Figure 3. More apoptosis in L. major–infected cells.

(A) In vivo quantification of cell death dynamics. DEVD, caspase-3 recognition site. (B) Rag1^–/– hematopoietic stem cells (HSC) transduced with caspase-3 reporter DEVD or noncleavable control (DEVG) were FACS-sorted and transferred into lethally irradiated hosts. (C) Z-projections of infected ear dermis of CFP-DEVD-YFP (left) and CFP-DEVG-YFP control (right) mice. Eighteen slices of 3-μm-spaced Z-stacks are projected. Scale bars: 100 μm. Gray, 3D surface plot of detected reporter-expressing cells; black, mapped cell positions; red, infected cells; blue, cells with increased CFP/FRET ratio; pink, infected cells exhibiting FRET loss (map only). (D) Z-projections (left) and xy/xz/yz sections (right) of Lm^DsRed-infected (top panels) and uninfected (bottom panels) apoptotic cells over time. Eleven and 14 slices (3-μm-spaced Z-stacks) taken longitudinally are projected. Scale bars: 10 μm. (E) Percentage of initial CFP and FRET fluorescence and normalized CFP/FRET ratio over time of cells in D. (F) Gating of cell tracks infected (left) or losing FRET (right), in infected (red) and uninfected (gray) cells. (G) FRET loss in infected (left panels) and uninfected (right panels) cells for CFP-DEVD-YFP (left) and CFP-DEVG-YFP control (right) mice. Fifty randomly selected tracks of at least 200 tracks analyzed per condition are shown. (H) FRET loss in infected and uninfected CFP-DEVD-YFP and CFP-DEVG-YFP control mice. Each symbol represents 1 track. Horizontal bars denote the median. (I) FRET loss in infected and uninfected cell populations from 4–5 animals imaged. Each symbol pair represents 1 mouse ear. (J) NucView405 (caspase-3 activity) in Lm^DsRed-infected (red) and uninfected (black) hMDMs 0 hours and 20 hours after incubation. Each symbol pair represents 1 donor. **P < 0.01, ****P < 0.0001 by Kruskal-Wallis with Dunn’s posttest in H, and paired 2-way ANOVA with Bonferroni’s posttest in I and J.