Figure 2.

Inactivation of PHDs during amino-acid starvation does not activate HIF. (a) Protein levels of HIF1α, REDD1, Glut1 and BNIP3 in U2OS cells incubated in the presence or absence of amino acids for the indicated time. A positive control of cells incubated under hypoxic conditions for 12 h (Hyp) was included. (b) U2OS cells expressing a construct containing either a wild-type HRE-luciferase reporter (black bars) or a mutant HRE-luciferase reporter unable to respond to HIF1α (white bars) were incubated in the presence or absence of amino acids for the indicated time and the luciferase intensity was analyzed using a luminometer (values represent average and s.e. of three independent experiments). A positive control of cells incubated in hypoxic conditions for 12 h was included (U=untransfected). (c) Protein levels of HIF1α and REDD1 in U2OS cells incubated in fed or starving conditions for 2 h followed by 30 min of incubation in the presence or absence of DMOG. (d) U2OS cells where incubated in hypoxia (1% O₂) in starving conditions for 2 h followed by 1 extra hour of incubation in the presence or absence of amino acids. HIF1α accumulation and S6 phosphorylation were then analyzed by western blot. (e, f) U2OS cells stably expressing NF-κB-reporter construct pGL4.32[luc2P/NF-kB-RE/Hygro] were incubated for the indicated time in KRBB either in the presence or absence of all amino acids or tumor necrosis factor-α (10 ng/ml). Activation of NF-κB was analyzed by luminescence analysis of the NF-κB-luciferase reporter (e) and by phosphorylation of IκB kinase (IKK) and IKβ-α by western blot (f). Graph depicts mean and s.d. of three independent experiments and represents fold activation compared with untreated samples.