Xiao et al. BMC Molecular and Cell Biology(2023) 24:9
https://doi.org/10.1186/s12860-023-00471-8

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Cyclic tensile force modifies calvarial
osteoblast function via the interplay between
ERK1/2 and STAT3
Xiaoyue Xiao1,2,3, Shujuan Zou1 and Jianwei Chen1*
Abstract
Background Mechanical therapies, such as distraction osteogenesis, are widely used in dental clinics. During thisprocess, the mechanisms by which tensile force triggers bone formation remain of interest. Herein, we investigated
the influence of cyclic tensile stress on osteoblasts and identified the involvement of ERK1/2 and STAT3.
Materials and methods Rat clavarial osteoblasts were subjected to tensile loading (10% elongation, 0.5 Hz) for
different time periods. RNA and protein levels of osteogenic markers were determined using qPCR and western blot
after inhibition of ERK1/2 and STAT3. ALP activity and ARS staining revealed osteoblast mineralization capacity. Theinteraction between ERK1/2 and STAT3 was investigated by immunofluorescence, western blot, and Co-IP.
Results The results showed that tensile loading significantly promoted osteogenesis-related genes, proteins andmineralized nodules. In loading-induced osteoblasts, inhibition of ERK1/2 or STAT3 decreased osteogenesis-related
biomarkers significantly. Moreover, ERK1/2 inhibition suppressed STAT3 phosphorylation, and STAT3 inhibition
disrupted the nuclear translocation of pERK1/2 induced by tensile loading. In the non-loading environment, inhibition
of ERK1/2 hindered osteoblast differentiation and mineralization, while STAT3 phosphorylation was elevatedafter ERK1/2 inhibition. STAT3 inhibition also increased ERK1/2 phosphorylation, but did not significantly affectosteogenesis-related factors.
Conclusion Taken together, these data suggested that ERK1/2 and STAT3 interacted in osteoblasts. ERK1/2-STAT3
were sequentially activated by tensile force loading, and both affected osteogenesis during the process.
Keywords Osteoblasts, Cyclic tensile stress, Osteogenesis, ERK1/2, STAT3*Correspondence:
Jianwei Chen
chenjianwei2017@vip.163.com
1
State Key Laboratory of Oral Diseases and National Clinical ResearchCenter for Oral Diseases, Department of Orthodontics, West China
Hospital of Stomatology, Sichuan University, Chengdu, China
2
Chongqing Key Laboratory of Oral Disease and Biomedical Sciences,
Stomatological Hospital of Chongqing Medical University, Chongqing,
China
3
Chongqing Municipal Key Laboratory of Oral Biomedical Engineeringof Higher Education, Stomatological Hospital of Chongqing Medical
University, Chongqing, China© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Xiao et al. BMC Molecular and Cell Biology(2023) 24:9

Introduction
Mechanical therapies have emerged as common choices
in dental clinics, such as distraction osteogenesis (DO),orthodontic tooth movement, and maxillary advancement/expansion. It is well known that tensile force stimulates osteogenic activity, thus driving bone formation.
In recent years, researchers have been paying increasing
attention to mechanotransduction, by which mechanicalforce is translated into biochemical signals and thereby
initiates osteogenesis.
Osteoblasts, which are derived from mesenchymal
stem cells, are the main bone-forming cells in response tomechanical loading [1]. It has been reported that osteoblasts are involved in bone formation from the synthesisof matrix proteins to mineralization [2, 3]. In addition,
osteoblastic cells such as MC3T3-E1 display mechanosensitive characteristics [4] and their function influences tension-induced osteogenesis [5]. Therefore, it isof critical importance to clarify mechanotransduction in
osteoblasts, which could help develop mechanism-based
adjuvant methods in favor of bone formation.
Extracellular signal-regulated kinase 1/2 (ERK1/2)
and signal transducer and activator of transcription 3(STAT3) are two key mechanosensory molecules. ERK1/2
reportedly mediates the conversion of external mechanical stimulation to intracellular biochemical signals [6, 7]
and then triggers cellular response. The mechanosensitive properties of STAT3 have also been confirmed inprevious studies. Its deletion impairs load-driven boneformation in long bones [8], as well as bone remodeling
during orthodontic tooth movements [5].
A number of biological processes have been proposed
to involve crosstalk between signaling pathways. ERK1/2
and STAT3 may coordinate in various ways to modulate
physiological processes. In mechanical loading-responsive myocardial cells, ERK1/2 and STAT3 were the downstream effectors of mechanical signal transmission [9].
Crosstalk between ERK1/2 and STAT3 plays a criticalrole in osteogenic lineage cells as evidenced by a growing body of research. Hyperactivation of ERK1/2 following STAT3 deletion enhances the proliferation abilityof bone mesenchymal stem cells (BMSCs) exposed togranulocyte colony-stimulating factor [10, 11]. Additionally, in MC3T3-E1 treated with IL-6/IL-6 soluble receptor, ERK1/2 and STAT3 played antagonistic actions in
MC3T3-E1 differentiation and the inhibition of STAT3
activated ERK1/2 [12]. It should be noted that there is noconclusive interaction between the two signaling pathways, and they could counteract or compensate for eachother to coordinate biological events. Thus, we speculate that crosstalk between ERK1/2 and STAT3 exists inosteoblasts under mechanical loading and contributes tothe osteogenic response.

