Cell isolation and culture

Murine primary osteoblasts were obtained as described in previous research [24, 25]. Briefly, the calvarial bone was carefully dissected from one-day-old neonatal Wistar rats and washed with PBS solutions containing 100 U/ml penicillin and 100 µg/ml streptomycin thrice. The semi-transparent calvarial bone was incubated with 0.25% trypsin at 37 °C for thirty minutes, then sliced and incubated with 0.1% collagenase I at 37 °C overnight. The isolated osteoblasts were collected from the final digests and resuspended in the complete medium consisting of α-MEM, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured in the humidified atmosphere of 5% CO2.

Mechanical stress application

Cells were seeded onto collagen I-coated six-well BioFlex® plates (Flexcell Int. Corp, Hillsborough, NC, USA) at a density of 4 × 105 cells/ml. When the cells reached 80% confluence, cells were subjected to cyclic tensile stress at 10% magnitude and 0.5 Hz frequency for various durations (6, 12, 24, and 48 h). Cells not exposed to tensile stress served as the control group. After mechanical loading, cells were harvested and tested for changes in the expression of various genes and proteins [26].

Inhibitors preparation

ERK1/2 inhibitor (U0126 solution): U0126 powder was dissolved in dimethyl sulfoxide (DMSO) to 50µM concentration as the storage solution.

STAT3 inhibitor (Stattic solution): Stattic powder was dissolved in DMSO to 20µM concentration as the storage solution.

Real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to instructions. Then cDNA synthesis and subsequent amplification were performed in compliance with the manufacturer’s protocols (Takara, Tokyo, Japan). Gene-specific primers (Table 1) were designed by Shanghai Sangon Biotechnology Co., Ltd.

Table 1 Primer sequences and product sizes for quantitative polymerase chain reaction Western blot (WB) assays

Total cellular protein (Signalway Antibody LLC, USA) and nuclear protein (Shanghai Sangon Biotechnology Co., Ltd, China) were extracted using the corresponding kit. The sample concentration was determined by a BCA protein assay kit (Beyotime, China). After being mixed with loading buffer, the protein sample was boiled at 95 °C for 5 min. The protein sample was separated by SDS polyacrylamide gel electrophoresis and transferred to PVDF membrane. Later, the blots were blocked in 5% fat-free skimmed milk for one hour at room temperature, followed by incubation with primary antibodies (Huabio, China) against Osterix (1:1000, ER1914-47), COL-I (1:1000, ET1609-68), OCN (1:1000, ER1919-20), Runx2 (1:1000, ET1612-47), ALP (1:1000, ET1601-21), ERK1/2(1:5000, ET1601-29), pERK1/2 (1:1000, ET1603-22), STAT3(1:1000, ET1607-38), pSTAT3(1:1000, ET1603-40), and GAPDH (1:1000, ET1601-4 ) overnight at 4 °C. After a three-time wash in TBST solution, membranes were probed with goat anti-rabbit IgG conjugated secondary antibody (Hubio, China) for one hour at room temperature. The blots were cut prior to hybridization with antibodies during blotting. The membranes were processed with enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and visualized.

Immunofluorescence/Co-Immunofluorescence

After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, penetrated with 0.5% Triton X-100 for 20 min, blocked with 0.5% BSA for one hour, and incubated with primary antibodies (Huabio, China) at 4 °C overnight. After washing thrice, cells were processed with rhodamine-labeled anti-rabbit secondary antibodies and fluorescein isothiocyanate-labeled anti-mouse secondary antibodies for one hour. Cell nuclei were stained with DAPI and images were observed with the fluorescence microscope (Leica, Germany).

Co-immunoprecipitation (Co-IP)

The total protein was extracted as mentioned before. Co-IP was conducted as described previously [27]. 10% of the supernatants were prepared for Input, and the rest were incubated with normal IgG, anti-ERK1/2 (dilution 1:20), or anti-STAT3 (dilution 1:20) at 4 °C overnight with gentle end-over-end mixing, followed by pre-washed Protein A/G magnetic beads (Shanghai Epizyme Biomedical Technology Co., Ltd), mellow agitation, and overnight attachment. Protein A/G magnetic beads conjugates were separated by a magnetic rack. Proteins were eluted by boiling with loading buffer and then detached from beads by the magnetic rack.

Alkaline phosphatase (ALP) staining and alizarin red (ARS) staining

The mineralization solution was prepared as previously described [28] (α-MEM containing 10% FBS, 50 µM ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate). For ALP staining, cells were incubated in mineralization media for 7 days. ALP activity was performed using an ALP staining kit (Beyotime, Shanghai, China). Briefly, cells were rinsed with PBS three times and fixed in 4% paraformaldehyde at room temperature for 15 min. They were then incubated in ALP staining solution for 15 min.

For ARS solution, cells were cultured in mineralization media for 15 days. After washing with PBS, the cells were fixed with 4% paraformaldehyde and stained with ARS solution (Sigma-Aldrich, St. Louis, MO, USA; Solarbio, Beijing, China). The quantitative analysis of Alizarin red staining images was performed by Image J software according to the standard protocols as described in our previous research [29].

Statistical analysis

All quantitative data from three experiments were expressed as mean ± standard deviation and analyzed by GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Tests for normality and homoscedasticity were performed in GraphPad Prism according to the instruction guide. Data were assessed through two way-ANOVA (for factorial designed experiments) or one-way ANOVA (for three or more groups) followed by post-hoc Tukey test. P < 0.05 was considered to be statistically significant.