2.10 Western blot analysis
Homogenized brain hippocampus tissues were lysed by protein extraction solution (PRO-PREP, iNtRON, Kyungki-do, Korea) and the total protein concentration was determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA). 40 µg of extracted protein were separated by SDS/PAGE and transferred to Immobilon® PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature, followed by incubation with specific primary antibodies for overnight at 4 °C. The membranes were washed with TBST and incubated with diluted HRP-conjugated secondary antibodies for 1 h at room temperature. After washes, binding of antibodies to the PVDF membrane was detected using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Bedford, MA, USA). The band intensities were measured using the Fusion FX 7 image acquisition system (Vilber Lourmat, Eberhardzell, Germany) and quantified using Image J software. To detect target proteins, specific primary antibodies against iNOS, IBA-1, GFAP, APP, and BACE1 (1:1000; Abcam, Inc., Cambridge, UK), COX-2 (1:1000; Novus Biologicals, Inc., CO, USA), ERK 1/2, p-ERK 1/2, JNK, p38, p-p38, IκBα, and p-IκBα (1:000; Cell signaling Technology, Inc., MA, USA), p-JNK and β-actin (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and PTX3 (1:1000; Invitrogen, Waltham, MA, USA) were used. The corresponding conjugated secondary antibodies such as anti-mouse, anti-rabbit and anti-goat purchased from Abcam (Cambridge, UK).
The brain fixed in a 10% formalin solution was embedded in paraffin wax, and then the brain was cut into Sect. 5-µm-thick slices. Immunohistochemistry was performed as descrived previously . To detect target proteins, specific antibodies against GFAP, IBA-1, iNOS (1:250; Abcam, Inc., Cambridge, MA, USA), and COX-2 (1:100, Novus Biologicals, Inc., CO, USA) were used. Brain sections were visualized by a chromogen diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Finally, brain sections were mounted with mounting medium Cytoseal XYL (Thermo Scientific, Waltham, MA, USA), and evaluated on a light microscope (Microscope Axio Imager. A2; Carl Zeiss, Oberkochen, Germany; × 50 and × 200).
2.12 quantitative real-time PCR
The mRNA level was measured by quantitative real-time polymerase chain reaction(qRT-PCR). Total RNA was extracted using RiboEX (Geneall biotechnology, Seoul, Korea) from hippocampus tissue and cDNA was synthesized using High-Capacity cDNA Reverse Transcription kit (Thermo Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed on a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) for custom-designed primers and β-actin was used for house-keeping control using HiPi Real-Time PCR SYBR green master mix (ELPIS biotech, Daejeon, Korea). Cycling conditions consisted of a initial denaturation step of 3 min at 94 °C, a denaturation step of 30 s at 94 °C, an annealing step of 30 s at 60 °C and an extension step of a minute at 72 °C followed by 40 cycles. The values obtained for the target gene expression were normalized to β-actin and quantified relative to the expression in control samples. Each sample was run with the following primer pairs shown in the supplementary material (Supplementary table S1).
2.13 BV-2 microglial cell culture
Microglial BV-2 cells were obtained from the American Type Culture Collection (Rockville, Maryland, United States). Microglial BV-2 cells were maintained with serum-supplemented culture media of DMEM supplemented with FBS (10%) and antibiotics (100 units/mL). The microglial BV-2 were incubated in the culture medium in a humidified incubator at 37 °C and 5% CO2. The cultured cells were treated with several concentrations (0.5, 1, 2 µM) of K284 -6111, 2 h before Aβ (5 µM) treatment. The cells were harvested after 24 h.
BV-2 cells were transiently transfected with siRNA (20 nM/well/6-well plate) or using the Lipofectamine® RNAiMAX transfection reagent in Opti-MEM, according to the manufacturer’s specification (Invitrogen, Waltham, MA, USA). BV-2 cells were transiently transfected with pcDNA3.1(+)-6 × Myc-CHI3L1 vector or control vector using the Lipofectamine® 3000 transfection reagent in OPTI-MEM, according to the manufacturer’s specification (Invitrogen, Waltham, MA, USA). Negative control (NC), PTX3 siRNA were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). pcDNA3.1(+)-6 × Myc-CHI3L1 vector was cloned from Bionics (Seoul, Republic of Korea).
2.15. Gene network analyses
The gene network of CHI3L1 was analyzed using the web-based analysis tool, String (https://string-db.org), based on the publicly available biological datasets.
2.16 Statistical analysis
The data were statistically analyzed using the GraphPad Prism software (Version 4.03; GraphPad software, Inc., San Diego, CA, USA). Data are presented as mean ± S.E.M. The group differences in all data were assessed by Student’s t test or one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison test. A value of p < 0.05 was considered statistically significant. *, Significantly different between two groups (p < 0.05). **, Significantly different between two groups (p < 0.01). ***, Significantly different between two groups (p < 0.001).