Fig. 2: GBP1 is required for Salmonella- and LPS-induced caspase-4 activation to induce pyroptosis in epithelial cells.

a Immunoblots for GBP1, caspase-4 and GAPDH (loading control) in cell lysates from IFNγ-primed wild-type or GBP1^–/– HeLa. b, c Release of LDH from naive or IFNγ-primed wild-type or GBP1^–/– HeLa after Salmonella infection (b) or after 5 h transfection with E. coli LPS (2.5 µg / 50,000 cells) (c). d, e Immunoblots for full length (p43) and cleaved (p32) caspase-4 in combined supernatants and cell lysates from naive or IFNγ-primed wild-type and GBP1^–/– HeLa, upon transfection with E. coli LPS for 5 h (d) or Salmonella infection (e). f Release of LDH from IFNγ-primed wild-type or GBP1^–/– HeLa, 3 h after electroporation with LPS (300 ng/50,000 cells). g Release of LDH in IFNγ-primed HBEC3-KT or HaCaT cells treated with non-targeting control siRNA (NT) or with siRNAs targeting CASP4, GSDMD or GBP1, after E. coli LPS transfection. Cells were treated with siRNAs for 24 h and transfected with LPS (2.5 µg / 50,000 cells) for 5 h. Graphs show the mean ± SD, and data are pooled from two (c, f), three (g) or four (b) independent experiments performed in triplicate, or representative of three independent experiments (d, e). ***P < 0.001; ns, not significant; two-tailed t-test.