Dysregulated phagosomal maturation in Lyst-mutant Bg-J cells. (A and B) WT and Bg-J BMDCs were exposed to LPS beads (3–5 µm diameter). After 15 (A) and 60 (B) min of incubation, cells were fixed, permeabilized, and stained for EEA1 and Rab7. Nuclei were counterstained with DAPI. Arrowheads indicate beads. Insets show enlargements of bead-containing phagosomes. Bars, 5 µm. (Right) Line profiles from confocal microscopy images are shown. The fluorescence signal intensity (FL intensity) of EEA1 (red) and Rab7 (green) was quantified along the yellow line drawn across internalized LPS beads (shown in the overlay). Percentages of bead-containing phagosomes in A that were positive for EEA1 at 15 min: WT, 47.9% ± 27.4 (n = 18); Bg-J, 53.3% ± 13.9 (n = 6; P = 0.316; Mann-Whitney U test). Percentages of bead-containing phagosomes in B that were positive for Rab7 at 60 min: WT, 49.9% ± 27.9 (n = 21); Bg-J, 0% ± 0 (n = 12; P < 0.0001; Mann-Whitney U test). a.u., arbitrary units; DIC, differential interference contrast; rel., relative. (C) WT and Bg-J BMDMs were exposed to LPS beads (2 µm diameter). After 30 min of incubation, cells were fixed, permeabilized, and stained for EEA1 and Rab7. Nuclei were counterstained with DAPI. Bars, 5 µm. BF, bright field. (Right) Graphs show quantification of percentages of bead-containing phagosomes in WT and Bg-J cells that were positive for EEA1 (top) or positive for Rab7 (bottom). Number of evaluated bead-containing phagosomes: WT, n = 70; Bg-J, n = 127. **, P < 0.01 (Mann-Whitney U test). Error bars represent SD. All data are representative for at least three independent experiments.