Fig. 5. Effect of activation of M on the stabilization of HIF1. (A) The time course of HIF1 stabilization following activation and (B) the corresponding densitometry values normalized with -tubulin and relative to non-activated controls. Cobalt chloride (CoCl₂) was used as a positive control for HIF1 in A. (C) Cells were transfected with HIF1 siRNA or scrambled siRNA as a control. After 24 hours they were treated with the agents indicated and incubated for 12 hours. Treatment with IFN + LPS resulted in stabilization of HIF1 under normoxic conditions. This was reduced in the cells in which HIF1 had been silenced, and in those treated with SEITU. Densitometry values, normalized with -tubulin and relative to that of control transfected M (in the case of HIF1) and control transfected and activated M (in the case of iNOS) are shown below each lane. (D) Silencing HIF1 reduced the generation of NO and (E) partially preserved mitochondrial oxygen consumption in activated M. (F) The upregulation of glycolysis in activated M was attenuated by silencing HIF1 and by treatment with SEITU; it was completely abolished by a combination of the two treatments. (G) The cellular ATP content was reduced in M in which HIF1 had been silenced and was reduced further by activation, an effect that was prevented by SEITU. ^*, Significantly different from control transfected values. Results are mean±s.d., n=3-5, and representative western blots are shown.