RSPO3-LGR4 Regulates Osteogenic Differentiation Of Human Adipose-Derived Stem Cells Via ERK/FGF Signalling

The four R-spondins (RSPOs) and their three related receptors, LGR4, 5 and 6, have emerged as a major ligand-receptor system with critical roles in development and stem cell survival. However, the exact roles of the RSPO-LGR system in osteogenesis remain largely unknown. In the present study, we showed that RSPO3-shRNA increased the osteogenic potential of human adipose-derived stem cells (hASCs) significantly. Mechanistically, we demonstrated that RSPO3 is a negative regulator of ERK/FGF signalling. We confirmed that inhibition of the ERK1/2 signalling pathway blocked osteogenic differentiation in hASCs, and the increased osteogenic capacity observed after RSPO3 knockdown in hASCs was reversed by inhibition of ERK signalling. Further, silencing of LGR4 inhibited the activity of ERK signalling and osteogenic differentiation of hASCs. Most importantly, we found that loss of LGR4 abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. Collectively, our data show that ERK signalling works downstream of LGR4 and RSPO3 regulates osteoblastic differentiation of hASCs possibly via the LGR4-ERK signalling.

Knockdown of rspo3 causes ventral oedema and vascular defects in Xenopus 20 . Rspo3-null mice suffer from severe vascular defects and are embryonic lethal 21 . Recently, R-spondins were identified as ligands of the leucine-rich repeat-containing G-protein coupled receptors (LGRs), including LGR4, 5 and 6 14,15,21 . RSPO-LGR was demonstrated play critical roles in development and stem cell survival. However, the exact roles of this ligand-receptor system in osteogenesis remain largely unknown.
In the present study, we first identified that RSPO3 is a negative regulator of hASCs osteogenic differentiation. RSPO3 silencing leads to activation of ERK signalling pathway, which is essential for osteoblast differentiation of hASCs.
LGR4 positively regulates osteoblast differentiation of hASCs via ERK signalling pathway. Moreover, loss of LGR4 attenuates the enhanced osteogenesis induced by RSPO3 silencing. Together, our findings suggested that RSPO3 functions as a negative regulator of osteogenesis possibly through a LGR4-ERK dependent mechanism.

Results
Downregulation of endogenous RSPO3 increases the osteogenic differentiation of hASCs in vitro. To evaluate the potential role of RSPO3 in the process of osteogenic differentiation, we first investigated the expression of RSPO3 in hASCs after osteogenic induction. As shown in Supplementary Fig. S1A,B, RT-qPCR showed that increased expression of RSPO3 was accompanied by upregulation of the osteogenic marker RUNX2. We next generated a stable cell line with lentiviruses expressing an RSPO3 shRNA. The knockdown efficiency was confirmed by immunofluorescence, western blotting, and RT-qPCR ( Fig. 1A-D). In addition, we examined the expressions of RSPO1, 2 and 4 by RT-qPCR after RSPO3 silencing. There was no significant difference between the RSPO3 knockdown cells and cells transfected with a scrambled shRNA ( Supplementary Fig. S1C,D). After culturing the hASCs in osteogenic media (OM) for 7 days, alkaline phosphatase (ALP) activity was detected as being increased significantly by RSPO3 knockdown (Fig. 1E,F). Moreover, the extracellular matrix mineralization, as determined by Alizarin Red S staining and quantification, was also augmented in RSPO3 knockdown cells at 2 weeks after osteogenic induction (Fig. 1G,H). To confirm that RSPO3 depletion promoted osteogenic differentiation, we investigated several osteogenic markers in osteogenically-stimulated hASCs. As shown in Fig. 1I-K, in contrast to the control cells, knockdown of RSPO3 resulted in significantly increased mRNA expression levels of RUNX2, ALP and OCN (encoding osteocalcin). Furthermore, we investigated the proliferation levels of the RSPO3-silenced cells. The growth curve revealed that RSPO3 silencing had no effects on the proliferation of hASCs, as determined by a CCK-8 assay ( Supplementary Fig. S1E). In addition, the osteogenic differentiation of hASCs could also be blocked with another independent RSPO3 shRNA fragment, but not with a random shRNA, excluding the possibility of off-target effects ( Supplementary Fig. S2A-K). Taken together, these data indicated that downregulation of RSPO3 promoted osteogenic differentiation in vitro.

