Hf hydrobromide

C57BL/6J mice were treated with intraperitoneal injection of 0.2 mg/kg [17 (link)] HF hydrobromide (Sigma-Aldrich), reconstituted in DMSO, and diluted in PBS for 3 d prior to DSS administration as described earlier [2 (link)]. Acute colitis was induced in mice by adding 5% DSS (MW36,000–50,000; MP Biomedicals) in drinking water for 7 d followed by normal drinking water for 3 d [4 (link)]. Mice were euthanized on d 10; colon tissues were examined for histological analysis, and IL-1β expression was analyzed in tissue lysates by immunoblotting.

HF hydrobromide, MDC, EBSS, CQ, Pierce (R) BCA Protein Assay Kit and SBI-0206965 were obtained from Sigma-Aldrich (Munich, Germany). Antibodies against SQSTM1/p62, LC3-II, phosphor-ULK1 (Ser317), phosphor-ULK1 (Ser757), phosphor-mTOR (Ser2448), Glut1, hexokinase II, PCK2, PCB, phosphor-AMPKα, AMPKα, phosphor-ACCα (Ser79), ACCα, ATG7, phosphor-LKB1, LKB1, phosphor-CaMKKβ (Ser511), and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against phosphor-ULK1 (Ser777), CaMKKβ, and goat anti-mouse IgG-HRP secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-goat anti-rabbit secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA).

DR-Wildtype (ATCC CRL2977) and GCN2-KO-DR (ATCC CRL2978) MEFs, purchased from American Type Culture Collection (ATCC), were maintained in DMEM containing 10% (vol/vol) FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptomethanol. Sodium thioglycolate (Sigma-Aldrich) was used to generate thioglycolate-elicited peritoneal macrophages. LPS (Sigma-Aldrich) and ATP (Sigma-Aldrich) were used at a concentration of 500ng/ml and 5mM, respectively, to stimulate BMDMs, peritoneal macrophages, or J774A.1 cells cultured in DMEM containing 10% (vol/vol) FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, and 1 mM sodium pyruvate. HF hydrobromide (trans-[±]-7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4[3H])quinazolinonemonohydrobromide (Sigma-Aldrich); 3-MA (Sigma-Aldrich), and Wortmannin (Sigma-Aldrich) were used at a concentration of 5 mM or 5 μM. Cycloheximide and Act-D (Sigma-Aldrich) were used at 1 μg/ml and 10 μg/ml. Rapamycin (Sigma-Aldrich) was used at a concentration of 200 nM. Lipofectamine 3000 was from Life Technologies (L3000015) and Sepharose G beads from Sigma-Aldrich. pCMV6 IL-1β was purchased from Origene.