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. 2020 Aug 11;32(6):108008. doi: 10.1016/j.celrep.2020.108008

Figure 5.

Figure 5

Caspase-1-Driven GBP1 Proteolysis Regulates Cell Death during Salmonella, but Not Toxoplasma, Infection

(A) Representative immunofluorescence images and quantification of recruitment of GBP1 to STm in IFNγ-primed and Dox-treated THP-1 ΔGBP1+Tet-mCH-GBP1 or mCH-GBP1D192E cells infected with STm SL1344-GFP (MOI = 30) for 2 h. Red: mCH-GBP1; Gray: STm; Blue: Nuclei. Scale bar, 10 μm.

(B) Representative immunofluorescence images as rainbow intensity diagram of GBP1 WT or D192E recruitment to STm SL1344-GFP at 2 h p.i. (left) and quantification of fluorescence intensity and coat thickness measured as full-width half maximum (FWHM) of the radial intensity distribution surrounding the STm centroid (yellow asterisks; right). Scale bar, 1 μm. N = number of quantified GBP1+ bacteria in the respective condition.

(C) Left: IL-1β ELISA from the indicated THP-1 cells primed with IFNγ and Dox and infected with STm SL1344 (MOI = 30) at 4 h post-infection. Middle: real-time propidium iodide (PI) uptake assay from IFNγ-primed THP-1 cells of the indicated genotypes and infected with STm SL1344 for 4 h. Right: LDH release assay to measure cell death at 4 h p.i. with STm SL1344.

(D) Representative immunofluorescence image of mCherry-tagged GBP1 fragments in IFNγ- and Dox-primed THP-1 ΔGBP1+Tet cells expressing the indicated GBP1 fragment infected with STm SL1344 (MOI = 30) for 2 h. Red: mCH-GBP1; white: STm; blue: nuclei. Scale bar, 10 μm.

(E) Area under the curve (AUC) from 4 h live PI uptake cell death assay in THP-1 WT or THP-1 ΔGBP1+Tet-GBP1 cells expressing either GBP1 fragment 1-192 or 193-592, pre-stimulated with IFNγ only, with IFNγ and Dox to induce GBP1 expression or left UT and infected with STm SL1344 (MOI = 30).

(F) Representative immunofluorescence images and quantification of recruitment of GBP1 in IFNγ-primed and Dox-treated THP-1 ΔGBP1+Tet-mCH-GBP1 or mCH-GBP1D192E cells infected with type I or type II Tg for 6 h. Red: mCH-GBP1; gray: Tg; blue: nuclei. Scale bar, 10 μm.

(G) AnnV-Glo assay of THP-1 WT and ΔGBP1 cells stably reconstituted with Tet-GBP1 WT or GBP1D192E as indicated, infected with type I or type II Tg for 18 h. Plotted as AUC from real-time assays.

Graphs in (A), (C), (E), (F), and (G) show mean ± SEM from n = 3 independent experiments and in (B) representative of n = 3 independent experiments. ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 for indicated comparisons in (B) from nested Student’s t test comparing means of the n = 3 experiments, in (A) from one-way ANOVA and in (C), (E), and (F) from two-way ANOVA following adjustment for multiple comparisons; ns, not significant.

See also Figure S5.