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. 2016 Oct 13;12(10):e1005910. doi: 10.1371/journal.ppat.1005910

Fig 6. Caspase-8 self-cleavage is necessary for apoptosis but not cytokine responses.

Fig 6

(A) Schematic of strategy used to generate Casp8 DA/DA mice using CRISPR/Cas9. GuideRNA is in blue. (B) Casp8 +/+, Casp8 DA/+, Casp8 DA/DA and Ripk3 -/- Casp8 -/- BMDMs were infected with YopJ-deficient (ΔYopJ) and wild type Yersinia and caspase-8 processing was measured by western analysis. (C) BMDMs were pretreated with GSK’ 872 or vehicle control 1 hr prior to infection with Yersinia. Cytotoxicity was measured by LDH release 4 hrs post-infection. (D) Cleaved caspase-3 was measured by flow cytometry in BMDMs 2 hrs post-indicated treatments. (E-G) Casp8 +/+, Casp8 DA/+, Casp8 DA/DA and Ripk3 -/- Casp8 -/- BMDMs were treated with PAMPs and cytokine production was measured after 6 hrs by flow cytometry. Representative flow plots of IL-12p40 production in response to LPS (100 ng/mL) (E), quantification of percentage of IL-12p40+ in response to LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) (F), IL-1β+ and IL-6+ in response to LPS (100 ng/mL) (G) (H) BMDMs were treated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) for 6 hrs and TNF production was measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s unpaired two tailed t-test. Representative of 3–5 independent experiments.