2.1 Cells culture
Murine macrophage lines RAW264.7 were purchased from American Type Culture Collection (ATCC, USA) and maintained in α-MEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Lonsera, Uruguay) and 1% antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml，) providing atmosphere of 37℃ and 5% CO2. In order to research macrophages polarization, cells were treated with different concentrations of AGEs (Bioss, China), 1μg/ml LPS (Solarbio, China) and 20ng/ml IL-4 (Sino Biological, China). To induce macrophages to differentiate into osteoclasts, α-MEM containing 50ng/ml RANKL (Novoprotein, China) and 30ng/ml M-CSF (Novoprotein, China) was used to culture cells for 6 days. The first 3 days of osteoclasts differentiation were considered to be the early stage, and the last 3 days were considered to be the late stage. Three repeat holes were set for each sample.
2.2 Cells viability and toxicity test
Cell Counting Analysis Kit (CCK-8, Beyotime, China) for cell viability detection was performed according to the manufacturer's instructions. Briefly，RAW 264.7 cells were seeded in a 96-well plate at a density of 5×103 /well . After 24h, the medium was replaced with fresh α-MEM medium containing AGEs at concentrations of 0, 100, 200, 400 mg/L for 12, 24, 36, and 48 h. Then, after the supernatant was discarded and cells were washed with PBS twice, 10μL CCK-8 solution was added to each well and cultured at 37 °C for 3 h. The absorbance was measured by enzyme labeling instrument (PerkinElmer, USA) at 450 nm wavelength.
An Annexin V‐FITC apoptosis assay kit (Beyotime, China) was used to estimate the apoptosis rate of RAW264.7 cells. Briefly, cells in logarithmic growth phase were inoculated in 6-well plates and cultured for 24 h. Then the medium was replaced with fresh α-MEM medium containing AGEs at concentrations of 0, 100, 200, 400 mg/L for 24 h. After washed with PBS twice, cells were stained with Annexin V-FITC and propidium iodide (PI) in binding buffer at 4˚C. Samples were detected with flow cytometer (BD Influx, USA) and the results were statistically analyzed by FlowJo 7.6.1.
2.3 Griess assay
First, a standard curve was drawn using NO standards according to the instructions of the Griess kit (Beyotime, China). After the cells were treated with 100mg/L AGEs for 24 h, the cell supernatant of each group was extracted and added to a 96-well plate at 50 μl/well. According to the instructions, 50 μl of Griess Reagent I and 50 μl of Griess Reagent II were added to each well, respectively. Then, the absorbance was measured at 540 nm wavelength with a microplate reader and the NO concentration in the supernatant was calculated using the standard curve.
2.4 Quantitative real-time PCR (qRT-PCR)
For gene expression analysis, cells were washed twice by PBS and lysed in RNAiso plus (Takara, Japan). Total RNA was extracted using the TRIzol method according to the manufacturer’s instructions and the RNA concentration was measured using NANO Drop2000 UV spectrophotometer (Thermo Scientific, USA). Then 2 µg of total RNA was reverse transcribed into cDNA using a 20 μl system reverse transcription kit (Promega, USA). Quantitative real-time PCR (qRT-PCR) was then performed with CFX Connect real-time PCR detection system (Bio-Rad, USA) using qPCR Master Mix kit (Promega, USA). The relative expression of target genes was calculated and analyzed with the 2-ΔΔCT method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and all reactions were performed in triplicate. The sequences of the primers (Sangon Biotech, China) are listed in Table 1.
2.5 Western blot analysis
Cells were lysed with RIPA buffer (Santa Cruz Biotechnology, USA) containing 1 mM phenylmethylsulfonyl fluoride (Beyotime, China) after washed by cold PBS three times. The total protein concentration was quantified with an enhanced bicinchoninic acid (BCA) protein analysis kit (Beyotime, China). For western blot analysis, 30 μg of protein extracts were loaded onto gels respectively and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, China). Then these proteins were transferred to a polyvinylidene fluoride membrane, after which the membrane was blocked with fat-free milk buffer for 2 h and blotted overnight at 4°C with primary antibodies (1:1000, Cell Signaling Technology, USA) against monoclonal anti-iNOS, anti-Notch1, anti-cleaved Notch1, anti-RBP-J, anti-GAPDH and anti-Histone H3. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abbkine, USA) at room temperature for 1 h. After washing, the Western blot bands were treated by an ECL Plus chemiluminescence reagent kit (Beyotime, China) and visualized using ChemiDoc Imaging System (Bio-Rad, USA). All images were analyzed by ImageJ software (NIH, USA).
2.6 Flow cytometry analysis for macrophages polarization
Surface antigen expressions on Raw264.7 cells were determined by FCM. Briefly, cells were collected and suspended at 1×106 cells/ml with PBS containing 3% FBS. Then cell suspensions were incubated with PE anti-CD86 (BD Biosciences, USA) on a shaker at 4°C for 30 min in the dark, during which time it was stirred every 5 min to avoid cell precipitation. After the incubation, unbound primary antibody was discarded and cells were washed twice with 500 µl of PBS containing 3% FBS by centrifugation at 500 g for 5 min. Then the cell suspensions were filtered into flow cytometer tubes with a sieve and analyzed by flow cytometer (BD Influx, USA). The data were statistically analyzed by FlowJo 7.6.1.
2.7 Tartrate resistant acid phosphatase (TRAP) staining
According to the protocols of the TRAP staining kit (Sigma, USA), cells were washed three times by PBS, then fixed with 4% paraformaldehyde at room temperature for 20 minutes and washed again with PBS for three times. Then the staining solution mixture prepared according to the instructions was used to incubate cells for 60 min in the dark, after which the cells were rinsed three times with deionized water to avoid nonspecific staining. Then, mature osteoclasts, which manifested as positively stained cells with more than three nuclei, were counted under a light microscope (Leica DMI4000B, Germany).
2.8 Treatment with DAPT
DAPT (Abmole, USA), as a new type of γ-secretase inhibitor, is usually used as an inhibitor of the Notch signaling pathway. While treating the cells with different stimuli, 5 μmol/mL DAPT was added to the cell culture medium to inhibit the transmission of Notch signal, and then the polarization changes of the cells were detected.
2.9 Gene interference with RBP-J siRNA
Small interfering RNA (siRNA) targeting RBP-J (sense: 5’-GCCGAAACAAUGUACAGAUTT-3’; anti-sense: 5’-AUCUGUACAUUGUUUCGGCTT-3’) or negative control siRNA (sense: 5’-UUCUCCGAACGUGUCACGUTT-3’; anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’) were synthesized (Gene Pharma, China). Transfection was performed by Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Scientific, USA) according to the given protocols. Briefly, RAW264.7 cells were seeded in 6-well plates. Cells at 70% confluent were transfected respectively with RBP-J siRNA or scramble siRNA using RNAiMAX Transfection Reagent. After 24 h of transfection, the transfection medium was replaced by fresh α-MEM medium. Then, qRT-PCR and Western blot were performed to test the efficiency of knockdown after 24 or 48h.
2.10 Statistical analysis
All statistical analysis were performed by GraphPad Prism 8.0.2 (GraphPad Software Inc., USA). All Data are presented as the mean ± standard deviation (SD). The results were statistically analyzed with Student’s t-test for two-group comparisons and one-way ANOVA with Tukey’s post hoc test for multigroup comparisons. Data with P-values <0.05 were considered statistically significant.