Gcn2 ko dr

Liver human hepatoblastoma cells (HepG2), and Vero cells were cultured in complete DMEM (Gibco) media consisting of 10% FBS (GIBCO), 100 units/ml Penicillin, 1 μg/ml Streptomycin, L-Glutamine, and 1 mM sodium pyruvate. DR-Wildtype (ATCC CRL2977) and GCN2-KO-DR (ATCC CRL2978) MEFs were purchased from American Type Culture Collection (ATCC) and cultured in complete DMEM growth medium as described earlier (15 (link)). Halofuginone hydrobromide (trans-[±]-7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4[3H]) quinazolinonemonohydrobromide and Actinomycin-D were purchased from Sigma-Aldrich; Lipofectamine 2000 (Invitrogen, Life Technologies). pNifty-Luc was purchased from Invivogen and pKM2L-phCOX-2 plasmid was purchased from RIKEN Bioresource Center (DNA Bank), Japan. pCDNA3.1-Flag-GCN2 plasmid was a kind gift from Prof. David Ron's lab, Cambridge University.

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.