Detection of nitric oxide-mediated metabolic effects using real-time extracellular flux analysis
Fig 5
Differential kinetics of key iNOS inhibitors SEITU and 1400W.
(A) Griess nitrite assay on media supernatant from BMDCs stimulated for 24 hours with U, 100 ng/mL LPS +/- SEITU or 1400W. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 6 biological replicates for U & LPS-100 ng/mL, and 3 for LPS-100 ng/mL + SEITU or + 1400W groups, representative of at least three independent experiments. (B) Modified mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U, 100 ng/mL LPS +/- SEITU or 1400W and the iNOS inhibitors in the run media. Oligo is short for injection of oligomycin, ATP synthase inhibitor. Injection is indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (C) ATP-linked OCR calculated from kinetic traces in (B) by subtracting the average OCR post injection of oligomycin from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 for U, L100 group, n = 3 for SEITU and 1400W group, representative of three independent experiments. (D) Modified mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U, 100 ng/mL LPS. Rot/Ant and iNOSib are short for injections of Rotenone/Antimycin A; electron transport chain complex I and III inhibitors, and iNOS inhibitor SEITU or 1400W were indicated in the key. Injections are indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (E) iNOS-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of iNOS by SEITU or 1400W from average baseline OCR before any injections. n = 4 for SEITU group, n = 2 for 1400W group, representative of three independent experiments. No statistical testing performed due to n = 2 for 1400W group. (F) Percentage of live cells assessed by flow cytometry using a fixable Live/Dead stain on BMDCs treated with 100 ng/mL LPS +/- SEITU or 1400W for 24 hours. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (G) iNOS inhibitor kinetic assay OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with 100 ng/mL LPS. Injection is indicated by a vertical dashed line, iNOSib is either SEITU or 1400W were indicated in the figure key, n = 1 biological replicate, representative of at least three independent experiments. (H) Maximal iNOS-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of iNOS by SEITU or 1400W at 150 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, 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. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.