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

Fig 5. ASC is important for NLRC4/caspase-8-mediated restriction of L. pneumophila replication independently of caspase-1/11.

Fig 5

(A-D) Bone marrow-derived macrophages (BMDMs) from C57BL/6 (open circles), Nlrc4-/- (closed squares), Casp1/11-/- (closed triangles) and Asc/Casp1/11-/- (open triangles) mice were infected with motility-deficient mutants expressing flagellin (fliI-, A), with flagellin-deficient mutants (flaA-, B), L. gratiana (C) or with L. micdadei (D) at a MOI of 10. The cells were incubated for 24, 48 and 72 hours for CFU determination. Data show the average ± SD of triplicate wells. *, P<0.05 compared with Casp-1/11-/- BMDMs. #, P<0.05 compared with C57BL/6 BMDMs, ANOVA. (E-I) BMDMs from Casp1/11-/- and Asc/Casp1/11-/- mice were transduced with a retrovirus encoding shRNA sequences to target caspase-8 (Seq1, Seq2) and a non-target shRNA sequence (NT). (E) Caspase-8 silencing was confirmed by western blot analysis. Cell lysates were separated by SDS-PAGE, blotted and probed with anti-caspase-8 (pro-caspase-8 p55) and anti-α-actin. (F-I) Transduced Casp1/11-/- (F, H) and Asc/Casp1/11-/- (G, I) BMDMs were infected with fliI- (F, G) or flaA- (H, I) at a MOI of 10 and incubated for 24, 48 and 72 hours for CFU determination. Data show the average ± SD of triplicate wells. *, P<0.05 compared with NT shRNA, ANOVA. NT, non-target shRNA. Data are presented for one representative experiment of four (A), two (B-D) and one (F-I) experiments performed with similar results.