Fig. 2: Elevated ROS levels inside LAPosomes are maintained for several hours. | Nature Communications

Fig. 2: Elevated ROS levels inside LAPosomes are maintained for several hours.

From: Oxidation inhibits autophagy protein deconjugation from phagosomes to sustain MHC class II restricted antigen presentation

Fig. 2

A Human macrophages were stimulated with zymosan for 6 h, fixed and costained for gp91 (green) and LC3B (red). B Histogram shows the fluorescence intensity of gp91 (green) and LC3 (red) along the white segment in the merged fluorescence panel of A. Confocal images are representative of three different independent experiments. Scale bar is 5 µm, and inserts are zoomed 5× from white-framed regions of the immune fluorescence micrographs. C Quantitative analysis of the percentage of zymosan-containing vesicles displaying only LC3 or gp91, both or none. Bar graph represents the mean ± SD of three independent experiments, quantified by two independent investigators. One-way ANOVA test: ***p < 0.005. D The first pie chart shows the percentage of gp91+ phagosomes displaying LC3B. Second pie chart on the left shows the percentage of LAPosome (LC3B+) colocalizing with gp91, Mann–Whitney test, two-tailed: **p < 0.005. E Macrophages were stimulated with zymosan, Candida albicans extract, or beads for 1 up to 24 h, lysed and the change on p40-phox protein level were assessed by western blotting. One representative experiment of three is shown. Bars represent the mean of band intensity of p40-phox normalized to the loading control vinculin from three independent experiments (symbols represent one experiment). All conditions are normalized to the unstimulated condition (US), which is set to 1. One-way ANOVA test. F Macrophages were stimulated with OxyBURST-coated zymosan for 6 h, fixed and stained for LC3 (red). OxyBURST intensity levels were measured inside LAPosomes and LC3-negative phagosomes. Confocal images are representative of three different independent experiments. Scale bar is 5 µm, and inserts are zoomed 5× from white-framed regions of the immune fluorescence micrographs. G Bar graph represents OxyBURST intensity increase after 3 or 6 h of OxyBURST-coated zymosan stimulation inside phagosomes or LAPosomes. All conditions are normalized to the condition of phagosome after 3 h of stimulation, which is set to 1. Bars represent the mean ± SD of three independent experiments (symbols represent one experiment) and for each, more than 50 LAPosomes and phagosomes were analyzed. One-way ANOVA test. Source data are provided as a Source Data file.

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