Antibodies against phosphorylated (p-)PDGFRβ (cat. no. 3161), PDGFRβ (cat. no. 3175), p-Src (cat. no. 2105), Src (cat. no. 2110), p-JAK2 (cat. no. 3771), JAK2 (cat. no. 3230; all dilution, 1:1,000), GAPDH (cat. no. 5174), β-actin (cat. no. 8457; both dilution, 1:2,500) and Runt-related transcription factor 2 (RUNX2; cat. no. 98059; dilution, 1:2,000), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The OSTERIX (cat. no. ab22552) and anti-COL1α1 (cat. no. ab166606; both dilution, 1:2,000) antibodies were purchased from Abcam (Cambridge, UK). Dexamethasone, ascorbic acid, β-glycerophosphate, AG1295 and hexadecylpyridinium chloride monohydrate were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). SU6656 and AG490 were purchased from Calbiochem (Merck KGaA). PDGF-BB was purchased from Peprotech, Inc. (Rocky Hill, NJ, USA).

Murine osteoblast-like MC3T3-E1 cells (China Center for Type Culture Collection, Wuhan, China) were cultured in α-MEM complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37°C, in a humidified atmosphere of 95% air and 5% CO₂. The cell culture medium was changed every 2–3 days. Cells were divided into five groups, including bovine serum albumin (BSA), PDGF-BB, PDGF-BB +20 µmol/l AG1295, PDGF-BB + 2 µmol/l SU6656 and PDGF-BB + 10 µmol/l AG490 10 µmol/l). All PDGF-BB used in this research was 25 ng/ml.

The MC3T3-E1 cells were cultured in a 6-well plate, grown to 70–80% confluence, and then starved in serum-free medium for 24 h. The cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich; Merck KGaA) at 4°C for 30 min. Equal amounts of protein quantified by BCA (60 µg) were loaded onto 10% SDS-polyacrylamide gels. Following electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Blots were blocked with 5% skimmed milk (Difco; BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 1 h, then incubated with the aforementioned primary antibodies overnight at 4°C. Afterwards, the blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Beijing TDY Biotech Co., Ltd., Beijing China; dilution, 1:5,000) for 2 h. Blots were detected using an ECL kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer's protocol. Western blot bands were quantified by densitometric analysis using Photoshop CS6 (Adobe Systems, Inc., San Jose, CA, USA).

Murine osteoblast-like MC3T3-E1 cells were cultured at 5×10⁴ cells/cm² in osteogenic medium containing 10% FBS, 0.1 µM dexamethasone, 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid for 21 days. At days 3 and 7, cells were harvested for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis or western blotting. Cells were fixed and alkaline phosphatase (ALP) staining was performed at days 3 and 7, whereas Alizarin Red staining was performed at days 14 and 21.

Total RNA was extracted from cells with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (1 µg) was used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis kit (Eppendorf, Hamburg, Germany). qPCR was performed in triplicate using SYBR Green Master mix (Takara Biotechnology Co., Ltd., Dalian, China) on an Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 31 sec and 95°C for 15 sec, then 60°C for 1 min and 95°C for 15 sec. The qPCR results were automatically analyzed using the Applied Biosystems 7500 system. The 2^−ΔΔCq method (14) was used to calculate the relative gene expression level. The primers used are presented in Table I.

Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, rinsed with deionized water and stained with a 5-bromo-4-chloro-3′-indolyphosphate/nitro-blue tetrazolium ALP color development kit (Beyotime Institute of Biotechnology, Haimen, China) for 12 h away from direct light, according to the manufacturer's protocol. Images were subsequently obtained.

Cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 10 min. They were then stained with 50 mM Alizarin Red S (pH 4.2) for 10 min at room temperature, and images were obtained. In order to determine the extent of calcium deposition, the mineralization of calcium nodules was quantified. After staining, the cells were washed three times with PBS. Hexadecylpyridinium chloride monohydrate (10%) was added and incubated for 20 min at room temperature. The absorbance of the supernatant was measured at 562 nm in triplicate using a Multiskan EX plate reader (Power Wave XS₂; Thermo Fisher Scientific, Inc.). Finally, cells were washed in PBS and lysed with RIPA buffer. and protein content quantified by BCA and calcium levels were normalized to the total protein content.

