Mouse calvaria osteoblasts, MC3T3-E1, were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in α-Minimum Essential Medium supplemented with 10% of fetal bovine serum. The culture medium was refreshed every three days thereafter.
The sense sequence for Orai1 small interfering RNA (siRNA) was 5`-CGTGCACAATCTCAACTCG-3`. Orai1 siRNA was dissolved in the serum-free α-Minimum Essential Medium with the Lipofectamine 2000 reagent for 20 min. Then, 40 nM Orai1 siRNA (or control siRNA) was added into 6-well plates containing MC3T3-E1 cells. The cells were collected for further analysis after 24 h.
Western Blot Analysis
Western blot analysis
The pretreated cells were lysed, and the total protein concentration was determined using the BCA Protein Assay Kit (Pierce, Illinois, USA). Equal quantities of cell lysate samples were mixed with loading buffer and then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in a 10% gel for 90 min. After that, the proteins were electrophoretically transferred from the gel onto a polyvinylidene fluoride microporous (PVDF) membrane for 60 min in a semidry chamber. Anti-mouse Orai1 antibodies and GAPDH antibodies were purchased from Invitrogen (Carlsbad, CA, USA). The following anti-mouse antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): cyclin D1 antibodies, cyclin E antibodies, Ras antibodies, Ras-GRF antibodies, p65-NF-κB antibodies, and phospho- (p-)p65-NF-κB antibodies. Anti-mouse CDK4 and CDK6 antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). These primary antibodies were diluted 1:800 to 1:1000 in Tris-buffered saline containing 0.1% of Tween 20 (TBST) and incubated with the PVDF membranes at 37°C for 60 min. To detect the primary antibody, the PVDF membranes were probed for 1 h with a horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG antibody diluted 1:5000 to 1:10000 in TBST. Immunoreactive protein bands were visualized with the Enhanced Chemiluminescence (ECL) Reagent Kit (Millipore, USA) and documented using a Western-Light Chemiluminescent Detection System (Applied Biosystems, USA). Densitometric analyses of the protein bands were performed in the ImageJ software (version 1.48, National Institutes of Health, USA). All fold changes of band densities were determined with normalization to GAPDH, an endogenous control. Relative protein expression was calculated as relative density of a protein band normalized to the endogenous control. Each experiment was conducted in triplicate and repeated three times independently.
Real-time Pcr Analysis
Total RNA was extracted from the pretreated cells using the RNA Extraction Kit (Toyobo, Japan). Then, the total RNA was reverse-transcribed into cDNA by means of the cDNA Reverse Transcription Kit (Invitrogen, USA). The forward primer for Orai1 expression analysis was 5`-TACTTAAGCCGCGCCAAG-3`, and the reverse primer was 5`-ACTTCCACCATCGCTACCA-3`. The forward primer for cyclin D1 expression analysis was 5`-TTTCTTTCCAGAGTCATCAAGTGT-3`, and the reverse primer was 5`- TGACTCCAGAAGGGCTTCAA-3`. The forward primer for cyclin E expression analysis was 5`-TTTCTGCAGCGTCATCCTC-3`, and the reverse primer was 5`-TGGAGCTTATAGCTTCGCACA-3`. The forward primer for CDK4 expression analysis was 5`- TCAGTGGTGCCAGAGATGG-3`, and the reverse primer was 5`-GGAAGGCAGAGATTCGCTTA-3`. The forward primer for CDK6 expression analysis was 5`- GCCCTTACCTCGGTGGTC-3` and the reverse primer was 5`-ACAGGGGTGGCATAGCTG-3`. Real-time PCR was used to measure mRNA expression levels of Orai1, cyclin D1, cyclin E, CDK4, and CDK6. Real-time PCR was carried out with the SYBR green PCR reagent (Takara, Japan) on a Stratagene M63005P Multiplex Quantitative PCR System (Agilent Technologies, Germany). GAPDH served as an endogenous control. The real-time PCR cycling conditions were as follows: denaturation at 95°C for 10 min; 35 cycles of denaturation at 95°C for 10 s, annealing at 59°C for 15 s, and extension at 72°C for 20 s; and final extension at 72°C for 15 min. The reaction was terminated by cooling at 4°C. Each experiment was conducted in triplicate and repeated three times independently.
Cell Proliferation Assay
Cell proliferation was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Approximately 104 cells per well were added into a 96-well cell culture plate. After the cells were cultured for 24, 48, 64, or 72 h, 50 µL of MTT (Sigma-Aldrich, USA ) and 150 µL of the culture medium were added into each well of the cell culture plate. Then, the cell culture plates were incubated at 37°C for 4 h. MTT was dissolved in 200 µL of DMSO in each well for 30 min. Finally, the absorbance of each well was measured at 570 nm on a microplate reader (BioAssay Systems, USA). All fold changes of cell proliferation were normalized according to the following formula: (Sample absorbance - Blank absorbance)/(Control absorbance - Blank absorbance). Each experiment was carried out in triplicate and repeated three times independently.
Cell Apoptosis Assay
Apoptosis of osteoblasts was detected using a DeadEnd Fluorometric Tunel System (Promega, USA) that end-labels fragmented DNA from apoptotic cells via modified terminal deoxynucleotidyl transferase dUTP nick end labeling (Tunel) staining. Briefly, the cells were fixed with a 4% methanol-free formaldehyde solution and rinsed with PBS twice, then the fragmented DNA of the apoptotic cells was end-labeled with fluorescein-12-dUTP, and the slides were incubated with 4,6-diamidino-2-phenylindole (DAPI) to stain nuclei. The cells were immediately analyzed under a fluorescence microscope with a standard fluorescein filter set (Olympus, Japan) to examine green fluorescence of apoptotic cells at 520 nm and blue fluorescence of DAPI-stained nuclei at 460 nm. Each experiment was carried out in triplicate and repeated three times independently.
Cell Cycle Assay
The cells were cultured in the serum-free medium for 24 h. Then, the cells were cultivated in the medium with 10% of FBS. After 24 h, the cells were collected into a centrifuge tube and resuspended in 1 mL of anhydrous ethanol. The cells were incubated overnight at 4°C. Next, the cells were collected into a centrifuge tube and resuspended in 500 µL of a propidium iodide (PI) solution (Absin, China). The cells were stained in at 4°C (in a refrigerator) for 60 min. Finally, the cell cycle was examined by flow cytometry, and the data were analyzed statistically. Each experiment was conducted in triplicate and repeated three times independently.
We employed SPSS 16.0 software to analyze the data statistically. All values are expressed as mean ± standard error from at least three independent experiments. Multigroup comparisons were analysed using one-way (ANOVA) followed by Tukey's post hoc significance difference tests. Student’s t test was used for pairwise comparisons. The results were considered statistically significant when a p value was < 0.05.