(A) Noninfected hMDMs were stained with Hoechst to identify their nuclei and challenged with FCCP (10 μM) for 18 hr, while Annexin-V Alexa Fluor 647 was added to the cell culture to monitor cell death. Percentage of living cells (Annexin-V negative) is shown. These values served as reference to interpret the results shown in Figure 4A. (B) hMDMs were infected with GFP-expressing bacteria or left uninfected (noninfected), and then were treated or not with BTB (50 μM). At 24 hr post-infection (hpi), the nuclei of host cells were stained with Hoechst and Annexin-V Alexa Fluor 647 was added to the cell culture to monitor cell death. Percentage of Annexin-V+ cells at 24 hpi is shown. *p<0.1; **p<0.01. ****p<0.0001; ns, nonsignificant (Mann–Whitney U test). (C) hMDMs were infected with Lpp-WT-GFP, the nuclei of host cells were stained with Hoechst, and Annexin-V-647 was added to the cell culture to monitor early cell death from 1 to 18 hpi in nontreated or BTB-treated hMDMs. Single-cell analysis of Annexin-V intensity at 18 hpi is shown. (D) hMDMs were infected as in (A), and Hoechst intensity in the nucleus was analyzed in single cells at 12 hpi. (E) HEK-293 cells stably expressing the FcγRII receptor were transfected with a control plasmid (pGFPmax, Lonza), with a plasmid expressing LpSpl WT (harboring an Xpress tag) or with a plasmid expressing a catalytically inactive LpSpl protein (*K366A, also harboring an Xpress tag). At 24 hr post-transfection, cells were labeled with Hoechst and Annexin-V-647 to monitor cell death, and 1 μM STS was added. At 6 hr, cells were fixed, permeabilized, blocked, and stained with primary mouse antibodies against Xpress tag and secondary anti-mouse Alexa Fluor 488 antibodies. Representative confocal images of transfected cells. Blue, nucleus (Hoechst, Nuc); yellow, Annexin-V-647; green, GFP or anti-Xpress (i.e., LpSpl). Bar: 20 μm. (F) Percentage of Annexin-V-positive cells in each condition, upon addition or not of STS during 6 hr. (G) Single-cell data of Annexin-V mean fluorescence intensity. More than 400 transfected cells were analyzed in each condition. *p<0.1; **p<0.01; ***p<0.001; ****p<0.0001; ns, nonsignificant (Mann–Whitney U test).