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In this study, we first examined the effects of cyclic tensile stress on osteogenesis-related factors in rat primaryosteoblasts. Next, inhibition of ERK1/2 or STAT3 wascarried out on osteoblasts to determine their roles andpotential interdependences.

Results
Osteogenesis-related factors were upregulated by cyclic
tensile loading in a time-dependent manner

Cyclic tensile loading (10% elongation, 0.5 Hz) was
imposed on osteoblasts using the Flexcell tension systemfor 6, 12, 24, and 48 h. Osteoblasts not subjected to load
served as the control group. To investigate the effects
of tensile loading on osteogenesis in osteoblasts, theexpression of osteogenesis-related genes was examined
by qPCR and western blots. Compared with the control
group, mRNA levels of Osterix, COL-I, OCN, Runx2,
and ALP demonstrated varying degrees of increase afterstrain load. Osterix mRNA level was significantly upregulated as early as 6 h of strain loading. Runx2 and ALP
mRNA expression peaked at 24 h and then decreased at
48 h. COL-I and OCN mRNA expression increased overtime, peaking at 48 h (Fig. 1A). Figure 1B showed that
their protein expression changes were in agreement withthe changes in the corresponding mRNA.
ERK1/2 and STAT3 were phosphorylated and translocatedto the nucleus in response to tensile loading

We analyzed the protein levels of ERK1/2, STAT3, andtheir phosphorylation after different time periods ofloading using western blots. ERK1/2 and STAT3 protein
expression was not significantly altered overall. However, the protein level of pERK1/2 was greatly increased
at 6 h of strain loading and reached its maximum at 24 h.
After 24 h of mechanical loading, pSTAT3 expression
increased significantly, followed by further increases after
48 h (Fig. 2A and B).
Immunofluorescence was used to determine the subcellular localization of ERK1/2 and STAT3. As shown in
Fig. 2C, ERK1/2 protein in the control group was mainly
located in the cytoplasm, whereas 24 h of tensile stressloading induced ERK1/2 into the nucleus (Fig. 2C).
STAT3 protein was found in both the cytoplasm andnucleus in the control group. The accumulation of STAT3
in the nucleus was also observed after 24 h of loading(Fig. 2D).
ERK1/2 inhibition suppressed osteoblast differentiationand mineralization-related factors

To assess ERK1/2 involvement in osteogenesis, we incubated the cells with the ERK1/2 inhibitor U0126 (10µM)
for 12 h, followed by 24 h of mechanical loading. The
mRNA levels of osteogenesis-related factors were measured using qPCR. There was a significant increase inXiao et al. BMC Molecular and Cell Biology(2023) 24:9

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Fig. 1 The osteogenic gene and protein expression of primary osteoblasts under mechanical tensile loading for 6, 12, 24, and 48 h. (A) The relative gene
expressions of Osterix, COL-I, OCN, Runx2, and ALP were increased over time. (B) The protein levels of Osterix, COL-I, OCN, Runx2, and ALP were determinedby western blots and their densitometric analysis. Bars represent mean ± SD, *p < 0.05, compared with the control group (0 h group)

