Articular cartilage and costal cartilage are both of permanent hyaline cartilage tissues, which have to supply cartilage matrix components and should never go beyond prehypertrophic stages in chondrocytic differentiation, except under pathological conditions. C-28/I2, the immortalized chondrocyte cell lines, transduced with simian virus 40 (SV 40) containing the large T-antigen were originated from rib cartilage of a 15-year-old female(14). Cells were seeded at a density of 100,000 cells/cm2 in 25 cm2 flasks and cultured in DMEM/F-12 medium (HyClone SH30023.01) containing 10% fetal calf serum (PAN Biotech P30-3302) at 37℃ and 5% CO2.
Overexpression and siRNA transfection of ANKH
Briefly, the chondrocytes were transfected with pEX-4-ANKH or siRNA (GenePharma) which were added to the total amounts of transfected DNA samples using Lipofectamine2000 according to the manufacturer’s protocol. Controls use nontransfected cells in the same way of transfection. Cells were cultivated for 24h and then collected for further experiments.
Pi uptake by chondrocytes.
PiPer phosphate assay kit (Molecular Probes, Eugene, Oreg) measured the intracellular Pi concentration of chondrocytes. Cells were washed, and the cytoplasmic fraction was obtained by ultracentrifugation as described below(15). According to the manufacturer’s instructions, the Pi concentration was measured by ten microliters of the cytoplasmic fraction.
Alkaline phosphatase staining
The chondrocytes were inoculated into six-well plates at a density of 1 × 105 cells per well. After the cells reached 70% confluence, the alkaline phosphatase (ALP) secretion was measured by the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime) staining the samples according to the manufacturer's instructions.
Alizarin red S staining
For estimation of mineralization event, confluent cultures of chondrocytes were preincubated in DMEM/F-12 containing 0.5% FBS for 24 h and then incubated in the same medium with SDF-1α(PeproTech), AMD3100(Sigma) or IKKβVI(Sigma). PBS was added to the control culture. Cells were stained with ALIZARIN RED S assay kit (GENMED) according to the manufacturer's instructions. To solubilize and release calcium-bound alizarin red into solution, the Alizarin red S stained cultures were incubated with 100 mM cetylpyridinium chloride for 2 h. Spectrophotometer was used to measured the absorbance of the released alizarin red S at 570 nm. Data are expressed as units of alizarin red S released per milligram of protein in each culture.
Real-time polymerase chain reactions (RT-PCR)
Total RNA was extracted by the TRIzol (Solarbio) method. According to the manufacturer’s instructions (TaKaRa), RNA was reverse-transcribed to cDNA. The TB Green Premix Ex Taq™ II (TaKaRa) was used to measured the expression levels of ANKH, col X, colⅡ and β-actin at the mRNA level. The following cycling conditions of RT-PCR were utilized: 95℃ for 30s and 40 cycles at 95℃ for 10s and at 60℃ for 30s. The primers for RT-PCR are listed in Table1. The expression of mRNA was calculated by 2-∆∆ct method, and ACTB was used as a reference gene.
Western blot analysis
RIPA lysis buffer (Solarbio) was used to extract cellular protein, and protein concentration was measured with the BCA Protein Assay kit (Solarbio). The extracted cellular protein was loaded on SDS-PAGE to electrophoresis, then it was transferred to a nitrocellulose membrane and incubated with 4% BSA in TBST for 60 min to block non-specific binding. The membrane was then incubated with the primary antibodies overnight at 4℃ in 4% BSA/TBST. The primary antibodies included anti-TAK1(1:1000, 4505, Cell Signaling Technology), anti-p-TAK1(1:1000, 4537, Cell Signaling Technology), anti-IKKβ(1:1000, 2684, Cell Signaling Technology), anti-p-IKKβ(1:1000, 2694, Cell Signaling Technology), anti-IkBα(1:1000, 9242, Cell Signaling Technology), anti-p-IkBα(1:1000, 9247, Cell Signaling Technology), anti-p-NF-kB p65(1:1000, 3031, Cell Signaling Technology), anti-ANKH(1:1000, ab129001, Abcam) and anti-GAPDH(1:1000, ab8245, Abcam). The membranes were incubated with anti-rabbit or anti-mouse secondary antibody for 60 min at room temperature. Finally, we measured the proteins on the membranes by a chemiluminiscent signal generated using Western blot detection reagents (Fdbio science). Western blot was visualized by enhanced chemiluminescence with Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY).
One-way analysis of variance (ANOVA) was used for multifactorial comparisons in this study. Experiments were performed three times. Data are presented as mean ± SD. Values of P < 0.05 were considered statistically significant. All data analysis was conducted with SPSS 22.0 analysis software.