Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection
Fig 4
Card19lxcn BMDMs retain intracellular alarmin HMGB1 following activation of cell death.
(A) Primary BMDMs from B6 (+) or Card19lxcn (-) mice were treated with indicated treatments or infections, and TCA precipitated supernatants (Sups) or whole cell lysates (Lysates) were harvested at 1 or 4 hours post-infection and analyzed by western blotting for HMGB1, CARD19, and actin (loading control). ATP indicates cells that were primed with LPS for 3 hours and treated with ATP for 1 or 4 hours. S.Tm–Salmonella Typhimurium; Yptb Yersinia pseudotuberculosis. Sts–staurosporine. (B) Confocal microscopy images of untreated and staurosporine (Sts) treated B6 and Card19lxcn BMDMs fixed at indicated times post-Sts treatment and stained for HMGB1, Actin and DNA (Hoescht). White arrows indicate HMGB1 clouds. Scale bar 20 microns, inset 10 microns. (C) Quantification of nuclear HMGB1. n = 25–50 cells per field, 5–8 fields per condition, per timepoint. (D) Quantification of HMGB1 clouds in B6 and Card19lxcn BMDMs after staurosporine treatment. 5–8 fields quantified per condition, per timepoint. Mean ± SEM is displayed. *** p < 0.001, ** p < 0.01, * p < 0.05. n.s. not significant. 2-way ANOVA with Bonferroni multiple comparisons post-test. Representative of 3 (A-C) or 2 (D) independent experiments.