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

Figure 2.

Figure 2

GBP1 Only Targets Already Cytosolic Salmonella and Recruits Caspase-4

(A) Representative immunofluorescence images at 2 h p.i. and quantification of the proportion of cytosolic Salmonella Typhimurium (STm) from differentially permeabilized, IFNγ-primed THP-1 WT or ΔGBP1 cells infected with STm SL1344 (MOI = 30) at indicated time p.i. Cells are outlined by the white, dashed line. Gray: STm; green: pseudo-colored cytosolic and extracellular STm; blue: nuclei. Scale bar, 10 μm.

(B) Representative immunofluorescence images at 2 h p.i., and quantification of GBP1 recruitment to cytosolic and intra-vacuolar STm in IFNγ-primed and Dox-treated THP-1 ΔGBP1+Tet-mCH-GBP1, infected with STm SL1344 (MOI = 30) at indicated times p.i. from differentially permeabilized cells stained for cytosolic STm and total STm. Cells are outlined by the white, dashed line. Red: mCH-GBP1; gray: STm; green: cytosolic STm; blue: nuclei. Scale bar, 10 μm.

(C) Quantification of galectin-8 (Gal-8) recruitment to STm SL1344-GFP in IFNγ-treated THP-1 WT or ΔGBP1 at the indicated times post infection.

(D) Representative immunofluorescence images at 1 h and quantification of Gal-8 recruitment to STm in IFNγ- and Dox-treated THP-1 ΔGBP1+Tet-mCH-GBP1 infected with STm SL1344-GFP (MOI = 30) at the indicated time post infection. Red: mCH-GBP1; gray: STm; green: Gal-8; blue: nuclei. Scale bar, 10 μm.

(E) Representative quantification of CellMask fluorescence intensities surrounding STm in infected THP-1 ΔGBP1+Tet-mCH-GBP1 pre-treated with IFNγ and Dox to induce GBP1 expression at 4 h p.i. plotted depending on whether STm were decorated with GBP1 (+) or not (−). N = number of STm quantified.

(F) Fold-change of intracellular STm colony forming units (CFUs) at 18 h p.i. normalized to 2 h p.i. of IFNγ-primed THP-1 WT or ΔGBP1 cells measured using gentamicin infection-protection assays.

Graphs in (A), (B) (C), (D), and (F) show mean ± SEM from n = 3 independent experiments and in (E) representative of n = 2 experiments. ∗p ≤ 0.05 or ∗∗∗p ≤ 0.001 in (E) from nested Student’s t test comparing GBP1+ to GBP1− objects from the two experiments following adjustment for multiple comparisons and in (A), (C), and (F) from two-way ANOVA following adjustment for multiple comparisons; ns, not significant.

See also Figure S3.