Figure 2.
IFNγ-induced increases in OCR are unique to type II interferon and not dependent on total mitochondrial mass, glucose uptake, or substrate-specific catabolism. (A) Real-time changes OCR measured by Seahorse following treatment with media alone or high (50 ng/mL) or low (5 ng/mL) dose of IFNγ, IFNα, or IFNβ for 24 hours prior to the start of the assay. Basal OCR was measured, then PMA (100 ng/mL) was injected during the assay, and OCR was monitored (n = 5 technical replicates). Data in panel A are representative tracings, with bar graphs indicating basal and PMA-stimulated OCR of 3 independent experiments. (B-C) Primary human monocytes were stimulated with media alone, LPS, IFNγ, or LPS plus IFNγ prior to the start of the assay, then (B) total mitochondrial mass was measured using MitoTracker Green staining, and (C) glucose uptake was measured using 2-NBDG by flow cytometry, and the geometric mean fluorescent intensities are indicated in the bar graphs. Data in panels B-C were analyzed by 1-way ANOVA with Dunnett’s multiple comparisons test. Error bars are mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, ****P < .0001. (D) Schematic representation of the primary mitochondrial fuel sources and their respective inhibitors used in panel E. Primary human monocytes were stimulated with or without IFNγ and in the presence of absence of metabolic inhibitors according to the Mito Fuel Flex Test protocol; healthy control monocytes were left untreated or treated with etomoxir (4 µM), UK5099 (2 µM), or Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (3 µM) throughout the 24-hour IFNγ-stimulation period. Oxygen consumption was assessed by Seahorse assay according to a modified Mito Fuel Flex Test protocol (Agilent, 103260-100). Basal OCR measurements were captured after the 24-hour stimulation. PMA (100 ng/mL) was injected during the assay, and PMA-stimulated OCR measurement were collected. Rotenone and antimycin A were then injected, and OCR measurements were collected. For data in panels A and E, n = 5 technical replicates, and the tracings are representative of 3 independent experiments. TCA, tricarboxylic acid.

IFNγ-induced increases in OCR are unique to type II interferon and not dependent on total mitochondrial mass, glucose uptake, or substrate-specific catabolism. (A) Real-time changes OCR measured by Seahorse following treatment with media alone or high (50 ng/mL) or low (5 ng/mL) dose of IFNγ, IFNα, or IFNβ for 24 hours prior to the start of the assay. Basal OCR was measured, then PMA (100 ng/mL) was injected during the assay, and OCR was monitored (n = 5 technical replicates). Data in panel A are representative tracings, with bar graphs indicating basal and PMA-stimulated OCR of 3 independent experiments. (B-C) Primary human monocytes were stimulated with media alone, LPS, IFNγ, or LPS plus IFNγ prior to the start of the assay, then (B) total mitochondrial mass was measured using MitoTracker Green staining, and (C) glucose uptake was measured using 2-NBDG by flow cytometry, and the geometric mean fluorescent intensities are indicated in the bar graphs. Data in panels B-C were analyzed by 1-way ANOVA with Dunnett’s multiple comparisons test. Error bars are mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, ****P < .0001. (D) Schematic representation of the primary mitochondrial fuel sources and their respective inhibitors used in panel E. Primary human monocytes were stimulated with or without IFNγ and in the presence of absence of metabolic inhibitors according to the Mito Fuel Flex Test protocol; healthy control monocytes were left untreated or treated with etomoxir (4 µM), UK5099 (2 µM), or Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (3 µM) throughout the 24-hour IFNγ-stimulation period. Oxygen consumption was assessed by Seahorse assay according to a modified Mito Fuel Flex Test protocol (Agilent, 103260-100). Basal OCR measurements were captured after the 24-hour stimulation. PMA (100 ng/mL) was injected during the assay, and PMA-stimulated OCR measurement were collected. Rotenone and antimycin A were then injected, and OCR measurements were collected. For data in panels A and E, n = 5 technical replicates, and the tracings are representative of 3 independent experiments. TCA, tricarboxylic acid.

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