Fig. 4.

PHF8 and KDM3A differentially regulate select NED markers and PHF8 regulates H3K9me2 and H3K4me3. A and B. LNCaP cells that stably express doxycycline-inducible Ctrl or PHF8- or KDM3A-shRNAs were cultured under normoxia (N or Nor) or hypoxia (H or Hyp; 1% O₂) for six days. Expression of the indicated proteins and mRNAs was quantified by western blotting and RT-PCR, respectively. In B, the expression of genes was compared: 1) between normoxia and hypoxia in the control RNAi cells; 2) between the PHF8 RNAi or KDM3 RNAi and the control RNAi under hypoxia. The S.D. was obtained from at least three independent experiments. The Student’s t-test was used to calculate the significance. *: p < 0.05; **: p < 0.01. *: β-Actin and γ Tub were used as loading controls for PHF8 and KDM3A RNAi experiments, respectively. #: precursor CgA, ##: cleaved CgA. C and D. LNCaP-ctrl or PHF8 shRNA cells were treated as in A and Band subjected to ChIP assay with the indicated antibodies. PCR amplicons for CHGA and TUBB3 are illustrated above C and D, respectively. Amp 3: enhancer; Amp 1: proximal promoter/transcription start site; Amp 2: non-specific control loci. The ChIP-PCR data were interpreted as percentage of input DNA (1%) and the comparisons were done as described in Fig. 3. The S.D. was obtained from at least three independent experiments. Significance was calculated using two-way ANOVA in the case of multiple comparisons, and the Student’s t-test in the case of paired comparisons. *: p < 0.05; **: p < 0.01.