Figure 5: mTORC1 and HIF-1α signalling is required for iNOS activity in LPS-activated GM-DCs.

GM-DCs were left unstimulated (Unstim) or stimulated with LPS (100 ng ml⁻¹) for 8 h, washed and then cultured in media containing either 10 mM glucose (Glu) or 10 mM galactose (Gal) for 20 h prior to the measurement of (a) OXPHOS levels by seahorse analysis and (b) nitrite production by the Greiss reaction. (c,d) GM-DCs were left unstimulated or stimulated with LPS (100 ng ml⁻¹)+/−rapamycin (20 nM) for 20 h and analysed for (c) nitrite production by the Greiss reaction (upper panel) and by immunoblot analysis (lower panel) for protein levels (phosphorylated p70 S6-kinase, p-S6K; protein kinase B, PKB) or (d) OXPHOS levels. (e,f) Hif1a^flox/flox (WT) or Hif1a^flox/flox VavCre (Hif1a^−/−) GM-DCs were left unstimulated or stimulated with LPS (100 ng ml⁻¹) for 20 h, then analysed (e) by qPCR for Nos2 mRNA expression (upper panel) and by immunoblot analysis (lower panel) for protein levels (Structural Maintenance Of Chromosomes protein, SMC1) or (f) for nitrite production by the Greiss reaction. (g,h) GM-DCs were left unstimulated or stimulated with LPS (100 ng ml⁻¹)+/−rapamycin (20 nM)+/−DMOG (200 μM) for 20 h and analysed for (g) nitrite production by the Greiss reaction or (h) by qPCR for Nos2 mRNA expression (upper panel) and immunoblot analysis for protein levels (lower panel). Data are mean±s.e.m. of at least three separate experiments performed in quadruplicate (a,d) or triplicate (b,c,e–h). Representative immunoblots of at least two separate experiments are shown. Data were analysed using a one-way analysis of variance with Tukey’s post test (*P<0.05, ***P<0.001). OCR, oxygen consumption rate.