Overexpression of RSPO3 inhibits the osteogenic differentiation of hASCs in vitro.
To further confirm the important function of RSPO3 in osteogenesis, the recombinant human RSPO3 protein (rhR-SPO3) was used for rescue experiments. At a concentration of 800 ng/ml, rhRSPO3 inhibited the upregulation of ALP activity and mineralization in RSPO3 knockdown cells as well as in Scrsh group cells ( Fig. 2A-D). Consistently, treatment with rhRSPO3 resulted in decreased mRNA expression levels of RUNX2 and OCN (Fig. 2E,F). Furthermore, we established RSPO3 overexpression cells by lentivirus transfection in hASCs (Supplementary Fig. S3A-C). After osteogenic differentiation for 7 days, ALP activity was decreased in RSPO3 overexpressing cells ( Supplementary Fig. S3D-E), and extracellular matrix mineralization also decreased, as assessed by Alizarin red staining at day 14 ( Supplementary Fig. S3F-G). In addition, RT-qPCR analysis revealed that RSPO3 overexpression decreased RUNX2 and OCN mRNA levels ( Supplementary Fig. S3H-I). Taken together, these results indicated that RSPO3 inhibited osteogenic differentiation in vitro.

Downregulation of RSPO3 enhances hASC-mediated bone formation in vivo. To verify our
in vitro findings, we examine whether RSPO3 affected hASC-mediated bone formation in vivo. As shown in Fig. 3A, haematoxylin and eosin (H&E) staining showed that RSPO3 knockdown cells formed more bone-like tissues compared with control cells. Quantitative measurements demonstrated that the area of bone formation was increased significantly in hASC/RSPO3sh cells (P < 0.05) ( Supplementary Fig. S4A). Consistent with the observations from H&E staining, the osteogenic differentiation potential was markedly increased for hybrids containing hASCs/RSPO3sh compared with hASCs/Scrsh, as detected by Masson' trichrome staining (Fig. 3B). Most importantly, we found that the osteogenic marker OCN was highly expressed in RSPO3sh cells, as determined by immunohistochemical (IHC) staining (Fig. 3C). Taken together, these results indicated that RSPO3sh hASCs could promote bone formation in vivo.

Downregulation of endogenous RSPO3 enhances ERK1/2 signalling pathway in hASCs.
To determine the molecular mechanism by which RSPO3 regulates osteogenic differentiation of hASCs, we screened several signalling pathways and key regulators of hASCs differentiation. Interestingly, we found that RSPO3 was responsible for the inhibition of ERK signalling in hASCs. We detected that the level of phosphor-ERK1/2 in RSPO3sh cells was increased significantly, as indicated by western blotting and immunoreactive band quantification (Fig. 4A,B). In contrast, the other MAPK cascades, p38 and JNK, were not affected by knockdown of RSPO3 (Fig. 4C,D). It is well established that FGF promotes osteogenic differentiation of mesenchymal cell through the ERK1/2 signalling pathway. Therefore, we next examined the expression of FGF pathway genes in RSPO3 knockdown cells. As shown in Fig. 4E,F, we observed increased FGF4 and FGFR2 gene expressions in RSPO3sh hASCs. This suggested that downregulation of endogenous RSPO3 leads to activation of the ERK signalling pathway.

