Fig. 5: GBP1/4 are sufficient to recruit caspase-4 to Salmonella and together with GBP3 activate the non-canonical inflammasome in naive human epithelial cells.

a Fluorescence confocal microscopy of naive and IFNγ-primed HeLa co-expressing caspase-4-eGFP (green) and mCherry-GBP1 (red) and infected with Salmonella for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. b Fluorescence confocal microscopy of IFNγ-primed or naive HeLa cells co-expressing mCherry-GBP1 (red), Dox-inducible eGFP-GBP1, −2, −3 or −4 (green) and caspase-4-V5 (gray), and infected with Salmonella for 1 h. DNA was stained with Hoechst (blue). eGFP-GBPs were expressed by inducing cells with 1 µg/mL Dox for 16 h. Caspase-4-V5 was visualized by immunostaining with an anti-V5 antibody. Representative confocal images are shown and scale bar corresponds to 10 µm. c Percentage of caspase-4-V5 positive Salmonella at 1 h p.i., quantified out of the mCherry-GBP1-positive bacteria. At least 50 GBP1-positive bacteria were counted per coverslip. d Percentage of cell death in HeLa cells co-expressing constitutive mCherry-GBP1 and Dox-inducible eGFP or eGFP-GBP1, −2, −3 or −4. FLAG-GBP3 and HA-GBP4 were constitutively expressed together using a bicistronic plasmid. Cells were transfected with the indicated plasmids for 24 h. eGFP-GBPs were induced for 16 h with 1 µg/mL Dox, whereas eGFP was induced for 3 h. Cells were then transfected with E. coli-derived LPS (2.5 µg/50,000 cells) for 6 h and cell death values were normalized considering IFNγ-primed HeLa as 100% and naive cells co-expressing mCherry-GBP1 and eGFP as 0%. Graphs show the mean ± SD, and data are pooled from two independent experiments performed in duplicate (c), pooled from three independent experiments performed in triplicate (d) or are representative from two (b) or three (a) independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA.