Figure 6.

NLRX1 promotes LAP through association with TUFM-ATG5-ATG12 complex. (A) Macrophages from WT mice were stimulated with or without H. capsulatum. Cell lysates were collected at 30 min after stimulation and used for immunoprecipitation with anti-NLRX1, anti-TUFM or isotype control antibodies, followed by immunoblotting with indicated antibodies. IP, IB, and WCL denote immunoprecipitation, immunoblotting, and whole-cell lysate, respectively. (B) Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 60 min. Cells were fixed and stained for NLRX1 (red), TUFM (green), F-actin (violet), and nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. Box areas are shown at higher magnification in the bottom left corner of the corresponding image. The intensity of different fluorochromes along the white arrow in the merged image is shown as histogram on the right. (C) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. After stimulation, cell lysates were collected and assessed by Western blotting. (D) Macrophages from WT and Nlrx1^−/− mice were treated with or without DPI (5 μM) 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. Data shown in the lower panel of (C,D) are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown (n = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (C,D)].