Fig 2: GBP1 KO cells were significantly more sensitive to paclitaxel and docetaxel. (A,B) Show real-time 2D color images and corresponding inhibition rate histograms of the DU145 and PC3 cells treated with docetaxel on the left and paclitaxel on the right, respectively. (C,D) Show the results of colony formation assay of the cells treated with docetaxel in the upper part, and the results of paclitaxel treatment in the lower part, respectively. The histograms of the colony formation assay results are shown on the right of (C,D). The data is presented as means from three independent experiments (mean ± S.D). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

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-γ-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-γ-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-γ-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-γ-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-γ-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).

Fig 4: Targeting of GBP1 to S. flexneri is dependent on its functional G domain, its CaaX box, and a C-terminal triple-arginine motif. (A) Schematic depiction of critical protein motifs, domains, and specific residues within the structure of human GBP1. (B) A549 cells were transfected with the mCherry construct (Control), mCherry-tagged wild-type GBP1, and mCherry-tagged GBP1 variants defective in GTP hydrolysis (R48A), nucleotide binding (K51A and S52N), or farnesylation (Δ589–592 and C589A), as indicated. Cells were infected with GFP+ S. flexneri at an MOI of 50 and assessed for colocalization with mCherry at 3 hpi inside mCherry-expressing cells. (C and D) HEK 293T cells were transfected with the mCherry construct (Control), mCherry-tagged wild-type constructs (GBP1 and GBP2), and chimeric (GBP1/2CTSD, GBP1/2CaaX, GBP2/1CTSD, GBP2/1CaaX), deletion mutant (GBP1ΔPBM), triple mutant (R584–586A), or insertion mutant (GBP2+PBM, GBP2+PBM+GBP1-CaaX) constructs. Cells were infected with GFP+ S. flexneri at an MOI of 50 and assessed for colocalization with mCherry at 3 hpi inside mCherry-expressing cells. CTSD, C-terminal subdomain; PBM, polybasic protein motif. Combined data from 3 independent experiments are shown. Per experiment, >200 bacteria were scored. Error bars indicate SEM. Significance was determined by one-way ANOVA relative to results for GBP1 (B) or as indicated (C and D). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

Fig 5: Ectopically expressed human GBP1 colocalizes with Gram-negative S. flexneri and B. thailandensis but not Gram-positive L. monocytogenes in human A549 cells. A549 cells were transfected with the indicated GBP paralogs fused to mCherry at their N termini or transfected with the mCherry control. Cells were primed with IFN-γ at 200 U/ml overnight or left untreated. (A) Cells were infected with GFP-positive S. flexneri at a multiplicity of infection (MOI) of 50, and microscopy images were taken at 1 and 3 hpi (1-hpi images are shown). (B) Cells were infected with GFP-positive B. thailandensis at an MOI of 100, and images were acquired at 8 hpi. (C) Cells were infected with GFP-positive L. monocytogenes at an MOI of 5 and monitored at 1 hpi and 3 hpi (1-hpi image and 1-h data are shown). (A to C) Combined data from 3 independent experiments are shown. Per experiment, >200 bacteria were scored in transfected, i.e., mCherry-positive, cells. Error bars indicate SEM. Significance was determined by two-way ANOVA. ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant; n.d., not detectable.