Human aβ1 42

The Aβ used in this study was human Aβ1-42 (China Peptides, Shanghai, China). The Aβ powder was dissolved, incubated with dimethyl sulfoxide (Gibco) and then diluted to a stock solution with phosphate buffered saline (Boster Biological Technology, Wuhan, China) [35 (link)]. After cell culture, microglial cells were treated with 10 μM Aβ1-42 for 24 h [35 (link)]. Aβ1-42-stimulated microglial cells served as the Aβ1-42 group. Microglial cells without Aβ1-42 treatment acted as the control (Con) group.

Indomethacin was purchased from Chengdu Aikeda Chemical Reagent Co., Ltd. (Chengdu, Sichuan, China). Bay-u3405 was obtained from Adooq Bioscience (Nanjing, Jiangsu, China). Human Aβ_1–42 was purchased from ChinaPeptides (Suzhou, Jiangsu, China). PGD₂ was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Antibodies against β-actin, LRP1, Aβ, ADAM10, p-AKT, AKT, p-ERK1/2, ERK1/2, p-mTOR, p-TSC2, p-4EBP1 and p-S6K were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies specific for COX-2 were purchased from Abcam (Shanghai, China). Antibodies specific for A2M, L-PGDS and CRTH2 were purchased from Santa Cruz Biotechnology (Shanghai, China). All reagents used in the qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories (Shanghai, China). All other reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) unless specified otherwise.

DMEM/F-12 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Waltham, MA, USA). The Aβ used in these experiments was human Aβ_1–42 (ChinaPeptides, Shanghai, China). The Aβ powder was dissolved and incubated in DMSO, and was then diluted to a stock solution (500 μM) with phosphate buffered saline (PBS; Boster Biological Technology, Wuhan, China). Before the experiments, we incubated the mixed stock solution at 37 °C for 24 h. Zerumbone (Zer, > 98% of Purity, Fig. 1) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (Munich, Germany). Zerumbone was dissolved in DMSO to produce a 20 μM stock solution. Same volume or amount of DMSO and/or PBS was applied as vehicles. For the in vitro experiments, stock solutions of Aβ or Zerumbone were further diluted to different working concentrations with culture media. After 6 h of pretreatment of Zer or vehicle, we treated the cells with the 10 μM Aβ_1–42 for 12 h. The dose of amyloid-beta used in cell culture experiments is based on our previous studies and is consistent with the majority of previous in vitro studies on AD. We tested serial concentrations of amyloid-beta from 1 to 30 μM and 10 μM was an appropriate concentration, which did not harm the cellular activity in our cell culture.
Fig. 1

Molecular structure of Zerumbone