Fig. 4.

GBP1 enhances LPS release from intracellular Shigella but is not essential for the induction of pyroptosis of infected cells. (A–H) WT or GBP1^−/− HeLa or HCT8 cells unprimed or primed overnight with 10 ng/mL IFNγ were infected with the indicated Shigella strains that each carry pNG162-AfaI at an MOI of 3. (A–F) Two hours p.i., cells were lysed with 0.005% digitonin and separated into cytosolic and residual fractions. LPS levels in filtered cytosolic extracts were quantified using a LAL assay (A and D); fractionation efficiency and GBP1 levels were assessed by immunoblotting fractions with designated antibodies (B and E), and numbers of bacteria within the residual and filtered supernatant fractions were enumerated (C and F). (G and H) Thirty minutes p.i., gentamicin, PI, and Hoechst were added to the medium, and cells were imaged using an automated fluorescent microscope. Time courses of cell death PI⁺/Hoechst⁺ cells are shown. Values shown are the mean ± SEM of three experimental replicates. Where indicated, statistical significance was assessed using two-way ANOVA with Tukey’s post hoc test. ****P < 0.0001, ns = nonsignificant.