Fig. 3.

Lipid trafficking from host membranes to Ehrlichia inclusions depends on endocytic and autophagic pathways. RF/6A cells were seeded on coverslips in a 12-well plate for 1 d and then infected with E. chaffeensis. At 1 dpi, cells were treated for 1 d with 5 μM Dynole 34-2 (A), 100 μM monodansylcadaverine (MDC, B), or 5 mM 3-methyladenine (3-MA, C). Cells were labeled with 5 μM DiI for 6 h, fixed and stained with Hoechst 33342 (pseudocolored green), then observed under a DeltaVision microscope. The boxed area in the merged images is enlarged 3× on the right. Arrows indicate E. chaffeensis inclusions not labeled by DiI. Images are representative of at least three independent experiments. (Scale bars, 10 μm.) (D) Percentage of DiI-labeled membranes of Ehrlichia inclusions or individual bacteria among total inclusions and (E) number of bacteria per host cell were quantified by counting at least 50 cells per group from three independent experiments. Results are shown as the mean ± SD; asterisk (*), significantly different from the control (CTL) group shown in Fig. 2C (P < 0.05, ANOVA).