Figure 7

5-dAzaC effect on PHD3 transcript (A) and protein (B) levels in HCT116 and DLD-1 CRC cells. HCT116 and DLD-1 cells were cultured in DMEM for 6, 24 and 48 h either in the absence or in the presence of 5-dAzaC at a concentration of 1.00 or 5.00 μM under hypoxic or normoxic conditions. After incubation the cells were used for total RNA isolation and protein isolation. Total RNA was reverse-transcribed, and PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of the respective controls. Each sample was determined in triplicate and results are presented as the mean ± SE from three experiments *P < 0.05. The cell protein was separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio PHD3 to GAPDH for control was assumed to be 1.