Article Figures & Data
Figures
- FIGURE 1.
IgE/Ag-activated mast cells undergo a rapid increase in glycolysis. (A and B) WT BMMCs were sensitized with IgE for 3 h and activated in-Seahorse with the indicated concentrations of DNP32 and DNP5 for 2 h. Data in (A) show the change in glycolysis (ECAR), whereas (B) shows mitochondrial respiration (OCR). (C and D) WT and Tim-3 knockout (KO) BMMCs were sensitized with IgE for 3 h and stimulated with indicated Ags directly in-Seahorse for 2 h. Arrows indicate when Ag was injected. Data are representative of three independent experiments.
- FIGURE 2.
Prestimulated mast cells increase both glycolytic potential and mitochondrial respiration. WT BMMCs were sensitized with 1 μg/ml IgE overnight and stimulated with DNP32 (high) and DNP5 (low) valency for 2.5 h prior to stress test by Seahorse flux analyzer. (A) SRC was calculated as the difference between FCCP-uncoupled and basal OCR, whereas in (B) glycolytic reserve (GR) was calculated as the difference between oligo-stimulated and basal ECAR. Results are representative of three independent experiments.
- FIGURE 3.
Mast cells require glycolysis for Ag-induced degranulation and cytokine production. (A and B) WT BMMCs were loaded with Lysotracker Deep Red and sensitized with IgE prior to stimulation with Ag alone, Ag together with DMSO or the indicated concentrations of DCA. Degranulation was assessed by flow cytometry analysis of loss of positive Annexin V staining and decreased Lysotracker staining. A representative degranulation assay is shown in (A), and the combined results of three experiments are shown in (B). (C) BMMCs generated from Nur77GFP Tg mice were sensitized with IgE and stimulated with Ag, plus varying concentrations of DCA, or with vehicle control. Nur77GFP expression was measured as an indication of Ag-induced FcεRI activation. Line colors correspond to the bars in (D). (D) IL-6 release was analyzed by ELISA of culture supernatant harvested after 6 h of stimulation. Results are representative of three independent experiments. *p < 0.05, **p < 0.005.
- FIGURE 4.
Mast cells require oxidative phosphorylation for Ag-induced degranulation and cytokine production. (A) WT BMMCs were stimulated as shown, together with DMSO or the indicated concentrations of rotenone. IL-6 release was analyzed by ELISA of culture supernatant harvested after 6 h of stimulation. (B) WT BMMCs were loaded with Lysotracker Deep Red and sensitized with IgE prior to stimulation with Ag alone. Degranulation was assessed by flow cytometry analysis of loss of positive Annexin V staining and decreased Lysotracker staining. (C and D) IL-6 secretion and degranulation were measured as above, in the presence of DCA or rotenone alone, or the two together. The combined results of three experiments are shown. *p < 0.05, **p < 0.005.
- FIGURE 5.
Fatty acid oxidation is dispensable for mast cell activation. (A) WT BMMCs were stimulated as shown, together with DMSO or the indicated concentrations of etomoxir. IL-6 release was analyzed by ELISA of culture supernatants harvested after 6 h of stimulation. (B) BMMCs generated from Nur77GFP Tg mice were sensitized with IgE and stimulated with Ag, along with varying concentrations of etomoxir, or with vehicle control. Nur77GFP expression was measured as a readout of Ag-induced FcεRI activation. Line colors correspond to the bars in (A). (C) WT BMMCs were loaded with Lysotracker Deep Red and sensitized with IgE prior to stimulation as indicated. Degranulation was assessed by flow cytometry analysis of loss of positive Annexin V staining and decreased Lysotracker staining.
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