COL-I, ALP, and Runx2 mRNA expression after tensile stress loading compared to the control group. It wasobserved that inhibition of ERK1/2 greatly reduced theexpressions of COL-I, ALP, and Runx2 regardless of loading presence (Fig. 3A). Consistent with mRNA alteration, protein expressions of COL-I, ALP, and Runx2 weremarkedly increased after loading, but were reversed by
ERK1/2 inhibition. In the control + U0126 group, Runx2
protein level was significantly reduced, whereas that of
COL-I and ALP was not affected (Fig. 3B).
The mineralization capacity was further examined by
ALP and ARS staining. As illustrated in Fig. 3C, alizarin red staining revealed that more mineralized nodules
were observed after loading, but they were reduced when
ERK1/2 was inhibited. The formation of mineralized nodules in the control + U0126 group was less pronounced
than in the control group. According to Fig. 3D, ALP
activity was enhanced by tensile stress, while ERK1/2
inhibition reduced its intensity.

STAT3 regulated the promoting effects of mechanicalstrain on osteoblast differentiation and mineralization

To assess STAT3 involvement in osteogenesis, we treatedthe cells with STAT3 inhibitor Stattic (5µM) for 6 h, followed by 24 h of loading. There was no significant difference between the control group and the control + Stattic
group in terms of the mRNA levels of COL-I, ALP, andRunx2. In response to mechanical loading, mRNA
expression of COL-I, ALP, and Runx2 increased, but
STAT3 inhibition dramatically reduced it (Fig. 4A). As
shown by western blot analysis, Runx2, ALP, and COL-I
protein expression did not differ significantly between
the control group and the control + Stattic group. STAT3
inhibition, however, attenuated their increased expression induced by mechanical loading (Fig. 4B).
ARS and ALP staining were also performed to identify
mineralization capacity. Tensile stress loading greatly
enhanced ALP staining intensity and mineralized nodule
formation. In the tensile stress + Stattic group, ALP activity and mineralized nodule formation were significantly
decreased compared with those in the tensile stressgroup. It was noteworthy that STAT3 inhibition did notXiao et al. BMC Molecular and Cell Biology(2023) 24:9

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Fig. 2 ERK1/2 and STAT3 were activated after mechanical tensile loading. (A) Densitometric analysis of ERK1/2, pERK1/2, STAT3, and pSTAT3 protein levels
in osteoblasts under tensile loading for 6, 12, 24, and 48 h. (B) Representative images of ERK1/2, pERK1/2, STAT3, and pSTAT3 protein changes under tensileloading for 6, 12, 24, and 48 h. (C-D) Immunofluorescence assay showed nuclear translocation of ERK1/2 and STAT3 protein induced by tensile loading for
24 h. Scale bar: 25 μm, Bars represent mean ± SD. *p < 0.05, compared with the control group (0 h group)

Fig. 3 The osteoblast differentiation and mineralization of primary osteoblasts after ERK1/2 inhibition in the presence or absence of tensile loading. Osteoblasts were treated with U0126 for 12 h, followed by mechanical loading of 24 h. (A) The mRNA expressions of COL-I, ALP, and Runx2 were detected by
qPCR. (B) Western blots analysis of the osteogenic markers COL-I, ALP, and Runx2. (C) Alizarin red staining showed the formation of mineralized nodulesof osteoblasts after ERK1/2 inhibition and (D) ALP activity (scale bar: 200 μm). Bars represent mean ± SD. *p < 0.05

Xiao et al. BMC Molecular and Cell Biology(2023) 24:9

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Fig. 4 The osteoblast differentiation and mineralization of primary osteoblasts after STAT3 inhibition in the presence or absence of tensile loading. Osteoblasts were treated with Stattic for 6 h, followed by mechanical loading for 24 h (A) The mRNA expressions of COL-I, ALP, and Runx2 were detected by
qPCR. (B) Western blots analysis of the osteogenic markers COL-I, ALP, and Runx2. (C) Alizarin red staining showed the formation of mineralized nodulesof osteoblasts after STAT3 inhibition and (D) ALP activity (scale bar: 200 μm). Bars represent mean ± SD. *p < 0.05

significantly impact ALP staining and mineralized nodule
formation in the control + Stattic group (Fig. 4C and D).
ERK1/2 and STAT3 had interaction and their activationinfluenced each other

