Male Sprague-Dawley rats (age, 6-8 weeks; weight, 200–250 g, n=24) used in the present study were purchased from the Laboratory Animal Center of the Tongji Medical College (Wuhan, China). All rats were kept in ventilated filter-top cages under standard laboratory conditions: 12-h light/dark cycle and a constant temperature of 24°C with 60% humidity. Rats were given ad libitu access to conventional rodent chow and water. All experimental animals were sacrificed via cervical dislocation. Prior to cervical dislocation, rats were anesthetized by intra-peritoneal injection of 2% pentobarbital sodium (35 mg/kg body weight). Animal death was confirmed by monitoring the heartbeat and body temperature. The present study was approved by the Experimental Animal Ethics Committee of Tongji Medical College. Briefly, rASCs were isolated, cultured and characterized as described in our previous study (16). Adipose tissues from the epididymis of male Sprague-Dawley rats were harvested, finely minced and digested with 0.1% type I collagenase (Wuhan Boster Biological Technology, Ltd., Wuhan, China) at a 1:1 volume ratio at 37°C for 1 h, followed by centrifugation at 100 × g for 10 min at 37°C. The supernatant layer was discarded and the remaining cells were collected and resuspended in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Undigested debris was removed by filtering through a sterile 75-mm nylon mesh. Finally, cells were cultured with DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in an atmosphere containing 5% CO₂. Cells at passage 3 were used for subsequent experiments.

For osteogenic and adipogenic differentiation, ASCs at passage 3 were plated into 6-well plates at a density of 1×10⁴ cells/cm² and cultured at 37°C in an atmosphere containing 5% CO₂. After 24 h, the cell culture medium was removed and replaced with adipogenic or osteogenic differentiation medium. The adipogenic differentiation medium comprised DMEM supplemented with 10% FBS, 1 µM dexamethasone (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 10 mg/ml insulin (Invitrogen; Thermo Fisher Scientific, Inc.), 0.5 mM methyl-isobutyl-xanthine (Sigma-Aldrich; Merck KGaA) and 100 mM indomethacin (Sigma Aldrich; Merck KGaA). The osteogenic differentiation medium consisted of DMEM supplemented with 10% FBS, 10 mM β-glycerophosphate (Sigma-Aldrich; Merck KGaA), 0.1 µM dexamethasone and 0.1 mM ascorbate-2-phosphate (Sigma-Aldrich; Merck KGaA).

rASCs at passage 3 were seeded at varying densities (1,500 or 12,000 cells/cm²) and cultured for 2 days in an atmosphere containing 5% CO₂ at 37°C with DMEM/F12 supplemented with 10% FBS. Cells were harvested and the gene expression of CTGF and Ankrd1 was evaluated via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To evaluate the role of the actin cytoskeleton in YAP activity, cells were seeded at a density of 1,500 cells/cm² and cultured for 2 days in an atmosphere containing 5% CO₂ at 37°C with DMEM/F12 supplemented with 10% FBS. After treatment with 1 µg/ml Latrunculin B for 30 min, cells were harvested and the expression levels of CTGF and Ankrd1 were evaluated by RT-qPCR.

rASCs were seeded in 6-cm dishes at a density of 1,500 cells/cm² and incubated in serum-free DMEM/F12 medium for 24 h at 37°C prior to treatment with various reagents. LPA at 10 µmol/l and S1P at 10 µmol/l were used in the present study. Latrunculin B (LatB; 1 µg/ml) was used to disrupt F-actin fiber organization. All reagents were purchased from Sigma-Aldrich (Merck KGaA).

To evaluate the effects of contact-inhibited proliferation on YAP subcellular localization, rASCs were plated on coverslips at varying densities (1,500 or 12,000 cells/cm²) and cultured for 2 days in an atmosphere containing 5% CO₂ at 37°C with DMEM/F12 supplemented with 10% FBS. To evaluate the effects of the actin cytoskeleton on YAP subcellular localization, cells were seeded on coverslips at a density of 1,500 cells/cm² with DMEM/F12 supplemented with 10% FBS and treated with 1 µg/ml Latrunculin B at 37°C for 30 min. Cells were subsequently fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. After blocking with 5% bovine serum albumin (BSA; Wuhan Boster Biological Technology, Ltd.) for 1 h at 37°C, slides were incubated with YAP primary antibody (cat. no. 4912; Cell Signaling Technology, Inc., Danvers, MA, USA) diluted with 5% BSA (dilution 1:200) at 4°C overnight. After washing three times with PBS, slides were incubated with Alexa Fluor^® 594-labeled donkey anti-rabbit secondary antibodies (cat. no. R37119; Invitrogen; Thermo Fisher Scientific, Inc.; dilution 1:1,000) for 1 h at 37°C. For staining of F-actin, cells were incubated with fluorescein isothiocyanate-conjugated phalloidin (cat. no. P5282; Sigma-Aldrich; Merck KGaA) 1 h after blocking at 37°C. After washing with PBS, cell nuclei were visualized with DAPI for 5 min. To reduce background fluorescence, slides were washed with PBS. Finally, cells were observed under a fluorescence microscope (FV500; Olympus Corporation, Tokyo, Japan).

