Detection of nitric oxide-mediated metabolic effects using real-time extracellular flux analysis
Fig 2
NO produced via iNOS in BMDCs suppresses mitochondrial respiration in a dose dependent manner.
(A) Nos2 gene transcription assessed by RT-qPCR from BMDCs stimulated for 5 hours with U or 100 ng/mL LPS. Signals normalized to β-actin as the housekeeping gene via the 2(ΔCt) method. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (B) Western blot for iNOS from protein lysates isolated from BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. β-actin serves as a loading control, n = 4 biological replicates, representative of at least three independent experiments. (C) Griess nitrite assay on media supernatant from BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (D) Mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. Oligo, FCCP, and Rot/Ant are short for injections of oligomycin; ATP synthase inhibitor, fluoro-carbonyl cyanide phenylhydrazone; mitochondrial membrane uncoupler, and rotenone/antimycin A; electron transport chain complex I and III inhibitors, respectively. Injections indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (E) Mitochondrial-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of the electron transport chain by rotenone/antimycin A from average baseline OCR before any injections. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (F) Mitochondrial-linked OCR calculated from kinetic traces during a standard mitochondrial stress test on BMDCs treated with a titration of LPS from 0 ng/mL up to 100 ng/mL, n = 2 biological replicates, representative of three or more independent experiments.