Fig. 4

LysoTracker staining after transfection of siRNA. ACA1_114460, ACA1_091500, or ACA1_362260-specific siRNA was inoculated into A. castellanii, which were subsequently co-cultured with L. pneumophila for 12 h (siRNA-A+L). Acanthamoeba and excreted vesicles containing Legionella were stained with LysoTracker. a A+L, b ACA1_114460 siRNA-transfected A+L, c ACA1_091500 siRNA-transfected A+L, d ACA1_362260 siRNA-transfected A+L, e excreted phagolysosome quantification. Arrows, excreted vesicles containing Legionella; black arrowheads, excreted vesicles containing Legionella without lysosomes; and white arrowheads, excreted vesicles containing Legionella and lysosomes. Images were acquired at ×1000 magnification