iNOS-derived NO blocks mitochondrial respiration in LPS-stimulated DCs. (A) Nitrite levels were determined in culture supernatants of DCs stimulated with medium or LPS for 24 hours in the presence or absence of specific NOS inhibitor SEITU. Data represent means ± SD of 3 experiments. (B) DCs were seeded in a Seahorse XF-24 analyzer, stimulated with medium or LPS for 24 hours in the presence or absence of SEITU, and real-time basal OCR was determined as well as in response to sequential treatments with oligomycin, FCCP, and antimycin-A/rotenone. Data represent means ± SD of triplicates. One of 3 experiments is shown. (C) iNOS^−/− DCs were stimulated with medium or LPS for 24 hours and analyzed as in panel B. Data represent means ± SD of triplicates. One of 3 experiments is shown. (D-E) Twenty-four-hour LPS-stimulated DCs were stained with hydroethidin to detect O₂⁻ production or DiOC₆ to determine mitochondrial membrane potential (Ψ_m). Bars represent geoMFI ± SD of 3 experiments and data are plotted relative to unstimulated cells. (F) DCs were stimulated for 24 hours with LPS in the presence or absence of SEITU and the NO donor SNAP as indicated, and analyzed as in panel B. Data represent means ± SD of triplicates. One of 2 experiments is shown. (G) DCs were stimulated with medium, LPS for 24 hours, or LPS for 48 hours, with the last 24 hours in the presence of SEITU, and analyzed as in panel B. Data represent means ± SD of triplicates and are shown as percentage of OCR before drug treatment. One of 2 experiments is shown. ant/rot indicates antimycin-A/rotenone; and oligo, oligomycin.