Fig. 8.

The electron transport chain is required for nominal reactive oxygen species production in peritoneal tissue-resident macrophages. a Bar graphs showing ATP and NADPH assays of peritoneal tissue-resident macrophages (pTRMØ). Cells were starved of glutamine (2 mM) and/or glucose (25 mM) 1 h before addition of zymosan (50 μg/ml) or control, extracts were taken after 2 h. Data were analysed by two-way ANOVA. For ATP, interaction p = 0.16, effects of zymosan or fuel p < 0.0001. For NADPH ratio, interaction p = 0.041. b Bar graphs showing ATP and NADPH assays of neutrophils. Cells were treated as in a, data were analysed by two-way ANOVA with Sidak’s post-tests for glutamine addition. For ATP, interaction p = 0.21, zymosan p = 0.15, fuel p < 0.0001. For NADPH ratio, interaction p = 0.85, zymosan p = 0.56, fuel p < 0.0001. c Bar graphs showing ATP and NADPH assays of pTRMØ. Cells were treated with inhibitors 30 min before addition of zymosan as in a. Oligomycin (1.26 μM), rotenone (200 nM), thenoyltrifluoroacetone (TTFA, 2 mM), antimycin A (1 μM), dehydroepiandrosterone (DHEA, 100 μM), 2-deoxyglucose (2-DG, 100 mM). Data were analysed by two-way ANOVA with Sidak’s post-tests vs control. For ATP, interaction p = 0.47, zymosan or drug were p < 0.0001. For NADPH ratio, interaction p = 0.036. Data a–c represent at least two independent experiments and show 3–4 separate wells per group. d Bar chart showing the median fluorescent intensity (MFI) of mitoSOX dye in Ncf1 ^−/− pTRMØ treated with phorbol–myristate–acetate (PMA, 1 μM) or control. Data (n = 4 separate wells per group) represent two independent experiments and were analysed by Student’s t-test. e Line graph (left) showing luminol luminescence against time in pTRMØ treated with zymosan 1 min before time 0. Bar chart (right) showing quantification of the area under the curve luminescence from the line graph in the presence of the indicated inhibitors (as in b + atpenin A5 (1 μM)). Data (n = 4–6 separate wells per group) were combined from two separate experiments, each treatment result is representative of at least two independent experiments. Data were analysed by one-way ANOVA with Tukey’s post-tests vs the matched control. All error bars denote mean ± SEM