Fig. 1.

GBP1 targeting of S. flexneri is reduced in A549 cells compared to HeLa cells, but pyroptosis levels are similar. (A) A549 and HeLa cells were infected with the indicated strains, fixed at 1.5 h post infection, and immunostained for GBP1. S. flexneri with GBP1 surrounding >50% of the bacterial surface were counted as GBP1 positive. (B and C) A549 and HeLa cells were grown with or without 100 U/mL IFNγ overnight, then lysed for western blotting. Membranes were probed with antibodies against the indicated GBPs and GAPDH (B), or CASP4, GSDMD, and GAPDH (C). (D–I) A549 and HeLa cells unprimed or primed with 100 U/mL IFNγ overnight were infected with the indicated S. flexneri strains expressing a bioluminescent reporter plasmid. Cell death was measured over time using sytox green fluorescence (D). 4 h timepoint of IFNγ primed cells was used for statistical analysis (E). Supernatant from IFNγ-primed cells infected with S. flexneri was removed at 3 hpi, and LDH levels (F) or IL-18 secretion (G) was measured. Bacterial luminescence was measured over time (H). Luminescence measurements from the 6 h timepoint were used to calculate the growth of each strain in primed cells relative to unprimed cells (I). Caspase-4 – CASP4. Graphs are averages from three independent experiments and are represented by mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test was used, all statistically significant comparisons are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.