Initially, we used Co-IP to determine whether STAT3
and ERK1/2 interacted, and the results suggested thatSTAT3 coimmunoprecipitated with ERK1/2 in all groups(Fig. 5A).
The subcellular localization of the two proteins was
further delineated by co-immunofluorescence (Co-IF). In
the control group, both ERK1/2 and STAT3 were locatedin the nucleus and cytoplasm. STAT3 was mostly foundin the nucleus in the control + U0126 group, suggesting
ERK1/2 inhibition impacted STAT3 localization. However, STAT3 inhibition did not significantly alter ERK1/2
localization in the control + Stattic group. As a result of
tensile loading, STAT3 and ERK1/2 were found to accumulate near nuclear regions. In contrast, in the tensilestress + U0126 group, ERK1/2 and STAT3 were more
localized in the cytoplasm. Furthermore, STAT3 inhibition decreased the loading-induced nuclear translocation of ERK1/2, as demonstrated in the in the tensilestress + Stattic group (Fig. 5B).
Western blot analysis further clarified the mutual relationship between STAT3 and ERK1/2 in a more quantitative sense. First, we detected the levels of STAT3 andpSTAT3 in whole cell lysates, finding that total STAT3
protein expression was not significantly different acrossgroups. The control + U0126 group displayed a significantly higher level of pSTAT3 protein than the control
group. However, loading-induced pSTAT3 upregulation was reduced after ERK1/2 inhibition (Fig. 5C). Our
next step was to examine ERK1/2 protein changes following STAT3 inhibition. ERK1/2 expression did not
differ significantly between groups. However, pERK1/2
expression was significantly higher in the control + Stattic group compared to the control group. In spite of this,Stattic administration did not impact the pERK1/2 protein expression between the tensile stress and the tensilestress + Stattic groups (Fig. 5D).
We then extracted nuclear proteins and used western
blots to measure nuclear pERK1/2 and pSTAT3 expression. As shown in Fig. 5E, pERK1/2 expression was
significantly higher in the control + Stattic group compared with the control group. STAT3 inhibition, however, greatly reduced loading-induced nuclear pERK1/2
expression. Regarding the impact of ERK1/2 on STAT3
expression, inhibition of ERK1/2 prohibited loadinginduced nuclear pSTAT3 protein. Moreover, the control + U0126 group demonstrated a higher level of nuclear
pSTAT3 protein than the control group.

Xiao et al. BMC Molecular and Cell Biology(2023) 24:9

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Fig. 5 ERK1/2 and STAT3 had interaction and their activation influenced each other. (A) Representative images of Co-IP. (B) Immunofluorescence assay
showed the subcellular location of ERK1/2 and STAT3 was affected by loading, U0126, and Stattic administration. (C) Western blot analysis of STAT3 andpSTAT3 protein expression in whole cell lysates. (D) Western blot analysis of EKR1/2 and pERK1/2 protein expression in whole cell lysates. (E) Western blot
analysis was performed to examine the protein levels of pERK1/2 and pSTAT3 in nuclear fractions. Bars represent mean ± SD. *p < 0.05

Discussion
A variety of mechanical therapies have been developed
to correct craniofacial skeletal or/and dental problems,
including distraction osteogenesis and orthodontic tooth
movement. Molecular mechanisms by which externalmechanical stimulation is translated into cells and thenaffects cell activities are always of interest. Currently, thepurpose of our study was to examine the effect of tensileloading on calvarial osteoblasts, as well as the involvement of ERK1/2 and STAT3.
The Flexcell tension system is widely used to apply
stretch forces to cell populations. The strain regimes varydepending on the type of cell population studied, and the
responses induced by stretch forces vary among different types of cells. In osteoblastic cells, elongation from
0.25% [13] to 24% [14] significantly influenced survival,
cytokine production, maturation, and differentiation. The
upregulation of osteogenic differentiation factors wasfound to be dependent on strain magnitude in the regionof < 18% elongation [15]. Additionally, osteoblast proliferation has been found to be stimulated by elongation
between 0–12% [16], but higher levels would adversely
impact it [17]. As our study focused mainly on osteoblastic function under tensile force, we exposed primary rat
osteoblasts to 10% elongation as a way to enhance differentiation without compromising cell viability.

