Figure 6: iNOS-induced NO is required for HIF1α activity in LPS-activated GM-DCs.

(a) GM-DCs were left unstimulated or stimulated with LPS (100 ng ml⁻¹) for 20 h in the presence or absence of the iNOS inhibitor SEITU (500 μM), then analysed by qPCR for Phd3 mRNA expression (upper panel) and by immunoblot analysis (lower panel) for protein levels (phosphorylated p70 S6-kinase, p-S6K; protein kinase B, PKB—loading control). (b–e) GM-DCs were left unstimulated or stimulated with LPS (100 ng ml⁻¹) for 20 h in the presence or absence of the amino acid arginine (Arg), then analysed (b) for nitrite production by the Greiss reaction, (c) by qPCR for Phd3 mRNA expression (upper panel) and by immunoblot analysis (lower panel) for protein levels (phosphorylated S6 ribosomal protein, p-S6; Total p70 S6-kinase, S6K), (d) for rates of glycolysis and (e) by qPCR for the mRNA expression of the glucose transporter Slc2a1 and glycolytic enzymes phosphofructokinase (Pkf) and Pyruvate kinase 2 (Pkm2). (f,g) GM-DCs were generated from either wild-type (WT) or iNOS knockout mice (Nos2^−/−), then left unstimulated or stimulated with LPS (100 ng ml⁻¹) for 20 h+/−the NO donor S-nitroso-N-acetylpenicillamine (250 μM) (f) or+/− BMDMs stimulated with LPS+IFNγ and added to the wells in a transwell insert (g) each for the last 4 h of the stimulation. The GM-DCs were then analysed by qPCR for Phd3 mRNA expression (upper panels) and by immunoblot analysis for HIF1α and β-actin protein levels (lower panels). (h) WT and Nos2^−/− GM-DCs were stimulated with LPS (100 ng ml⁻¹) for 20 h with LPS+IFNγ-stimulated BMDMs added to the wells in a transwell insert for the last 4 h of the stimulation+/−SEITU (500 μM). Cells were analysed by immunoblot analysis for HIF1α and β-actin protein levels. Data are mean±s.e.m. at least three separate experiments performed in quadruplicate (d) or triplicate (a–c,e–g). Representative immunoblots of at least three separate experiments are shown. Data were analysed using a one-way analysis of variance with Tukey’s post test (*P<0.05, **P<0.01, ***P<0.001). ECAR, extracellular acidification rate.