Cell lines and reagents
Human adipose-derived stem cells (hADSCs) were isolated and purified from fresh human adipose tissues donated from healthy adults less than 40 years of age. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA) containing 10% fetal bovine serum and cultured at 37 °C in a humidified atmosphere of 5% CO2. Osteogenic induction medium was purchased from SALILA (SALILA, Guangzhou,China). The medium was changed every 2 days. To inhibit DNA methylation, hADSCs were treated by 5 µmol/L 5-aza-2′-deoxycytidine (DAC, Sigma Aldrich, Munich, Germany). To inhibit NOTCH signaling, cells were treated with 2 µM DAPT(Selleck), protein deacetylation was inhibited by 20 µM OSS_128167 (Selleck). For the cycloheximide (CHX)-chase assay, cells were treated with 100 µg/mL CHX (Sigma-Aldrich) for the indicated hours in the absence or presence of 5 µg/ml actinomycin D (Sigma-Aldrich) and western blot analysis was performed.
hADSCs were lysed on ice for 15 mins using RIPA lysis buffer (BeyoTime, China) supplemented with protease inhibitors cocktail (Roche). The protein concentration of the lysate was then measured using the BCA Protein Assay Kit (Beyotime, China) according to the manufacturer's protocol. Protein sample were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA), which were blocked with 5% fat-free milk and then incubated with specific primary antibodies overnight at 4 °C. An anti-rabbit-HRP (horseradish peroxidase) secondary antibody was added, and the staining was visualized using an enhanced chemiluminescence detection system (Millipore, Billerica, USA). The primary antibodies used in this study were as follows: SIRT6, RUNX2, Sp7, COL1A1, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Jag1, HEY1(Abcam, UK), HA, Flag, GAPDH (Beyotime, China) DNMT1,Acetylated Lysine (Ac-K) ( Cell Signaling Technology).
Quantitative Reverse Transcription (RT)-PCR
Total RNA was extracted using Trizol reagent (Invitrogen). cDNA was synthesized using a Reverse Transcription Kit (Promega, Madison, WI). cDNA was applied for quantitative PCR reactions to determine the expression of specific genes with SYBR Green Real-Time PCR Master Mix Kit (Invitrogen) according to the manufacturer’s instructions. GAPDH mRNA was applied as endogenous controls. Primer sequences were shown in Supplementary Table 1.
Lentivirus, siRNAs and shRNAs transfection
The lentivirus particles for overexpression of knocking down genes were purchased from GeneChem (GeneChem, Shanghai, China）. hADSCs cells in 24-well plates were infected with the lentiviral vectors fixed with 10 mg/ml polybrene (GeneChem) in DMEM (Invitrogen, USA). Stable clones were selected using 0.5 μg/ml puromycin. Short hairpin RNAs (Supplementary Table 2) were synthesized by RiboBio(Guangzhou, China). hADSCs cells in 6-well plates were treated with 50nM siRNAs or 4 μg shRNAs using Lipofectamine 3000 reagent (Invitrogen) according to the manuscript and then harvested for assays.
ALP staining and activity
ALP staining was performed in cells seeded in 24-well plates. After treatment with osteogenic differentiation medium for 7 days, the cells were fixed in 70% ethanol and incubated with buffer a staining solution containing 0.1% naphthol AS-TR phosphate and 2% fast violet B (Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. The ALP activity was calculated quantitatively using a kit (Cell Biolab, San Diego, CA, USA) according to the manufacturer’s protocol
Alizarin red staining
hADSCs were incubated with osteogenic red S (pH 4.2, Sigma) for 10 min. For the quantitative assessment of the mineralization, the mineralized bone nodules were eluted by 10% (w/v) cetylpyridinium chloride (Sigma-Aldrich) for 1 h and determined by measuring the absorbance at 562 nm.
Masson’s trichrome staining
In vivo transplantation was previously described20. In brief, 4 weeks old NOD mice were purchased from the Provincial Animal Center (Guangdong, China). The osteogenic differentiation-induced hADSCs were loaded onto 5 mg hydroxyapatite/tricalcium phosphate (HA-TCP; Sigma, St Louis, MO) and subcutaneously inoculated into the right dorsal region of mice ( 5 mice per group). 4 weeks later, the mice were subjected to surgical procedures as previously described 20. The research proposal on animal experiments was approval by the local Ethics Committee for Animal Study. All procedures were approved by the Animal Care Committee of Southern Medical University. The samples were collected, fixed with 4% paraformaldehyde, and decalcified in 10% EDTA (pH 6.0) for 7 days. Paraffin sections were prepared and stained with hematoxylin and eosin, Masson's Trichrome stain (Sigma) according to the manufacturers' protocols. The research proposal on animal experiments was approved by the local Ethics Committee for Animal Study. All procedures were approved by the Animal Care Committee of Southern Medical University.
Luciferase reporter assay
The 2 kb region upstream of the transcription starting site of NOTCH1, NOTCH 2 and DNMT1 were synthesized and cloned into upstream of the luciferase gene in the pmirGLO luciferase vector (GeneChem, Shanghai, China). hADSCs and 293T cells were treated with the indicated Transfection particles using Lipofectamine 2000 (Invitrogen). Promoter activity was measured using the luciferase assay kit (Promega) and normalized to the Firefly and Renilla luciferase activities 48 h after transfection.
NP-40-containing lysis buffer supplied with protease inhibitor cocktail was used to lysed cells (Roche), the immunoprecipitated complexes were recovered with ChIP grade antibodies against acetylated lysine (Ac-K) (Cell Signaling Technology), HA epitope(Beyotime, Shanghai,China), Flag epitope (Beyotime, Shanghai, China), rabbit immunoglobulin (Ig)G control antibodies (Sigma-Aldrich), which were incubated with protein A/G Sepharose beads (Santa Cruz, USA) first, and then rinsed with wash buffer. The eluted immune complexes were denatured and subjected to western blot assay.
The data were presented as the mean± SD. All data were analyzed by one- or two-way ANOVA with Bonferroni post hoc tests. SPSS 18.0 was used to perform all analyses. Statistical significance was defined as p< 0.05.