The upregulation patterns of osteogenic markers in ourstudy were correlated with osteoblast differentiation profiling to some extent. Runx2 and Osterix are two mastertranscription factors that are involved at the early stageof osteoblast differentiation [18]. These two transcription factors were found to be upregulated as early as 12 hof tensile loading, which might drive the upregulationof other osteogenic markers. ALP, COL-I, and OCN aremarkers of mature osteoblasts [19] and in our study, they
were increased at 24 h of loading. This may imply Runx2and Osterix are the regulators to initiate loading-induced
differentiation, which then triggers activation of latestage markers such as ALP, COL-I and OCN.
It has been reported that ERK1/2 activation directly promotes the expression of Runx2 and OCN [20]. As mentioned above, Runx2 was the chief transcription factor at
the early stage, while OCN was the marker of mature osteoblasts. The ERK1/2 phosphorylation in our study was sustained from 6 to 48 h of tensile loading, which suggested
that ERK1/2 was involved through osteoblast differentiation
process in response to mechanical loading. To further clarify its role, U0126 was used to inhibit ERK1/2. Interestingly,
ALP and ARS staining revealed that ERK1/2 inhibition
impaired osteoblast mineralization regardless of mechanicalloading. However, ERK1/2 inhibition did not alter ALP protein expression when no mechanical loading was applied.
This inconsistency could be explained by the accumulation

Xiao et al. BMC Molecular and Cell Biology(2023) 24:9

of inhibitory effects and the presence of tensile forces. For
the ALP staining test, osteoblasts were incubated in osteogenic medium supplemented with U0126 for 7 days. In contrast, the duration of U0126 treatment in the western blot
was 12 h. Taken together, we concluded that tensile stressaccentuated the role of ERK1/2 in osteoblast differentiationand mineralization. It is possible that ERK1/2 inhibition
undermines osteoblast differentiation capacity in a timedependent manner in a non-loading environment.
Increasing evidence suggests that STAT3 plays a rolein osteogenesis depending upon cell lineages, maturation status, and external stimuli [21]. Accordingly, in ourstudy, STAT3 exerted different effects depending upon
whether loading was present or not. Inhibition of thisfactor reduced loading-enhanced osteogenesis-related
markers and mineralization activities, but no significant
interference was observed in the non-loading environment. To update, recent in vivo research [22] has shownthat STAT3 signaling is involved in load-augmented boneformation. Thus, as a result of our study, STAT3 was
implicated in loading-enhanced osteogenic differentiation in primary osteoblasts, but did not appear to exert
a significant effect when tensile loading was not applied.
In light of this discovery, we further examined whether
STAT3’s lack of engagement under non-mechanical loading conditions could be attributed to complicated interactions with ERK1/2.
Advances in decoding the crosstalk between ERK1/2
and STAT3 in specific settings may provide novel insights
into the mechanism behind cellular events. The inhibition of ERK1/2 in esophageal cancer activated STAT3 andthe activation of STAT3 reversed the therapeutic effect of
ERK1/2, resulting in drug resistance [23]. In our study, theinteraction between the two pathways varied depending on
the presence of tensile forces. ERK1/2 inhibition resulted
in STAT3 activation in non-loading environments and viceversa. We correlated this reciprocal relationship with osteogenesis. It should be noted that ERK1/2 inhibition remarkably impeded osteoblast differentiation and mineralizationin the absence of loading, despite STAT3 activation. By contrast, STAT3 inhibition did not impair osteogenesis with thecompensatory activation of ERK1/2. Based on these observations, we assumed that ERK1/2 played a more prominentrole in osteogenesis than STAT3 in non-mechanical loading
environments. The interaction between these two pathways
altered when tensile loading was applied. The activation of
ERK1/2 and STAT3 by tensile loading occurred in chronological order, with ERK1/2 activation followed by STAT3
activation. Furthermore, ERK1/2 inhibition completely
prevented STAT3 phosphorylation and nuclear translocation induced by loading. Although STAT3 inhibition did not
impact loading-induced ERK1/2 phosphorylation (total), it
disrupted phosphorylated ERK1/2 nuclear translocation.
Together, STAT3 might function as a downstream mediator

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of ERK1/2 during mechanical loading, and STAT3 activation is essential for pERK1/2 translocation.
However, the study had certain limitations. First, lateactivation of STAT3 at 24 h of tensile loading may involvethe synthesis of intermediate factors. Therefore, methods such as cycloheximide chase analysis could be used
to further explore potential protein synthesis or degradation. Second, a more thorough elucidation of their roles
in osteogenesis could be achieved by using silencing RNA
to directly target ERK1/2 and STAT3 gene expression.

