FIG 1.
P. acanthamoebae infection induces mitochondrial apoptosis in HeLa cells. HeLa cells were infected with P. acanthamoebae and analyzed for cell death and caspase-3 activation. (A) HeLa CTRL or Bax/Bak-deficient cells were analyzed for activation of caspase-3 by flow cytometry 24 and 48 h after infection. (B) DEVD (caspase-3 reporter cell line) and DEVG (control cell line) were infected for 24 h to analyze caspase activation. (C) CTRL and mutant cell lines (Bax/Bak or Noxa deficient; Bcl-XL overexpressing) were infected with P. acanthamoebae and stained for active caspase-3 after 24 h. (D) HeLa CTRL and Bax/Bak-deficient cells were infected with P. acanthamoebae (24, 48, or 72 h) and stained with a live/dead stain to determine percentage of dead cells in the population. (E) CTRL and mutant cell lines (Bax/Bak or Noxa deficient; Bcl-XL overexpressing) were infected with P. acanthamoebae and stained for active caspase-3 after 24 h and 48 h of incubation at 30°C. (F) DEVD and DEVG were infected for 24 h to analyze caspase activation (incubation at 30°C). The MOI was 25 in all experiments. p.c., positive control, treatment with Mcl-1 inhibitor S63845 (500 nM) and ABT-737 (1 μM) for 1 h; h.-t., heat treated (1 min at 95°C). Data are shown as means/standard errors of the mean (SEM) of at least three independent experiments. Experiments for positive controls were done separately. *, P < 0.05 (unpaired two-tailed t test) between control and mutant cells (A and C to E) or P < 0.05 (paired two-tailed t test) between uninfected and infected cells (B and F).