Fig. 5

Impaired recruitment of V-ATPase is associated with non-acidic Leishmania-PV in B-1P cells in vivo. (A) Representative images of either B-1P cells or peritoneal macrophages (PM) infected with Leishmania amastigotes and stained with AO. The images were acquired in the InCell Analyzer High Content Imaging System by using two fluorescence channels: FITC (DNA and RNA) and Cy3 (acidic compartments). The merge channel images are shown. N: cell nucleus; white arrows: Leishmania nucleus. Scale bar, 10 μm. See also Fig. S5 for additional data. (B) The scatter plot shows the mean number of parasites/infected cells. Each circle represents an individual field, and bars are mean ± SD of technical replicates. ***, p < 0.0001 (Mann-Whitney test). (C) The scatter plot shows the percentage of infection. Each circle represents an individual field, and bars are the mean ± SD of technical replicates. ***, p < 0.0001 (Mann-Whitney test). (D) The scatter plot shows vacuole mean fluorescence intensity (A.U.) in the Cy3 fluorescence channel. Each circle represents an individual vacuole, and bars are the mean ± SD of technical replicates. ***, p < 0.0001 (Mann-Whitney test). (E) Representative images showing infected cells containing Leishmania-PV (white circle) stained for V-ATPase (green) and for IgM (purple signal). Parasites (white arrows) inside PV (white circle) by phase contrast and immunofluorescence staining merge (including LAMP-1 staining in red) showing infected B-1P cells (IgM⁺) and PM (IgM⁻) from the same representative image is showed. Scale bar, 10 μm. (F) Leishmania-PV areas were selected in a random and blinded fashion for analysis of the ratio of the fluorescence intensity for V-ATPase/LAMP-1. Results correspond to the mean ± SD of replicates. ***, p < 0.0001 (Student’s t-test).