Proliferation activity was measured using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay. MC3T3-E1 cells were seeded into a 96-well plate at 2×10⁴ cells/cm². The medium was removed following adherence, and the cells were continuously cultured for 72 h. CCK-8 solution (10 µl) was added to 100 µl of medium in each well, and incubated with the cells for 2 h, before measuring the optical density (OD) at 450 nm. All experiments were performed in six replicates.

All quantitative data are expressed as the mean ± standard deviation. Analysis was performed with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons were evaluated with one-way or two-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated ≥3 times, and representative images are presented.

To evaluate whether PDGF-BB could activate PDGFRβ, Src and JAK2 in MC3T3-E1 cells, the cells were treated with PDGF-BB at a range of different durations, followed by the analysis of PDGFRβ, Src and JAK2 phosphorylation. It was identified that PDGFRβ, Src and JAK2 were rapidly phosphorylated by PDGF-BB at 5 min and their phosphorylation levels increased over time for the first 30 min (Fig. 1A-D). These results indicated that PDGFRβ, Src and JAK2 were expressed in MC3T3-E1 cells and could be activated by PDGF-BB in a time-dependent manner within 30 min.

Subsequently, it was investigated whether the phosphorylation of Src was performed directly by PDGFRβ, and whether Src was an upstream molecule of JAK2, in MC3T3-E1 cells. Therefore, the effects of low molecular mass inhibitors against PDGFRβ (AG1295), Src family kinases (SU6656) and JAK2 (AG490) on Src phosphorylation were determined. Inhibition of PDGFRβ and Src kinase activity resulted in the inhibition of JAK2 phosphorylation (Fig. 2A and D). However, the inhibition of JAK2 activity did not affect PDGF-BB-induced Src phosphorylation (Fig. 2A and C). SU6656 and AG490 inhibited the activity of PDGFRβ (Fig. 2B), while previous study showed that AG490 had no effect on PDGFRβ (9). Therefore, we hypothesized that Src and JAK2 may be involved in other pathways that affect the phosphorylation of PDGFRβ in MC3T3-E1 cells, and that Src was upstream of JAK2 in MC3T3-E1 cells.

In order to evaluate whether the Src/JAK2 signaling pathway served a function in MC3T3-E1 cell differentiation and proliferation, the effects of PDGF-BB were measured in MC3T3-E1 cells cultured in osteogenic differentiation media. As identified with, the expression of osteoblast differentiation-associated genes, including Runx2, a key transcription factor (15), and Osterix, ALP and Col1α1, increased in the PDGF-BB group compared with the control, PDGF-BB + AG1295 and PDGF-BB + SU6656 groups following culture in osteogenic differentiation media for 3 days (Fig. 3A). The same trend was observed for the expression of osteocalcin (OCN) and osteopontin (OPN) at 7 days (Fig. 3B). The expression levels of these genes increased 2–5 fold. In addition, western blot analysis indicated an increase in Runx2 and Col1α1 at 3 days (Fig. 4A and B) and 7 days (Fig. 4C and D) in the PDGF-BB group compared with the other groups. A significantly enhanced ALP signal was observed in the PDGF-BB group compared with the control, PDGF-BB + AG1295, PDGF-BB + SU6656 and PDGF-BB +AG490 groups at 3 and 7 days (Fig. 5A). The Alizarin Red staining intensity was greatly enhanced in the PGDF-BB group compared with the other groups at 14 and 21 days (Fig. 6A-C).

It was not previously determined whether PDGF-BB promotes the proliferation of MC3T3-E1 cells through the Src/JAK2 signaling pathway. Thus, the proliferation rate of cultured MC3T3-E1 cells was quantified with a CCK-8 assay in the present study. It was identified that the OD570 was markedly increased in the PDGF-BB group compared with the control, PDGF-BB + AG1295, PDGF-BB + SU6656 and PDGF-BB + AG490 groups (Fig. 5B).