Cell culture and transfection
PDLSCs (LMAI Bio, Shanghai, China) were maintained in osteogenic-inducing medium containing 10% FBS, 50 µM/mL ascorbic acid, 5 mM β-glycerophosphate and 100 nM dexamethasone at 37°C in a 5% CO2 humidified incubator. P. gingivalis-LPS (Pg-LPS; Sigma-Aldrich, MA, USA) was used to stimulate PDLSCs as previously described (22, 23). Overexpression plasmids pcDNA 3.1-Cullin3 was generated by GenePharma (Shanghai, China). 1.2 µg pcDNA 3.1-Cullin3 was transfected into PDLSCs at a density of 2 × 104 per well using Lipofectamine 3000 reagent (Invitrogen) according to the instruction.
PDLSCs were seeded into a 96-well plate at a density of 1 × 103 per well. Cell viability was estimated at 0 h, 24 h and 48 h at first, after which 10 µl CCK-8 solution (Dojindo, Kumamoto, Japan) was added into each well, followed by incubation for 1 h. The absorbance value was at last recorded at 450 nm.
The supernatants of PDLSCs were collected and centrifuged at 4°C (1000 g) for 10 min. The concentration of TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA kits (Beyotime, Jiangsu, China) according to the manufacturer’s instruction. The absorbance value was recorded at 450 nm.
Total protein from cells were extracted using RIPA lysis buffer (Solarbio, Beijing, China). And the protein concentration was determined by a BCA assay kit (Beyotime, Jiangsu, China). Afterwards, SDS-PAGE was prepared to separate proteins, which were then transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, MA, USA). PVDF membranes were incubated with primary antibodies, anti-Shh, anti-Gli1, anti-Cullin3 and anti-Nrf2 (Abcam, Cambridge, UK), which were removed the second day. And secondary antibodies (Sigma-Aldrich) were prepared for incubation with PVDF membranes. The protein bands were visualized and analyzed using a chemiluminescence system (Bio-Rad, CA, USA).
PDLSCs were seeded into a 24-well plate at a density of 2 × 104 per well. Osteogenic-inducing medium was prepared to culture PDLSCs for 7 d. The medium was refreshed every 3 days, which was removed later on the 7th day. PDLSCS were subsequently cultured with added fix solution (MKBio, Shanghai, China) for 4 min at room temperature. Then with 0.6 ml dye (MKBio) added into each well, the cells were cultured at room temperature for 10 min in the dark. Cell observation was done under a light microscope (Carl Zeiss, Jena, Germany) at last.
Alizarin red staining
The mineralization of PDLSCs was evaluated by Alizarin red S. PDLSCs were briefly seeded into a 24-well plate at a density of 2 × 104 per well. Osteogenic-inducing medium was prepared to culture PDLSCs for 21 d, after which PDLSCs were fixed by 4% paraformaldehyde for 15 min at 4°C. The cells were then incubated for 30 min with 0.2 % Alizarin Red S solution (Sigma-Aldrich) added into each well. Lastly, cell observation was done under a light microscope (Carl Zeiss, Jena, Germany).
Apoptotic cells were detected by a TUNEL staining kit (KeyGEN, Jiangsu, China). 4% paraformaldehyde was used to fix PDLSCs for 30 min at room temperature, after which Proteinase K was added for incubation for another 30 min at 37°C. Subsequently, treatment of 1% Triton X-100 was added for incubation for 5 min. The cells were then incubated likewise for 30 min at 37°C in the dark after Streptavidin-HRP was added to the plate. Color reaction was produced by adding DAB solution. And then the cells were re-dyed with hematoxylin. Finally, the cells were observed under a light microscope (Carl Zeiss, Jena, Germany).
Data was presented as mean ± SD and was analyzed by GraphPad Prism 6.0. Student’s t-test and one-way analysis of variance tests followed by Tukey’s post hoc test were used to compare differences between groups. P < 0.05 was considered as statistical significance.