Detection of nitric oxide-mediated metabolic effects using real-time extracellular flux analysis
Fig 6
Visualization of NO modulation of DC metabolic reprogramming in real time.
(A) Glycolytic burst assay ECAR kinetic trace of extracellular flux analysis on freshly harvested BMDCs. Cells were stimulated by injection with 100 ng/mL LPS +/- SEITU. Injection is indicated by dashed a vertical dashed line, n = 1 biological replicate, representative of at least three independent experiments. (B) NO-independent glycolytic burst calculated from kinetic traces in (A) by subtracting the average ECAR post stimulation with LPS at the highest point ~100 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA using pairwise comparisons by biological replicate, adjusted p values are reported (p value > 0.05 ns), n = 6 independent BMDC cultures from 3 mice, representative of at least three independent experiments. (C) NO-dependent glycolytic reprogramming calculated from kinetic traces in (A) by subtracting the average ECAR post stimulation with LPS at 600 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 6 independent BMDC cultures from 3 mice, representative of at least three independent experiments. (D) Glycolytic burst assay OCR kinetic trace of extracellular flux analysis on freshly harvested BMDCs. Cells were stimulated by injection with 100 ng/mL LPS +/- SEITU. Injection is indicated by dashed a vertical dashed line, n = 1 biological replicate, representative of at least three independent experiments. (E) NO-dependent respiration inhibition calculated from kinetic traces in (D) by subtracting the average OCR post stimulation with LPS at 600 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA using pairwise comparisons by biological replicate, adjusted p values are reported (p value > 0.05 ns), n = 6 independent BMDC cultures from 3 mice, representative of at least three independent experiments. If no p value is provided, the data is non-significant (p value > 0.05) and therefore, not included on the figure itself.