Pbqm100a 1

The human C-terminal 99-residue fragment (β-CTF) of amyloid precursor protein was introduced into the C6 rat glioma cell line using a lentivector (System Biosciences LV500A-1, Palo Alto, USA) as previously described [11 (link)]. The production of Aβ40 and Aβ42 peptides was induced by a Cumate-inducible gene expression system (System Biosciences, PBQM100A-1) [11 (link)]. The culture medium was Ham’s F12K (Thermo Fisher, 21127022) containing 2.5% FBS, 15% horse serum (Thermo Fisher, 26050088), and 1% penicillin-streptomycin (Thermo Fisher, 15140122). The cell line was kindly provided by Professor Yi-Cheng Chen from the Department of Medicine, Mackay Medical College, Taipei, Taiwan.

Cell transfection was performed using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacture’s instruction. AMBN-pcDNA3.1 plasmid into 143B-Luc cells, and the cells were treated with Zeocin (16.7 μg/mL; Invitrogen) for 2–3 weeks and some clonal colonies were selected and isolated, then stably AMBN overexpressing 143B-Luc cells were established. Also, AMBN was subcloned into a inducible vector, PBQM812A-1 (System Biosciences, LLC, Palo Alto, CA). And AMBN-inducible plasmid was transfected into 143B-Luc cells, and the cells were treated with puromycin (0.5 μM, Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 2–3 weeks and some clonal colonies were selected and isolated, then stably AMBN-inducible 143B-Luc cells were established. The cells were treated with Cumate solution (150–300 μg/mL, PBQM100A-1, System Biosciences, LLC, Palo Alto, CA) for 3 days, then AMBN expression was confirmed by western blot analysis.