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. 2018 Dec 3;9:2761. doi: 10.3389/fimmu.2018.02761

Figure 3.

Figure 3

NADPH oxidase-mediated ROS response to H. capsulatum is essential for LC3 conversion in macrophages. (A) Macrophages from WT mice were preloaded with CM-H2DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for indicated time. 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. Mean ± SEM are shown (n = 4). (B,C) Macrophages from WT mice were treated with or without DPI (5 μM), Mito-Tempo (10 μM) (B), and gp91ds-tat (10, 50, and 100 μM) (C) for 1 h prior to stimulation with H. capsulatum (MOI = 5). Cell lysates were collected after 1 h of stimulation and analyzed by Western blotting. (D) Macrophages from WT or Ncf1−/− mice were stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. Cell lysates were subjected to Western blotting. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin; mean ± SEM are shown. [n = 3 for (B,C); n = 4 for (D)]. (E,F) Macrophages from WT and Ncf1−/− mice were stimulated with H. capsulatum (MOI = 5) for 1 h. Cells were fixed and stained for LC3B (green), F-actin (red), and the nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus image point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as 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 4 independent experiments. (G) Macrophages from WT and Ncf1−/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells taking up H. capsulatum were analyzed by flow cytometry. Mean ± SEM are shown (n = 4). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 [ANOVA with Tukey post-hoc analysis (B,C) or Bonferroni's multiple comparisons post-hoc test (D); 2-tailed t-test (F,G)].