Fig 2: GBP1 promotes caspase-4-dependent pyroptosis and restricts multinucleated giant cell (MNGC) formation and B. thailandensis replication. (a) Percentage of PI uptake in naive or IFN-?-primed HeLa cells infected with B. thailandensis (MOI of 200). (b) Immunoblotting for caspase-4 processing, GBP1, and tubulin (loading control) of combined supernatants and cell lysates of naive or IFN-?-primed HeLa cells 6 h after infection with B. thailandensis (MOI of 200). (c) Representative fluorescence confocal microscopy images of IFN-?-primed wild-type and GBP1–/– HeLa cells treated with z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) (10 µM). Cells expressing caspase-4–eGFP were infected with B. thailandensis-mCherry (MOI of 100) for 6 h. DNA was stained by Hoechst stain. Bars, 10 µm. (d) Percentage of intracellular caspase-4–eGFP-positive B. thailandensis-mCherry bacteria in IFN-?-primed HeLa cells treated with z-VAD-FMK (10 µM) at 6 h p.i. At least 100 to 200 bacteria per well were counted. (e) Percentage of PI uptake in wild-type, GBP1–/–, CASP4–/–, and GSDMD–/– IFN-?-primed HeLa cells infected with B. thailandensis (MOI of 200). (f) Percentage of nuclei in MNGCs or in single cells, determined by counting the nuclei of naive or IFN-?-primed wild-type, GBP1–/–, CASP4–/–, and GSDMD–/– HeLa cells 20 h after infection with B. thailandensis (MOI of 0.3) in 6 fields of view under each experimental condition. (g) Representative phase-contrast images (magnification, ×40) of naive or IFN-?-primed wild-type, GBP1–/–, CASP4–/–, and GSDMD–/– HeLa cells 20 h after infection with B. thailandensis (MOI of 0.3). The corresponding DNA was stained with Hoechst stain (right), and clustered nuclei, indicating MNGCs, are highlighted by yellow outlines. Bars, 100 µm. (h) Assessment of bacterial invasion in naive or IFN-?-primed wild-type, GBP1–/–, CASP4–/–, and GSDMD–/– HeLa cells 8 or 12 h after infection with B. thailandensis (MOI of 100). Data are representative of results from at least three independent experiments (c, d, and g) or two independent experiments (b). Graphs show the means ± SD, and data are pooled from two independent experiments performed in duplicate (f) or triplicate (h) or are representative of results from at least two independent experiments performed in triplicate (a and e). For panels a and e, the area under the curve (AUC) under each experimental condition was calculated, and data were analyzed by a parametric t test (a) or one-way ANOVA (e). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by 2-way ANOVA [f and h]).

Fig 3: GBP1 does not impair actin-based motility or promote direct bacteriolysis of cytosolic B. thailandensis. (a) Percentage of B. thailandensis bacteria with actin tails at 5 h p.i. in naive and IFN-?-primed CASP4–/– HeLa cells (MOI of 30). At least 100 to 200 bacteria per coverslip were counted. (b) Representative fluorescence confocal microscopy images of naive and IFN-?-primed CASP4–/– HeLa cells expressing iRFP703-GBP1 and infected with B. thailandensis-mCherry (MOI of 30) for 5 h. DNA was stained by Hoechst stain, and F-actin was labeled with CellMask green actin tracking stain. Bars, 10 µm. (c) Percentage of B. thailandensis bacteria with actin tails that are GBP1 positive or negative in naive and IFN-?-primed CASP4–/– HeLa cells expressing iRFP703-GBP1. Cells were infected with B. thailandensis-mCherry for 5 h (MOI of 30) and fixed, and F-actin was labeled with CellMask green actin tracking stain. Between 100 and 300 bacteria were counted per coverslip. (d and e) Time-lapse fluorescence confocal microscopy images of naive (d) and IFN-?-primed (e) GSDMD–/– HeLa cells expressing N-terminally mCherry-tagged GBP1 and LifeAct-eGFP infected with B. thailandensis (MOI of 50). Images were acquired every 5 min. Bars, 10 µm. (f) Percentage of B. thailandensis bacteria with actin tails in naive and IFN-?-primed wild-type and GBP1–/– HeLa cells. Cells were treated with z-VAD-FMK (10 µM) and infected for 5 h at an MOI of 30. Between 100 and 300 bacteria were counted per coverslip. (g) Time-lapse fluorescence confocal microscopy of naive HeLa cells expressing N-terminally eGFP-tagged GBP1 and infected with B. thailandensis-mCherry (MOI of 50). MNGCs are indicated by dashed white lines. DIC, differential interference contrast. Images were acquired every 3 min. Bars, 10 µm. Data are representative of results from at least three independent experiments (b, d, e, and g). Graphs show the means ± SD, and data are pooled from three (c and f) or five (a) independent experiments performed in duplicate. ns, not significant (by a parametric t test).