4egi 1

Single-cell suspensions from the pooled spleens and lymph nodes were enriched for CD4⁺ T cells with CD4 microbeads (Miltenyi Biotech). Naive (CD4⁺CD25⁻CD44⁻ CD62L⁺) T cells were purified from enriched CD4⁺ T cells on a MoFlo cytometer (DakoCytomation). Naive T cells were stimulated with plate-bound anti-CD3 (145-2C11; BioXcell; 10 μg/ml) and soluble anti-CD28 (37.5; BioXcell; 10 μg/ml) at a density of 1×10⁶ cells/ml in complete RPMI medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 10 mM Hepes, 1× nonessential amino acids (Cellgro), and 50 μM β-mercaptoethanol. Rapamycin was purchased from Cell Signaling Technology and 4EGI-1 was purchased from Selleckchem.

HEK293 (human embryonic kidney) cell lines and SH-SY5Y (human neuroblastoma) cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HEK293 were maintained in DMEM (Gibco, Rockville, MD, USA) with 10% of fetal bovine serum (FBS, Hyclone). SH-SY5Y(Y5Y) were cultured in F-12 (Gibco, Rockville, MD, USA) supplemented with 10% of FBS. HEK-APP was generated from HEK293 stably expressing human full-length APP695 as previously described [34 (link)]. HEK-APP cells were cultured in DMEM supplemented with 10% of FBS and 200 mg/mL of G418 (Sigma-Aldrich, St. Louis, MO, USA). Y5Y-APP (SH-SY5Y cells stably expressing human full-length APP695) cells were cultured in F-12 with 10% of FBS and 200 mg/mL of G418 [35 (link)]. All cells were cultured in a humidified incubator at 37 °C and 5% CO₂.
The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA): lysosomal inhibitor chloroquine (CQ), protease inhibitor MG132, the transcriptional inhibitor actinomycin D (ActD), and the protein biosynthesis inhibitor cycloheximide (CHX). The eIF4E/eIF4G interaction inhibitor 4EGI-1 were from Selleck (Houston, TX, USA). 4EGI-1, MG132, ActD, and CHX were all dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was at least 1:2000. CQ was dissolved in sterilized double-distilled H₂O.