Fig. 1: IFNγ priming is required for LPS-induced caspase-4 activation in human epithelial cells.

a–c Intracellular bacterial fold-replication (a) and release of LDH (b, c) in naive or IFNγ-primed wild-type, CASP4^–/– or GSDMD^–/– HeLa cells, at 1 or 6-h post-infection (p.i.) with Salmonella. Cells in 96-well plates were infected for 30 min, washed and gentamicin was added to kill extracellular bacteria. At the indicated time points supernatant was collected to determine the release of LDH, and then cells were lysed and the number of viable intracellular bacteria was determined by counting colony forming units (CFUs). The bacterial fold-replication was calculated relative to 1 h p.i. d Percentage of CHQ-resistant cytosolic Salmonella in naive or IFNγ-primed HeLa at 1.5 h p.i. Cells were infected for 30 min as in (a) and then treated with gentamicin ± CHQ for an additional 1 h before cells were lysed and bacteria counted by CFUs. The percentage of cytosolic bacteria was calculated as the ratio of (CHQ + gentamicin^resistant / gentamicin^resistant). e–g Release of LDH from naive or IFNγ-primed cells, 5 h after transfection with LPS (2.5 µg/50,000 cells) or 3–4 h after electroporation with LPS (300 ng/50,0000 cells). h Western blot analysis of full length (p43) and cleaved (p32) caspase-4 in the supernatants and cell lysates from naive or IFNγ-primed HaCaT cells, upon transfection with E. coli LPS LPS (2.5 µg/50,000 cells). i Streptavidin pull-down assay of the binding of biotin-conjugated LPS to endogenous caspase-4 from the lysates of naive or IFNγ-primed HBEC3-KT. Cells in 6-well plates were transfected with LPS-biotin (10 µg) or left untransfected, and biotinylated substrate was pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by immunoblotting for caspase-4. Graphs show the mean ± SD, and data are pooled from two to six independent experiments performed in triplicate (a–g) or representative of two (h, i) independent experiments. *** P < 0.001; ns, not significant; two-tailed t-test.