Methods
Cell isolation and culture

Murine primary osteoblasts were obtained as describedin 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 andincubated with 0.1% collagenase I at 37 °C overnight. The
isolated osteoblasts were collected from the final digests
and resuspended in the complete medium consistingof 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 tensilestress at 10% magnitude and 0.5 Hz frequency for various durations (6, 12, 24, and 48 h). Cells not exposed totensile stress served as the control group. After mechanical loading, cells were harvested and tested for changes inthe 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.

Xiao et al. BMC Molecular and Cell Biology(2023) 24:9

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Table 1 Primer sequences and product sizes for quantitative
polymerase chain reaction
Target gene

Runx2
Osterix
OCN
COL-I
ALP
GAPDH
*

Sequences (5’-3’)*

F: ATCTACACCGTGGGATATTCCA
R: GCAGCATAAACGACAGGAACA
F: CCTCTTGAGAGGAGACGGGA
R: GGGCTGAAAGGTCAGCGTATF: GCACACCTAGCAGACACCAT
R: GTGTTGTCCCTTCCCCTCTG
F: GATGTGGAATACGAACTGGATG
R: TGGGAATGCTTGTGTCTGG
F: GAACAGACCCTCCCCACGAG
R: CCTGAGATTCGTCCCTCGC
F: ACAGCAACAGGGTGGTGGAC
R: TTTGAGGGTGCAGCGAACTT

Product
sizes
(bp)
130
157
135
134
104
136

F means forward primers; R means reverse primers

Western blot (WB) assays

Total cellular protein (Signalway Antibody LLC, USA) andnuclear 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 gelelectrophoresis 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 ) overnightat 4 °C. After a three-time wash in TBST solution, membranes were probed with goat anti-rabbit IgG conjugatedsecondary antibody (Hubio, China) for one hour at room
temperature. The blots were cut prior to hybridization with
antibodies during blotting. The membranes were processedwith enhanced chemiluminescence detection reagents(Applygen Technologies Inc., Beijing, China) and visualized.
Immunofluorescence/Co-Immunofluorescence

After treatment, the cells were washed with PBS, fixedwith 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 incubatedwith primary antibodies (Huabio, China) at 4 °C overnight. After washing thrice, cells were processed withrhodamine-labeled anti-rabbit secondary antibodies andfluorescein isothiocyanate-labeled anti-mouse secondary antibodies for one hour. Cell nuclei were stained withDAPI and images were observed with the fluorescencemicroscope (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 withgentle end-over-end mixing, followed by pre-washed
Protein A/G magnetic beads (Shanghai Epizyme Biomedical Technology Co., Ltd), mellow agitation, andovernight attachment. Protein A/G magnetic beads conjugates were separated by a magnetic rack. Proteins were
eluted by boiling with loading buffer and then detachedfrom 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, 50µM 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 ourprevious research [29].
Statistical analysis

All quantitative data from three experiments wereexpressed 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.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00471-8.
Supplementary Material 1

Xiao et al. BMC Molecular and Cell Biology(2023) 24:9

Acknowledgements
None.
Author Contribution
CJW initiated the study and revised the manuscript; ZSJ designed the study,
helped perform the analysis and reviewed the manuscript; XXY conducted
relevant experiments and drafted the manuscript.
Funding
This work was supported by the National Natural Science Foundation of China[ grant number 82071150] and the National Natural Science Foundation ofChina [ grant number 81901040]. And the authors declared no potential
conflicts of interest.
Data Availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethical approval and consent to participate
The procedures involving animals were in accordance with the guidelines of
the National Institutes of Health for the Care and Use of Laboratory Animals.
All the methods were approved by the experiment ethics committee of theState Key Laboratory of Oral Diseases of Sichuan University (Chengdu, China)(Reference number: WCHSIRB-D-2021-193) and in accordance with ARRIVE
guidelines.
Consent for publication
Not applicable.
Competing Interest
The authors declare that they have no competing interests.
Received: 1 December 2022 / Accepted: 2 March 2023

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