Fig. 3

Impaired recruitment of V-ATPase to the Leishmania-PV in B-1P cells. B-1P and RAW cells were infected with stationary-phase promastigotes and stained with acridine orange (AO) and Hoechst for DNA staining in live microscope imaging assays. (A) Representative images of B-1P cells containing Leishmania-PV and stained with AO and Hoechst (nuclei of cells) showing neutral/basic pH (low-AO signal) or acidic pH (high-AO signal). The panel at right represents a merged image of the AO red channel merged (at left) including the phase contrast image. White arrows and punctuated circles show Leishmania-PV. Scale bar, 10 μm. (B) Percentage of Leishmania-PV containing low/high pH identified in (A). Results correspond to the mean ± SD of three independent biological assays. ***, p < 0.0004 (two-way ANOVA). (C) Number of Leishmania-PV per 100 infected B-1P or RAW cells after 48 h of infection. No statistical significance (n.s). (D) Representative images of Leishmania-PV (PV) in B-1P cells (top images) and BMM (bottom images) showing V-ATPase (green) staining marker. In the inset, the phase contrast is shown with white arrows indicating the PV. V-ATPase image was merged with DAPI in the panel on the right. Punctuated yellow circles indicate Leishmania-PV; Scale bar, 5 μm. See also Fig. S3 for additional data. (E) Representative image of the fluorescence intensity analysis in the selected area in B-1P cells (yellow circles) corresponding to the Leishmania-PV, and are represented in arbitrary units (A.U.). (F) Quantification of the recruitment of V-ATPase or (G) LAMP-1 marker to the Leishmania-PV in B-1P cells and BMM. Results correspond to the mean ± SD of three independent biological assays. *, p < 0.0427 (Wilcoxon test); no statistical significance, (n.s.).