Inhibition of ERK1/2 signalling pathway blocked osteogenic differentiation in hASCs.
To further determine the role of ERK1/2 in osteogenic differentiation of hASCs, we first analysed the activity of ERK1/2 after osteogenic differentiation. As shown in Supplementary Fig. S5A,B, the phosphorylation level of ERK1/2 (B-D) Knockdown of RSPO3 was validated by western blotting and RT-qPCR. (E,F) RSPO3 knockdown increased the ALP activity in hASCs. Control or RSPO3 knockdown cells were treated with proliferation or osteogenic media for 7 days for ALP staining (E), and cellular extracts were prepared to quantify ALP activity (F). (G,H) Knockdown of RSPO3 increased mineralization of hASCs. Cells with or without RSPO3 knockdown were treated with proliferation or osteogenic media for 14 days, and then calcium deposition was observed using Alizarin Red S staining (G) and quantification (H). The knockdown of RSPO3 promoted the expression levels of RUNX2 (I), ALP (J) and OCN (K) in hASCs, as assessed by RT-qPCR detection. RUNX2 and ALP were detected at day 7 and OCN was detected at day 14 after osteoblast differentiation. All data are shown as the mean ± SD, n = 3. *P < 0.05 and **P < 0.01; PM: proliferation media; OM: osteogenic media.
was increased when cells were cultured in osteogenic media. Next, the small interfering RNA (siRNA) was used to knockdown the ERK1/2 in hASCs. Silencing of ERK1/2 expression decreased ERK mRNA and protein levels, whereas a control siRNA had no effect ( Fig. 5A and Supplementary Fig. S5C,D). To further confirm the knockdown efficiency of ERK1/2, the expression level of ERK1/2 was evaluated in proliferation medium (PM) at 7 days and 14 days, separately (Supplementary Fig. S5E-J). As shown in Fig. 5B,C, in contrast to the control siRNA, the ERK1/2 siRNA decreased ALP activity. Moreover, the extracellular matrix mineralization, as determined by Alizarin Red S staining and quantification, was also decreased in ERK1/2-silenced cells at 2 weeks after osteogenic induction ( Supplementary Fig. S5K,L). In addition, RUNX2 and ALP mRNA levels also decreased significantly in ERK1/2 siRNA cells, as shown in Fig. 5D,E. These data indicated that ERK1/2 was a positive regulator of osteogenesis. To further confirm the role of ERK in the osteogenic differentiation of hASCs, we next investigated the effects of pharmacological inhibition of ERK1/2 by U0126 on osteoblast formation in OM. Upon treatment of hASCs with the ERK1/2 inhibitor U0126, the activity of ERK decreased significantly (Supplementary Fig. S5M-N). As shown in Fig. 5F,G, U0126 blocked the osteogenic capacity of hASCs, as indicated by ALP staining and quantification. Moreover, extracellular matrix mineralization, as determined by Alizarin Red S staining and quantification, was also impaired by U0126 treatment (Fig. 5H,I). Furthermore, U0126 decreased the expressions of RUNX2, ALP, and OCN in hASCs, as determined by RT-qPCR analysis ( Fig. 5J-L). And most importantly, we detected that U0126 treatment had no effect on cell proliferation rate, as shown in Supplementary Fig. S5O. Collectively, the above data indicated the importance of ERK signalling in the osteogenic differentiation of hASCs.

RSPO3 regulates osteogenic differentiation in an ERK-dependent manner.
In light of the above observations, we hypothesized that inhibition of ERK1/2 signalling would abrogate the increased osteogenic capacity of RSPO3 knockdown hASCs. To verify this hypothesis, two sets of osteogenic differentiation assays in RSPO3sh hASCs were performed either in the presence of U0126 or in the ERK1/2-silenced cells. As shown in Fig. 6A and Supplementary Fig. S6A, ERK1/2 was effectively knocked down in RSPO3-silenced cells, as determined by western blotting. When cells were treated with OM, the increased osteogenic differentiation ability induced by RSPO3 knockdown was effectively reversed in the RSPO3 and ERK1/2 double knockdown cells, which was indicated by ALP staining and quantification (Fig. 6B,C). In addition, the increased expression of RUNX2 and ALP caused by RSPO3 deficiency was also blocked by ERK1/2 silencing (Fig. 6D,E). Next, we treated RSPO3   knockdown cells in the absence or presence of U0126 in OM. As shown in Fig. 6F,G, U0126 decreased ALP activity, as determined by ALP staining and quantification. Additionally, Alizarin Red S staining and quantification was also inhibited in RSPO3 knockdown cells treated with U0126 (Fig. 6H,I). Consistently, the increased expression of RUNX2, ALP and OCN induced by RSPO3 knockdown was blocked in the presence of U0126 (Fig. 6J-L). Taken together, these results suggested RSPO3 regulates osteogenic differentiation in an ERK-dependent manner.
Silencing of LGR4 impaired the osteogenic differentiation potential of hASCs significantly. Previous studies demonstrated that R-spondins are bona fide ligands of the leucine-rich repeat-containing G-protein coupled receptors (LGRs), including LGR4, 5 and 6 15,16,22 . To investigate the potential role of LGRs on RSPO3-regulated osteogenesis, we first detected the expression levels of LGR4/5/6 in the hASCs. As shown in Supplementary Fig. S7A,B, compared with LGR4, the expressions of LGR5/6 were almost undetectable in hASCs. In addition, the expression level of LGR4 decreased after osteogenic differentiation ( Supplementary Fig. S7C). To determine the role of LGR4 in osteogenic differentiation of hASCs, we first conducted a small interfering RNA (siRNA)-mediated knockdown experiment. As shown in Fig In addition, the growth curves revealed that LGR4 silencing had no effect on the proliferation of hASCs (Supplementary Fig. S7J). Most importantly, we found that LGR4 was a positive regulator of ERK signalling: the phosphor-ERK1/2 level in LGR4-silenced cells decreased significantly (Fig. 7J,K).

