Fig. 2.

The histone demethylase activity of PHF8 is required for its activation of HIF1α and its upregulation of target genes. A and B. LNCaP cell lines stably expressing a doxycycline-inducible control (Ctrl) or PHF8 shRNAs were incubated under normoxia (N or Nor) or hypoxia (H or Hyp;1% O₂) for 24 h. The protein and RNA levels of the indicated genes were quantified by western blotting and RT-PCR, respectively. For RT-PCR, gene expression in the control RNAi cells under hypoxia was compared to that in cells under normoxia. In the case of the PHF8 RNAi cells, gene expression under hypoxia was compared to that in the corresponding control. C and D. pOZ-empty (Mock), -wild-type (WT) or -mutant (F279S) PHF8 were stably expressed in the LNCaP-PHF8shRNA_1 cell line. The cells were treated as in A and B, with doxycycline treatment as indicated (Dox), and gene expression was assessed accordingly. For RT-PCR, in the PHF8 RNAi cells, gene expression under hypoxia was compared to that under normoxia. In the rescue experiments, gene expression in cells expressing wild-type or mutant PHF8 was compared with that in mock-treated cells. The S.D. was obtained from at least three independent experiments. The Student’s t-test was used to calculate significance. *: p < 0.05; **: p < 0.01.