PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27112