Loss of
LGR4 abrogates RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. We next examined the effect of LGR4 siRNA on the osteogenic differentiation of RSPO3 knockdown cells. As shown in Fig. 8A and Supplementary Fig. S8A, LGR4 was effectively silenced, as determined by western blotting analysis. When cells were treated with OM, the increase in osteogenic differentiation induced by RSPO3 knockdown was effectively reversed in the RSPO3 and LGR4 double knockdown cells, which was indicated by ALP staining and quantification (Fig. 8B,C). Moreover, the extracellular matrix mineralization, as determined by Alizarin Red S staining and quantification, was also impaired in the RSPO3/LGR4 knockdown cells compared with RSPO3-silenced cells (Fig. 8D,E). In addition, the increased expression of RUNX2 caused by RSPO3 deficiency was also blocked by silencing of LGR4 (Fig. 8F). These results suggested that LGR4 is involved in the RSPO3-regulated osteogenic differentiation. To further support this speculation, we next examined whether the knockdown of LGR4 affected ERK signalling. As shown in Fig. 8G,H, western blotting demonstrated that knockdown of LGR4 abrogated the increased level of phosphor-ERK1/2 in RSPO3sh cells. Taken together, these results indicated a novel role for RSPO3-LGR4 in osteogenesis by regulating the ERK signalling pathway.

