Analysis of HMGB1 translocation and release in AT-deficient and AT-infused mice. Mice were treated with control siRNA and AT siRNA, followed by infusion of saline, AT-WT, AT-4Mut, or AT-R425del at 24 and 48 hours of siRNA treatment. Mice were euthanized after 72 hours of siRNA treatment and perfused with PBS. (A) Perfused liver cryosections were fixed, permeabilized, and incubated with anti-HMGB1 (rabbit) and anti-CD31 (goat) antibodies followed by Alexa Fluor 555–conjugated anti-rabbit and Alexa Fluor 488–conjugated anti-goat antibodies. DAPI was used to stain the nucleus. Arrows indicate nuclear to cytoplasmic translocation of HMGB1. Inset boxes from each group are magnified. Scale bar, 50 μm. (B) The quantification of HMGB1 translocation from the nucleus to the cytoplasm. (C) Plasma level of HMGB1 was measured using commercial ELISA kit according to the manufacturer’s protocol. (D) Plasma level of IL-1β was measured using a commercial ELISA kit according to the manufacturer’s protocol. All data are presented as mean ± SEM. n ≥ 4. Statistical analysis was performed using Graph Pad Prism 10. P values are determined by 1-way ANOVA, followed by Bonferroni multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P ≤ .001. ns, not significant.