Phosphate buffered saline (pbs)

L-Cys, D,L-Hcy, L-GSH, and CysGly were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid (TCA) was obtained from Wako Pure Chemical (Osaka, Japan). Ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) was purchased from Dojindo (Kumamoto, Japan). TCEP was purchased from the Tokyo Chemical Industry (Tokyo, Japan). Phosphate buffered saline (PBS) was purchased from Takara Bio (Shiga, Japan). HPLC-grade acetonitrile was used. Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade.

HT22 cells were seeded in 96-well plates at 3000 cells/well and incubated at 37 °C with 5% CO₂ for 3 h [35 (link),36 (link)]. The cells in each well were transfected with 0.2 μg of the pEGFP-C1/Aβ₄₂ plasmid (originating from Professor Junsoo Park, Yonsei University, Seoul, Korea) with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After transfection for 10 h, the tested compounds dissolved in the medium were added to the seeded cells, and the cells were incubated for 24 h. Then, 20 μL of MTT solution (2 mg/mL) was added, and the cells were incubated for 3 h in the dark. The cells were subsequently washed with phosphate-buffered saline (PBS) (Takara, Kusatsu, Japan), and 100 mL of DMSO was added to solubilize formazan. The absorbance at 570 nm was measured with a microplate reader (VersaMaxTM, Randor, PA, USA). Fluorescence imaging was conducted by using a fluorescence microscope (Olympus ix70, Tokyo, Japan).

All reaction reagents
and solvents were obtained
from Nacalai Tesque, Fujifilm Wako, Tokyo Chemical Industry, Kanto
Chemical, and Aldrich and used without further purification. Workup
and purification procedures were carried out with reagent-grade solvents
under air. Optical spectra were measured with spectroscopic grade
solvents. Deionized water (filtered through a 0.22 μm membrane
filter, R > 18.2 MΩ cm) was purified using
a Milli-Q system from Millipore.
The reagents for cellular experiments
were as follows: phosphate-buffered saline (PBS; TaKaRa), Dulbecco’s
modified Eagle medium (DMEM; Nacalai Tesque), FluoroBrite DMEM (Nacalai
Tesque), Opti-MEM I Reduced Serum Medium (Gibco), Trypsin-EDTA (Nacalai
Tesque), MG132 (Peptide Institute, INC.), bafilomycin A1 (Merck),
Lipofectamine 2000 (Invitrogen), MitoTracker Red (Thermo Fisher Scientific),
Transferrin-Alexa 594 (Thermo Fisher Scientific), LumiTracker Lyso
Red (LysoTracker-Red; Lumiprobe), 35 mm single-well glass-base dishes
(Iwaki), and Easy iMatrix-511 for laminin coating (Nippi).

Mandibles and first molars were taken from Dmp1-EGFP neonatal mice and fixed overnight in 4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries, Ltd., Osaka). Fixed tissues were then decalcified with 10% ethylenediaminetetraacetic acid (EDTA) (MUTO PURE CHEMICALS Co., Ltd., Tokyo) for 1 week. After decalcification, they were dehydrated with a series (10%, 20%, 30%) of sucrose (Sigma-Aldrich Co. LLC, St. Louis) and phosphate buffered saline (PBS) (Takara Bio Inc., Shiga). Tissues were embedded in optimal cutting temperature compound (O.C.T., Sakura Finetek Japan Co., Ltd., Tokyo) and 10-μm sections were obtained using a cryostat (Leica, Wetzlar). Fluorescent imaging was performed using a fluorescence microscopy system (BZ-X700) (KEYENCE Corp., Osaka).

The 26 heparinized whole blood samples were used for the TB test, which was performed by IFN-γ release assay, as described previously [42 (link)]. Briefly, bovine purified protein derivatives (bPPD) (1000 international units per milliliter [IU/mL], diluted into 50 IU/mL) (China Institute of Veterinary Drugs Control, Beijing, China), avian purified protein derivatives (aPPD) (1000 IU/mL, diluted into 50 IU/mL) (China Institute of Veterinary Drugs Control, Beijing, China), Concanavalin A (ConA) (5 mg/mL, diluted into 100 μg/mL) (Sigma, California, USA) were used as the positive control, and phosphate-buffered saline (PBS) (10X, diluted into 1X) (Takara, Dalian, China) was used as the negative control to stimulate the whole blood overnight. Then, the supernatant plasma was collected and the IFN-γ concentration was measured using the standard curve of the IFN-γ according to the instructions of the commercial human/monkey IFN-γ quantification kit (MABTECH, Stockholm, Sweden). Based on the criteria previously established by this laboratory, a sample was classified as TB positive if the difference in the IFN-γ concentration between the bPPD- and aPPD-stimulated samples was equal to or greater than 114.5 pg/mL [43 (link)].