FIGURE 8.

Effect of PHD2 and PHD3 silencing on the stability of HIF-1α and HIF-2α in NP cells under normoxia and hypoxia. A, immunofluorescence analysis of NP cells transduced with lentivirus coexpressing GFP and ShRNA of either PHD2 (LV-Sh-PHD2) or PHD3 (LV-Sh-PHD3) shows good transduction efficiency. Magnification ×20. B, real-time RT-PCR analysis of cells transduced with LV-Sh-PHD2/3. The mRNA expression of PHD2 or PHD3 was suppressed robustly by respective ShRNAs compared with cells transduced with control lentivirus (LV-Sh-control) under both normoxic and hypoxic conditions. C and D, Western blot analysis of cells transduced with LV-Sh-PHD2/3. The expression of PHD2 or PHD3 was suppressed by respective ShRNAs compared with cells transduced with control lentivirus (LV-Sh-control). Note that accumulation of HIF-1α was seen in PHD2- but not PHD3-silenced cells under both normoxia (C) and hypoxia (D). KD, kilodalton. Accumulation of HIF-2α was not affected by silencing of either PHD2 or PHD3. E, densitometric analysis of multiple blots from experiment described in C and D above. Note that Relative HIF-1α level compared with LV-Sh-control was significantly increased with LV-Sh-PHD2 but not LV-Sh-PHD3 irrespective oxemic tension. Relative HIF-2α level was not affected by either LV-Sh-PHD2 and LV-Sh-PHD3. F, role of PHD3 on maintenance of HIF-1 transcriptional activity. NP cells were transfected with the HRE-luc reporter along with the Sh-PHD3 construct, and activity was measured under normoxic or hypoxic conditions. Note that hypoxic induction in HRE activity is significantly blocked by silencing of PHD3 expression. Data are represented as mean ± S.E. of three independent experiments performed in triplicate (n = 3). *, p < 0.05. ns, not significant.