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. 2017 Aug 3;13(8):e1006502. doi: 10.1371/journal.ppat.1006502

Fig 8. Naip5 is required for the functions of the NLRC4/ASC/Caspase-8 inflammasome independently of caspase-1/11.

Fig 8

Bone marrow-derived macrophages (BMDMs) generated from Casp1/11-/- and Asc/Casp1/11-/- mice were transduced with a retrovirus encoding shRNA sequence to target Naip5 and a non-target shRNA sequence (NT). Transduced BMDMs were infected with wild-type L. pneumophila (WT Lp), motility-deficient mutants expressing flagellin (fliI-) or with flagellin-deficient mutants (flaA-) at a MOI of 10. (A) 8 hours after infection the activity of caspase-8 was measured using the Caspase-8 Glo Assay. (B) Quantification of the Naip5 gene expression by Real Time qPCR. Gapdh gene was used as a control for normalization of expression levels. The number above the bars indicates the percentage of silencing compared to the NT shRNA (open bars). (C-J) Pore formation was assessed fluorometrically in real time by the uptake of propidium iodide. The RFU (%) represents the percentage of RFU estimated with cells lysed with Triton X-100. (K-N) The cells were infected with fliI- or flaA- for 24, 48 and 72 hours for CFU determination. Data show the average ± SD of triplicate wells. *, P<0.05, Student´s t test (A) and ANOVA (K-N). NS, not significant; RFU, relative fluorescence units; NI, uninfected. Data are presented for one representative experiment of two (A-J) and three (K-N) experiments with similar results.