Discussion
In several genome-wide association studies to identify genes associated with osteoporosis, RSPO3 was demonstrated as a novel loci associated with bone mineral density(BMD) variations [23][24][25] . The present study demonstrated that RSPO3 plays an important role in osteogenic commitment of hASCs. When cultured in osteogenic medium, we found that RSPO3 deficiency could promote osteogenic differentiation of hASCs significantly, with increased ALP activity, matrix mineralization capacity, and mRNA expression of RUNX2, ALP, and OCN. Previous studies have shown that RSPO1 and RSPO2 could promote osteoblastic differentiation in synergy with Wnt proteins in vitro [26][27][28][29] . It appears that RSPO3 plays a different role in osteogenesis of hASCs. Investigation of the molecular mechanism suggested novel crosstalk between RSPO3 and the ERK1/2 signalling pathway. ERK1/2, a component of the MAPK signalling pathway, has been associated with cellular survival, proliferation and differentiation 30,31 . Despite numerous studies, the role of ERK1/2 in osteogenic differentiation is still a matter of some controversy. Administration of the ERK1/2 inhibitor PD98059 has been reported to promote early osteoblastic differentiation and mineralization in BMP2-treated C2C12 and MC3T3-E1 cells 32,33 . However, conflicting results obtained from other investigations indicated that activation of ERK signalling promotes osteogenic differentiation of stem cells in a cell-type specific manner [34][35][36][37][38][39] . In this study, we revealed the importance of the ERK pathway in the osteogenic differentiation of hASCs. To clarify the potential role of the ERK1/2, we established ERK1/2 knockdown cells and showed that ERK1/2 deficiency significantly impaired the osteogenic differentiation of hASCs ( Fig. 5A-E). Moreover, pharmacological inhibition of ERK1/2 with U0126 also impaired the osteogenesis of hASCs significantly (Fig. 5F-L). This work enriched our knowledge of the mechanisms underlying the regulation of hASCs osteogenic differentiation by the ERK signalling pathway. To further clarify whether ERK activation is necessary for increased osteogenic differentiation of RSPO3-silenced hASCs, we blocked the ERK1/2 signalling pathway using U0126 or an siRNA for MAPK1/3. The results showed that the increased osteogenic capability of media in the presence or absence of U0126 (10 μ M) for 7 days for ALP staining (F), and cellular extracts were prepared to quantify ALP activity (G). (H,I) U0126 inhibited mineralization in hASCs. Cells in the presence or absence of U0126 (10 μ M) were treated with proliferation or osteogenic media for 14 days, and then calcium deposition was observed using Alizarin Red S staining (H) and quantified (I). (J-L) U0126 treatment inhibited the expressions of RUNX2 (J), ALP (K) and OCN (L) in hASCs as determined by RT-qPCR. RUNX2 and ALP were detected at day 7 and OCN was detected at day 14 after osteoblast differentiation. All data are shown as the mean ± SD, n = 3. *P < 0.05 and **P < 0.01; PM: proliferation media; OM: osteogenic media.
Scientific RepoRts | 7:42841 | DOI: 10.1038/srep42841   RSPO3-knockdown hASCs was almost completely abrogated by U0126 treatment or ERK1/2 silencing (Fig. 6F-L). Thus, ERK1/2 signalling is involved in the osteoblast differentiation of hASCs regulated by RSPO3. Despite the importance of ERK signalling in osteogenesis of hASCs, it is not the only pathway that is regulated by RSPO3. Numerous studies suggested that RSPO3 is associated with several important cell signalling and metabolic pathways. Rspo3 deletion blocks Wnt/Ca 2+ /NFAT signalling by upregulation the expression of Rnf213, Usp18, and Trim30α, thus leading to the vessel regression phenotype of Rspo3-iECKO mice 40 . Deletion of RSPO3 leads to loss of SHH signalling and impaired organ growth in adrenal glands 41 . RSPO3 also controls metabolic zonation of liver hepatocytes via beta-catenin signalling 42 . Perhaps other factors are involved in RSPO3-mediated osteoblast differentiation of hASCs.
Notably, we observed that RSPO3 negatively regulates the ERK signalling. R-spondins were first discovered as secreted Wnt agonists 43 and were identified as ligands of three related receptors LGR4-6 44 .
LGRs bound with high affinity to the furin domains of R-spondins and then activated Wnt signaling 15,16 . Interestingly, our data showed that knockdown of RSPO3 increased the phosphorylation of ERK1/2, while LGR4 silencing exhibited opposite effect.
LGR4 silencing blocked the increased phosphorylation of ERK1/2 induced by RSPO3 knockdown (Fig. 8G,H). LGR4 was identified as a key regulator of bone formation and resorption. Deletion of LGR4 in mice resulted in a dramatic delay in osteoblast differentiation and mineralization. LGR4 regulated bone formation and remodelling through the cAMP-PKA-Atf4 signalling pathway 45 . Silencing of LGR4 abrogated the osteogenesis in MC3T3-E1 in presence of BMP2 or RSPO2 46,47 . These studies suggested that multiple signalling pathways were involved in LGR4-regulated osteogenesis, not merely ERK signalling. Recently, another two molecules have been validated as the ligands of LGR4. Norrin, a secreted protein, also a known ligand for Fzl4, stimulated Wnt signalling specifically through LGR4 but not LGR5 and 6. Norrin also interacted directly with Fzl4 to promote LRP5/6 internalization and to increase beta-catenin levels. Moreover, Norrin bound to BMP2/4, blocking the activation of type I and II BMP receptors, thus leading to decreasement in downstream Smad activities 48 . An intriguing and important idea is that the functions of Norrin and RPSO3 are similar: first, disruption of them led to vascular defect 20,21,[49][50][51] ; in addition, both RSPO3 and Norrin can bind to LGR4 and act as a Wnt agonist 15,16,48 . Until recently, studies about Norrin mainly focus on its biomedical effect in retinal vascularization. Considering the important function of Wnt and BMP signalling in osteogenesis, one cannot rule out the possibility that Norrin may be a strong competitor to RSPO3 in regulation of osteogenesis. Further studies are needed to verify the presumption. Additionally, it was also reported that LGR4 acted as a second receptor for RANKL. Luo, J. et al. showed that RANKL-LGR4-Gα q signaling promoted GSK3β activation, leading to NFATc1 nuclear export that downregulate NFATc1 expression and ultimately impairing osteoclastogenesis. Besides, LGR4 also competed with RANK for RANKL binding and attenuated RANK downstream signaling including NFκ B pathway, calcium osscilation and AKT pathway. The study also demonstrated that RANKL competed with RSPO1 to interact with LGR4 in HEK293T cells, which prompted us that similar biological process may exist in hASCs 52 . In the present study, we confirmed that knockdown of LGR4 inhibited the osteogenic differentiation of hASCs. Most importantly, we confirmed that LGR4 was involved in ERK signalling activation. Loss of LGR4 abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. Although RSPO3 is reported as a ligand of LGR4, it is difficult for us to verify whether RSPO3 regulates ERK signalling directly through LGR4. There is a reasonable speculation that RSPO3 functions via LGR4 in regulating the activity of ERK signalling and osteogenesis, while some other related ligands and signalling pathways participate in this process, making it a complicated regulatory network.
In conclusion, we have shown that the RSPO3-LGR4 system plays an important role in osteogenic differentiation of hASCs. RSPO3 regulates osteogenesis possibly through the LGR4-ERK signalling pathway. This novel function of RSPO3-LGR4 might provide a new scientific rationale for the regulation of osteogenic differentiation of hASCs, and the regulatory control of ERK by RSPO3-LGR4 might extend beyond osteogenesis and have important implications in other cellular processes.

