Figure 5.

NLRX1 acts independently of ROS response to promote H. capsulatum-induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1^−/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown (n = 3) (A,B). (C,D) Macrophages from WT and Nlrx1^−/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C). Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3⁺ phagosome are shown as mean ± SEM of 3 independent experiments (D). (E) Macrophages from WT and Nlrx1^−/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry (n = 7). (G) Macrophages from WT and Nlrx1^−/− mice were preloaded with CM-H₂DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS⁺ cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells (n = 4). Bars represent the mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G); 2-tailed t-test (D,E)].