Figure 3. Inhibition of the ‘reverse mode’ of mitochondrial F_OF₁ ATPase reduces the Δψ_m of L. pneumophila-infected human monocyte-derived macrophages (hMDMs).

(A) hMDMs were infected with GFP-expressing bacteria (green), Lpp-WT or Lpp-ΔdotA, or left uninfected (noninfected). At 5.5 hr post-infection (hpi), cells were labeled with Hoechst to identify cell nucleus (Nuc, blue) and tetramethylrhodamine methyl ester (TMRM) (red) to quantify Δψ_m. At 6 hpi, BTB06584 (BTB, 50 μM), a specific inhibitor of the ‘reverse mode’ of the ATPase, was added and Δψ_m monitored. Representative confocal microscopy images of noninfected (top) and Lpp-WT-infected (bottom) hMDMs (6 hpi) at 5 min before the addition and at 5, 25, and 50 min after the addition of BTB. Bar: 20 μm. (B) Quantification of (C) before (baseline) and after the addition of BTB. Each dot represents the mean ± SD of three independent experiments with a total of six replicates. (C) Same infection conditions than (A) but TMRM values (SD/mean) at 50 min after BTB addition are shown. Data from three experiments with a total of six replicates (three replicates for Lpp-ΔdotA). (D) Single-cell analysis of Δψ_m in Lpp-WT-infected hMDMs treated with BTB (50 μM) or left untreated (nontreated). Single-cell data from one representative experiment *p<0.05; **p<0.01; ns, nonsignificant (Mann–Whitney U test).