Materials and Methods
All animal work in this study was approved by the Peking University Biomedical Ethics Committee Experimental Animal Ethics Branch, and was in accordance with the Guide for the Care and Use of the Laboratory Animals (National Academies Press, National Institutes of the Health Publication).

Culture of hASCs.
Primary hASCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). To induce osteogenic differentiation, hASCs were cultured in OM containing 100 mM/ml ascorbic acid, 2 mM β -glycerophosphate and 10 nM dexamethasone. All experiments were repeated three times using human ASCs from the three donors, respectively. Viral infection. Viral packaging and infection was prepared as described previously 53  Alkaline phosphatase (ALP) staining. After 7 days of culture, cells were washed with PBS three times and then fixed in 4% paraformaldehyde at room temperature (RT) for 10 min. Subsequently, the cells were washed in PBS three times, incubated with a BCIP/NBT staining kit (CWBIO, Beijing, China) solution for 15 min at RT and rinsed with water.
Quantification of ALP activity. After 7 days of culture, cells from six-well culture plates were washed three times with ice-cold PBS and then treated with 500 μ l/well of 1% Triton X-100 (Sigma, St. Louis, MO, USA) for 5 min at RT for cell lysis. Cells were collected with a cell scraper, sonicated on ice and then centrifuged at 4 °C for 30 min at 12000 rpm. The supernatants were used to determine the protein concentration using a BCA protein assay reagent (Prod#23225, Pierce Thermo Scientific, Waltham, MA, USA) and to measure the ALP activity. ALP activity in cell lysates was measured using an ALP assay kit (A059-2, Nanjing Jiancheng Bioengineering Institute, China) and normalized by the total protein content.
Alizarin Red staining and quantification. Analysis of mineralization was determined by Alizarin Red staining. Cells were induced for 2 weeks, fixed for 30 min in 70% ethanol at 4 °C and then rinsed with Milli-Q water. Calcium deposition was then visualized after incubation with 2% Alizarin Red S pH 4.2. Alizarin Red S was extracted by destaining with hexadecyl pyridinium chloride monohydrate. Mineral accumulation was quantified on a microplate reader at 562 nm and normalized by the total protein concentration detected in a duplicate plate.
Quantitative real-time reverse transcription PCR (RT-qPCR). Total RNA was extracted with the TRIZOL reagent (Invitrogen) and precipitated with ethanol. To exclude potential contamination of DNA, RNA was treated with DNase I for 30 min at 37 °C. cDNA was synthesized from 0.5-2 μ g-of RNA with oligo (dT) 18 primers using a Quantscript RT Kit (Tiangen, Beijing, China).
RT-qPCR was performed on an Eppendorf Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using the Fast-Start Universal SYBR Green Master Mix (Roche Applied Science, Mannheim, Germany). Reactions were carried out in a total volume of 20 μ l, containing 2 μ l of 1:10 diluted template cDNA, 10 μ l of 2 × SYBR green PCR Master Mix (Roche Applied Science, Mannheim, Germany) and 100 nM of each primer. The following amplification program was used in all PCRs: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The specificity of each amplified reaction was verified by a dissociation curve (melting curve) analysis after 40 cycles, which was carried out by heating the amplicon from 60 to 95 °C. Moreover, the specificity of the PCR product was further confirmed by 2% agarose gel electrophoresis. Each sample was analysed in triplicate wells, and no-template controls (without cDNA in the PCR) were included. Data were collected and analysed quantitatively using realplex software.
Western blotting. Cells were harvested and then lysed in RIPA lysis buffer with a complete protease inhibitor mixture (Roche). Lysates were sonicated and centrifuged at 12000 rpm at 4 °C for 30 min to obtain the supernatant. The Pierce BCA protein assay kit (Thermo Scientific) was used to measure the protein concentrations.