Figure 4
Inactive PHD2 caused by reactive oxygen species (ROS) participate in HIF-1α target glycolytic gene expression.
(A) H-rasWT or H-rasV12 expressing U2OS cells were incubated in the absence or presence of 3 mM of NAC for 16 h and then HIF-1α levels were detected by immunoblotting. (B) Cells were transiently transfected with indicated reporter plasmids in H-rasWT or H-rasV12 stably expressing U2OS cells. Transfected cells were further incubated with 3 mM of NAC or 50 units/ml of PEG-conjugated catalase for 24 h. Luciferase activity was measured using a luminometer and β-gal assay was performed to normalize transfection efficiencies. Values represent mean ± SD of independent experiments performed three times; **p < 0.01 versus H-rasWT vehicle and #p < 0.05 versus H-rasV12 vehicle. (C) H-rasWT or H-rasV12 stably expressing U2OS cells were cultured in the absence or presence of 3 mM of NAC for 48 h and then HIF-1α target genes or PHD isoforms were assessed by quantitative PCR. Values represent mean ± SD of three independent experiments performed in triplicate; *p < 0.05, **p < 0.01 versus H-rasWT vehicle and #p < 0.05 versus H-rasV12 vehicle. (D) H-rasWT or H-rasV12 stably expressing U2OS cells were cultured in the absence or presence of 50 units/ml of PEG-conjugated catalase for 48 h and then HIF-1α target genes were assessed by quantitative PCR. Values represent mean ± SD of three independent experiments performed in triplicate; **p < 0.01 versus H-rasWT vehicle and #p < 0.05 versus H-rasV12 vehicle. (E) U2OS cells were transfected with 40 nmol/L small interfering RNA (siRNA) targeting PHD2 and then endogenous PHD2 levels were detected by immunoblotting. (F) H-rasWT or H-rasV12 stably expressing U2OS cells were transiently transfected with 40 nmol/L of siRNA against PHD2 and then cells were further incubated for 48 h. After transfection and incubation, HIF-1α target genes were assessed by quantitative PCR. Values represent mean ± SD of three independent experiments performed in triplicate; *p < 0.05 and **p < 0.01.
