Gzd824

Abdominal sepsis model was induced by cecal ligation and puncture (CLP) as previously described^{26 (link)}. Briefly, animals were anesthetized and the abdominal wall was incised in the midline to mobilize the cecum. The cecum was filled with feces from ascending colon, ligated 75% of cecum (5-0 silk suture), soaked with PBS and punctured twice with a 21-gauge needle. The cecum was then returned to the peritoneal cavity and the abdominal incision was sutured. The specific c-ABL kinase inhibitor GZD824 (5 mg/kg, Selleck Chemicals, Houston, TX, USA) or vehicle (DMSO) was administered via the jugular vein 30 min prior to induction of abdominal sepsis. Sham mice underwent the identical laparotomy and resuscitation procedures, but the cecum was neither ligated nor punctured. Mice were then returned to their cages and reanesthetized for sample collection at 6 and 24 h after CLP induction. The left lung was ligated and excised for edema measurement. The right lung was used for collecting bronchoalveolar lavage fluid (BALF), in which neutrophils were quantified in a Burker chamber. Next, the lung was perfused with PBS, one part was fixed in formaldehyde for histology and the remaining lung tissue was snap-frozen in liquid nitrogen, and stored at −80 °C for later enzyme-linked immunosorbent assay (ELISA) as described subsequently.

RPMI-1640 and McCoy's 5A medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Lonza Milano SRL (Milan, Italy). ZSTK474, BYL719, TGX221, AS605240, CAL101, IPI145, Imatinib, Nilotinib and GZD824 were obtained from Selleck Chemicals (Houston, TX, USA). For cell viability determination, CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Milan, Italy). Annexin V/7-ADD detection kit was from Merck-Millipore (Darmstadt, Germany). Western blot antibodies for total Akt-1, Ser473 p-Akt-1 and Thr308 p-Akt-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody. Chloroquine, the mouse secondary antibody and the monoclonal β-Actin antibody were purchased from Sigma Aldrich (Milan, Italy). Signals were detected using ECL Plus reagent from Perkin Elmer (Boston, MA, USA).

RPMI-1640 medium, fetal bovin serum (FBS), penicillin and streptomycin were from Lonza (Lonza Milano SRL, Milan, Italy). Imatinib, Nilotinib, RAD001, GZD824, GSK690693, XL-147, MK-2206, ZSTK474, BGT226, KU0063794, CCI-779, AZD8055 and Torin-2 were provided by Selleck Chemicals (Houston, TX, USA). For cell viability determination, CellTiter 96(R) AQueous One Solution Assay (MTS) was purchased from Promega (Milan, Italy). Annexin V/7-AAD, Cell Cycle, Autophagy LC3 Activation detection kits were used for the analysis with the Muse™ Cell Analyzer from Merck-Millipore (Milan, Italy). All the antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody. The mouse secondary antibody was from Sigma Aldrich (Milan, Italy). Signals were detected with the ECL Plus reagent purchased from Perkin Elmer (Boston, MA, USA).