Cells were washed with PBS and treated with lysis buffer containing 50 mM Tris-HCl, 0.1 mM EDTA, 0.1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride for 30 min at 37°C. The proteins were prepared using commercial bicinchoninic acid kits (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's protocols. Aliquots containing equal amounts of protein (25 µg/lane) were separated by SDS-PAGE (7–15% gradient cross-linked polyacrylamide gels) and were then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After transfer, membranes were probed overnight at 4°C with anti-YAP rabbit polyclonal antibodies (cat. no. 4912; Cell Signaling Technology, Inc.; dilution 1:1,000), anti-RUNX2 rabbit polyclonal antibodies (cat. no. ab23981; Abcam, Cambridge, UK; dilution 1:200), anti-PPARγ rabbit polyclonal antibodies (cat. no. ab59256 Abcam; dilution 1:200) and anti-GAPDH rabbit polyclonal antibodies (cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.; dilution 1:200) followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C (cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.; dilution 1:10,000). Blots were developed by enhanced chemiluminescence (ECL) using Western ECL Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). The intensity of each band was semi-quantified using digital image analysis software (Quantity One, version 4.6; Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was selected as a protein loading control.

For relative mRNA expression analysis, cells in each group were washed with PBS and treated with TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) to extract total RNA, according to the manufacturer's protocol. RNA samples were then purified and reverse-transcribed to cDNA using the 1st Strand cDNA Synthesis kit (Toyobo Life Science, Osaka, Japan), as per the manufacturer's protocol. The cDNA samples were then subject to qPCR to determine the expression of various genes. qPCR was performed with cDNA samples in triplicate under the following conditions: 95°C for 5 sec, followed by 40 cycles at 94°C for 20 sec, 60°C for 20 sec and extension at 72°C for 10 sec. GAPDH was used as an internal control. Primers used in the present study were as follows: Connective tissue growth factor (CTGF), 5′-CGT TAG CCT CGC CTT GGT G-3′ (sense) and 5′-GGG AGC CGA AGT CGC AGA-3′ (antisense); ankyrin repeat domain 1 (Ankrd1), 5′-CGG CTC TTG ATG ACC TTC G-3′ (sense) and 5′-GCA TTC TCC TTG AGG CTG TC-3′ (antisense); YAP, 5′-CCC AAG GCT TGA CCC TCG T-3′ (sense) and 5′-CGT ATT GCC TGC CGA AAT AAC T-3′ (antisense); proliferating cell nuclear antigen (PCNA), 5′-AAG GGC TGA AGA TAA TGC TGA TAC-3′ (sense) and 5′-CAT ATA CGT GCA AAT TCA CCA GAT-3′ (antisense); RUNX2, 5′-TGG TAC TTC GTC AGC GTC CTA TC-3′ (sense) and 5′-GCT TCC ATC AGC GTC AAC ACC-3′ (antisense); PPARγ, 5′-ACA TCA GTG GGA ATT AAG GCA-3′ (sense) and 5′-TCA AAG GAA TGG GAGTGG TC-3′ (antisense); and GAPDH, 5′-GGC AAG TTC AAC GGC ACA G-3′ (sense) and 5′-CGC CAG TAG ACT CCA CGA CAT-3′ (antisense).

Cells were cultured under various conditions and subsequently seeded into 96-well plates at a density of 2×10³ cells/well. Cells were divided into three groups: Serum starvation (SS) group (DMEM/F12 medium only), SS + LPA group (DMEM/F12 medium supplemented with 10 µmol/l LPA) and SS + short hairpin (sh) RNA-targeting YAP (shYAP) + LPA group (cells transduced with shYAP lentivirus and cultured in DMEM/F12 medium supplemented with 10 µmol/l LPA). Cells were cultured for 1, 2 or 3 days at 37°C and each sample was assessed for cellular proliferation. A total of 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubated at 37°C in the dark for 2 h. Absorbance was read on a microplate spectrophotometer (Bio-Rad Laboratories, Inc.) at a wavelength of 490 nm.

rASCs were seeded in 6-well plates at a density of 1,500 cells/cm² with serum-free DMEM/F12 medium at 37°C. Cell cycle distribution and DNA content were analyzed by flow cytometry. Cells were divided into the following groups: SS group, SS + LPA group, SS + S1P group (DMEM/F12 medium supplemented with 10 µmol/l S1P), SS + shYAP + LPA group and SS + shYAP + S1P group (cells transduced with shYAP lentivirus cultured in DMEM/F12 medium supplemented with 10 µmol/l S1P). After 3 days treatment, cultured cells were rinsed with PBS twice and fixed with 70% cold ethanol at -20°C for 2 h. Fixed cells were treated with RNase A (50 µg/ml; cat. no. R4875; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min and then stained with propidium iodide (PI; 65 µg/ml; Sigma-Aldrich; Merck KGaA) in the dark for 30 min at 37°C. PI fluorescence of individual nuclei was measured using a flow cytometer (FACSort; BD Biosciences, Franklin Lakes, NJ, USA).

