aPC restricts inflammasome by suppressing mTORC1. (A-C) Expression of Raptor, and HK1 and phosphorylation (phospho) of ribosomal p70-S6 kinase (pS6K70) were analyzed in LPS-primed and ATP-challenged BMDMs (A) and mouse neonatal cardiac fibroblasts (B) or mouse neonatal cardiomyocytes subjected to hypoxia/reoxygenation (H/R) (C). aPC inhibits expression of Raptor and HK1 and p70S6K phosphorylation levels in LPS-primed and ATP-stimulated cells (A-B) or in H/R-injured primary cardiomyocytes (C). (D-E) Treatment of mice with aPC inhibits mTORC1 signaling; representative immunoblots showing cardiac Raptor and HK1 expression as well as total and phosphorylated pS6K70. Representative immunoblots (D) and bar graph summarizing results (E). (F-J) BMDMs from TSC1^LoxP/LoxP mice were transiently transfected with GFP- or Cre-expressing plasmids, resulting in loss of TSC1 expression after 48 hours. Representative immunoblots of TSC1, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (cont) (F). (G-H) In TSC1-deficient BMDM cells, aPC treatment fails to reduce Raptor or HK1 expression or phosphorylation of p70S6K (TSC1^LoxP/LoxP+Cre+aPC) when compared with PBS-treated cells (TSC1^LoxP/LoxP+Cre+PBS) Representative immunoblots (G) and bar graph summarizing results (H). (I-J) Likewise, aPC fails to reduce Nlrp3 expression or cl-Casp1 or cl–IL-1β in TSC1-deficient BMDMs. Representative immunoblots (I) and bar graph summarizing results (J). (I) Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (J) The active form was quantified. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments each with at least 2 technical replicates (A-J); GAPDH as loading control (A-D,F-G,I). **P < .01; analysis of variance (E,H,J).