Fig 9. Caspase-8 colocalizes with the NLRC4/ASC/Caspase-1 inflammasome, but it is only activated if caspase-1 or gasdermin-D is inhibited.

(A-C) Bone marrow-derived macrophages (BMDMs) generated from C57BL/6 mice were infected with motility-deficient L. pneumophila mutants expressing flagellin (fliI^-) at a MOI of 10 for 8 h. The cultures were stained with anti-caspase-1 (green) (A, C) or anti-ASC (green) (B), anti-caspase-8 (red). Cell nuclei were stained with DAPI (cyan). Images were acquired by multiphoton microscopy with a 63x oil immersion objective and analyzed using ImageJ software. The images are the maximal projection of a z project. Scale bars, 10μm. (C) The percentage of the caspase-1 puncta containing caspase-8 was determined using an epifluorescence microscope. (D) BMDMs were generated from C57BL/6, Casp1/11^-/- and Asc/Casp1/11^-/- mice and infected with fliI^- or flaA^-. (E) BMDMs were generated from Casp1/11^+/+, Casp1/11^+/- and Casp1/11^-/- littermate control mice and infected with WT, fliI^- and flaA^-. (F) BMDMs were generated from C57BL/6, Casp1^-/-Casp11^tg and Casp11^-/- mice and infected with WT, fliI^- and flaA^-. (G-H) BMDMs generated from C57BL/6 and Casp1/11^-/- mice were transduced with a retrovirus encoding shRNA sequences to target Gasdermin D (GSDMD) (Seq1, Seq2) and a non-target shRNA sequence (NT). (G) Transduced cells were infected with WT Lp, fliI^- and flaA^-. (D-G) Cells were infected with an MOI of 10 and after 8 hours the activity of caspase-8 was measured using the Caspase-8 Glo Assay. (H) GSDMD silencing was confirmed by Real Time qPCR. Gapdh gene was used as a control for normalization of expression levels. The numbers above the bars indicate the percentage of silencing compared to the NT sequence. Data show the average ± SD of triplicate wells. *, P<0.05, Student´s t test in relation to flaA^-. RLU, relative luminescence units; NI, non-infected. Data are presented for one representative experiment of two (A-C and E-H) and five (D) experiments with similar results.