Rapamycin rapa

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Ursolic acid (UA) was purchased from Nanjing Zelang Plant Extract Co., Ltd. (Nanjing, China). Epirubicin was purchased from Rhawn (Shanghai, China). Autophagy inhibitor, 3-Methyladenine (3-MA), was purchased from Selleck Chemicals (Houston, TX, USA). Protease inhibitor, ECL luminescence reagent, Cell lysis buffer, Cell Counting Kit-8, Caspase3 ELISA Kit, Bradford Protein Assay Kit and autophagy agonist, rapamycin (RAPA), were obtained from Solarbio (Beijing, China). Class I PI3K, AKT, Beclin-1, LC3-II/LC3-, Atg5, Atg7 and β-Actin antibodies were purchased from CST (USA). RIPA Lysis Buffer (RIPA) was purchased from Biosharp (Guangzhou, China). Monodansylcadaverine (MDC) and Transwell chambers were purchased from Sigma (Shanghai, China). BCA Protein Assay Kit (BCA) was purchased from Bioteke (Beijing, China).

The treatment was initiated after lymph node cell infusion and lasted for 10 days. Mice were randomly divided into 6 groups: AA group, mice were fed the control diet and orally administered sterile saline; TBI group, mice were fed the same as the normal group; CsA (Zhongmei Huadong Pharmaceutical Co. LTD, Hangzhou, China) group, mice were fed the same control diet and intraperitoneal injection daily administered 50 μg/g/d for 10 days; rapamycin (Rapa) (Solarbio, China) group, mice were fed the same control diet and intraperitoneal injection daily administered 2 μg/g/d for 10 days; Elt group, mice were fed the same control diet and intraperitoneal injection daily administered Elt (20 μg/g/d) for 10 days; and Rapa+Elt group, mice were fed the same control diet and intraperitoneal injection daily administered Rapa and Elt (20 μg/g/d) for 10 days. The mice were sacrificed on the 17th day after treatment. Mice were anesthetized by isoflurane anesthesia (2-3% isoflurane with oxygen supply). Peripheral blood samples were collected by removing the eyeballs, and BMCs were obtained by the femoral cavity flushing.

T-2 toxin was obtained from Cayman Chemical (Ann Arbor, MI, USA). MTT, AVPI, LDH, acridine orange (AO), Hoechst 33258 staining, and Bicinchoninic acid (BCA) protein assay kits were purchased from Beyotime (Nantong, China). AST, ALT, CAT, GSH-PX and MDA assay kits were purchased from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). Antibodies of mouse monoclonal anti-β-actin, rabbit monoclonal anti-Bcl-2, rabbit monoclonal anti-Bax, rabbit monoclonal anti-PARP-1, rabbit monoclonal anti-caspase-3, rabbit monoclonal anti-P62, rabbit monoclonal anti-Beclin 1, rabbit monoclonal anti-LC3, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were purchased from Proteintech (Rosemont, IL, USA). Rapamycin (RAPA) and chloroquine diphosphate salt (CQ) were obtained from Solarbio (Beijing, China). pGMLV-CMV-RFP-GFP-hLC3 lentivirus was purchased from Genomeditech (Shanghai, China).