Activation of DCs by LPS drives iNOS expression and NO production. (A) Presence of functional NADPH oxidase in DCs was tested by determining OCR in a Seahorse XF-24 analyzer in response to phorbol 12-myristate 13-acetate to activate the complex, and subsequently to specific NADPH oxidase inhibitor apocynin to block the complex.21 (B) DCs were seeded in a Seahorse XF-24 analyzer, stimulated with LPS or medium for 24 hours, and OCR was determined during sequential treatments with antimycin-A/rotenone (ETC inhibitors) and apocynin. Data represent means ± SD of triplicates. One of 2 experiments is shown. (C) Relative NOS2 mRNA expression was determined in DCs 8 hours after medium or LPS stimulation. Data represent means ± SD of 3 independent experiments. (D) Intracellular iNOS expression in 24-hour LPS-activated (black line) and unstimulated (gray histogram) DCs was determined by FACS. One experiment of 6 is shown. (E) iNOS expression was determined as in panel D at the indicated time points after LPS stimulation. Data represent means ± SD 2 independent experiments. (F) Nitrite levels were determined in culture supernatants at the indicated time points after LPS stimulation. Data represent means ± SD of 2 independent experiments. (G) DCs were seeded in a Seahorse XF-24 analyzer, stimulated with LPS or medium for 24 hours, and real-time OCR was determined during sequential treatments with antimycin-A/rotenone (ETC inhibitors) and the NOS inhibitor SEITU. Data represent means ± SD of triplicates. One of 3 experiments is shown.
