Figure 3. PPARγ expression regulates mitochondrial ROS generation.

(A) Amplex Red Assay was used to quantitate H₂O₂ generation in sorted lung CD11c⁺ cells from naïve WT mice in the presence or absence of RSG (10 µM) and tolerized WT mice in the presence or absence of Rotenone (10 µM), Antimycin A (100 µM) or L-NAME (50 µM). Complex I and III activities were measured to assess specificity and dose efficiency of Rotenone and Antimycin A, respectively. (B) Quantification of H₂O₂ generation in sorted lung CD11c⁺ cells from naïve and tolerized WT and PPARγ^ΔAPC mice. Data for panel A and B represents one of 3 independent experiments and are shown as mean ± SEM. (C) Intracellular H₂O₂ detection in sorted lung CD11c+ cells from tolerized WT and PPARγ^ΔAPC mice using the H₂O₂-specific probe PF6-AM. During the incubation with PF6-AM, OVA (10 µg/ml) or Der p1 (0.5 µg/ml) was included. In the absence of antigen in vitro, a similar profile was obtained with lower signal intensity in the WT cells and none detected in the PPARγ-deficient cells (data not shown). As a positive control, exogenous H₂O₂ (100 µM) was added to the CD11c⁺ cells in vitro ± catalase (10 µM). Representative immunofluorescence images (left) and fluorescence quantification (right) represent cytosolic H₂O₂ signal per cell per field (5–6 fields chosen randomly using DAPI as a guide and then assessed for PF6-AM-specific signal). Scale bar-100 µm and magnification 20X. Bar graphs show data as mean values ± SEM. (D–E) Lung CD11c+ cell from tolerized WT and PPARγ^ΔAPC mice were analyzed for (D) MnSOD and Cu/ZnSOD mRNA (left panel) and protein expression (right panel) and (E) SOD activity. Data are shown as mean values ± SD. (F) Representative immunofluorescence images (left) and sphericity and mitochondrial volume/cell estimation (right) of lung CD11c⁺ cells stained with Tom20 (green), DAPI (blue), and phalloidin (red). Scale bar- 10 µm; magnification 60X. (G) Quantification of mitochondrial DNA (mtDNA) and nuclear (nDNA) in purified lung CD11c⁺ cells from naïve and tolerized WT and PPARγ^ΔAPC mice. Data in panels (F) and (G) represent one of two independent experiments and shown as mean values ± SD. ND: Not detected, NS: Not significant, *p< 0.05, ***p< 0.001, ****p<0.0001. See also Figure S4.