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. 2019 Jun 3;38(13):e100926. doi: 10.15252/embj.2018100926

Figure 2. GBP1 promotes ASC‐ and CASP8‐dependent atypical apoptosis in Toxoplasma‐infected human macrophages.

Figure 2

  • A
    Propidium iodide (PI) uptake in wild‐type THP‐1 or ΔCASP1, ΔCASP4, ΔASC cells, or THP‐1 stably expressing GSDMD‐targeting miRNA (GSDMD miR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two‐way ANOVA following adjustment for multiple comparisons.
  • B
    Real‐time apoptosis assay (Annexin V (AnnV)‐Glo luminescence) from IFNγ‐primed primary human MDMs transfected with non‐targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units.
  • C
    Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre‐treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.
  • D, E
    Live time‐lapse imaging to visualize apoptotic cell death and measure caspase‐3/7 activities using a fluorescent caspase substrate. THP‐1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase‐3/7 activity from an 18‐h time course (mean ± SEM from n = 3 independent experiments). Representative phase‐contrast images overlaid with fluorescence signal for caspase‐3/7 activity (green) images are shown in (E) from IFNγ‐primed THP‐1 cells infected with type I Tg captured at indicated times post‐infection. Scale bar 20 μm.
  • F
    AnnV‐Glo assay from IFNγ‐primed THP‐1 transfected with siRNA against the indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real‐time AnnV‐Glo luminescence assays similar to those in (B) is plotted as mean ± SEM from n = 3 independent experiments. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 from two‐way ANOVA for indicated comparisons following adjustment for multiple comparisons; ns, not significant.
  • G
    AnnV‐Glo assay from IFNγ‐primed primary human MDMs transfected with siRNA against CASP8, CASP10, and non‐targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real‐time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors. ****P ≤ 0.0001 from two‐way ANOVA following adjustment for multiple comparisons; ns, not significant.

Source data are available online for this figure.