rASCs were seeded in 6-well plates at a density of 1×10⁴ cells/cm² and cultured at 37°C in an atmosphere containing 5% CO₂ in osteogenic medium. Cells were divided into four groups: Control group (DMEM/F12 supplemented with 10% FBS), Osteo group (cells cultured in osteogenic differentiation medium), Osteo + control shRNA (shControl) group (cells transduced with control lentivirus cultured in osteogenic differentiation medium) and Osteo + shYAP group (cells transduced with shYAP lentivirus cultured in osteogenic differentiation medium). Following 5 days of culture, cells were rinsed with PBS three times and fixed with 4% paraformaldehyde for 15 min at 4°C. Cells were rinsed again with deionized water and stained with naphthol AS-MX phosphate and fast blue RR salt (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C in the dark. Excess dye was removed with PBS and photomicrographs were captured under a light microscope.

rASCs were seeded in 6-well plates at a density of 1×10⁴ cells/cm² and cultured at 37°C in an atmosphere containing 5% CO₂ in adipogenic medium. Cells were divided into four groups: Control group, Adipo group (cells cultured in adipogenic differentiation medium), Adipo + shControl group (cells transduced with control lentivirus cultured in adipogenic differentiation medium) and Adipo + shYAP group (cells transduced with shYAP lentivirus cultured in adipogenic differentiation medium). Following 5 days of culture, cells were rinsed with PBS three times and fixed with 4% paraformaldehyde for 15 min at 4°C. Cells were rinsed again with deionized water and stained with Oil Red O for 30 min at 37°C. Excess dye was removed with PBS and photomicrographs were captured under a light microscope.

Specific restriction sites of YAP (NM_001034002; Shanghai GeneChem Co., Ltd., Shanghai, China) were inserted into a GV118 plasmid (Shanghai GeneChem Co., Ltd.). The target of shYAP#1 was GGC AAT ACG GAA TAT CAA T, whereas that of shYAP#2 was GAC AGT CTT CCT TTG AGA T. Linked products were identified by double digestion, and DNA sequencing confirmed accurate insertion. The lentiviral vector system included three parts: pGC-LV vector, pHelper 1.0 vector and pHelper 2.0 vector. pHelper 1.0 plasmid DNA (15 µg), pHelper 2.0 plasmid DNA (10 µg) and pGC-LV shYAP were co-transfected into subconfluent 293T cells (80% confluence) at 37°C for 8 h using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Supernatants were collected by ultracentrifugation at 4,000 × g for 10 min at room temperature and the viral pellet was resuspended in Hanks' balanced salt solution. The vector titers were determined by serial dilution. Lentiviral null vectors with scrambled shRNA (sequence: sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′) (Shanghai GeneChem Co., Ltd.) were used as the shControl group.

rASCs at the third passage were seeded at the density of 3×10⁴ cells/ml in 24-well tissue culture plates and cultured until cells reached 30% confluence. Following removal of the cell culture medium, rASCs were incubated with DMEM/F12 containing lentivirus (shYAP or shControl) and 5 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) for 5 h at 37°C. Multiplicity of infection (MOI) ranges between 10 and 200 were calculated. Following lentiviral vector infection, the medium was replaced with normal growth medium comprised of DMEM/F12 and 10% FBS. A total of 1 day post-transduction, reporter gene expression [green fluorescent protein (GFP)] was examined with fluorescence microscopy. ASCs were transduced with lentiviruses at a MOI of 100 and passaged for further study.

All data are presented as the means ± standard deviation. SPSS 13.0 was used for general statistical analysis (SPSS, Inc., Chicago, IL, USA). Significant differences in numerical data between two groups were determined using a Student's t-test. For multiple group comparisons, one-way analysis of variance was used with the Bonferroni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

YAP is a transcriptional co-activator, which has been reported to serve critical roles in cell proliferation and differentiation; however, the functions of YAP and its upstream regulating factors in rASCs have yet to be elucidated. The present study demonstrated that the subcellular distribution of YAP in rASCs was regulated by cell density and F-actin integrity. rASCs were seeded at varying densities in tissue culture plates (1,500 and 12,000 cells/cm²). When cultured at 1,500 cells/cm² (low density), cells showed no contact with neighboring cells (Fig. 1A). When cultured at a higher density of 12,000 cells/cm² (high density), a marked amount of contact was observed with neighboring cells (Fig. 1B). In addition, in the low density group, YAP expression was mainly localized to the nuclei of rASCs (Fig. 1A). Conversely, nuclear YAP expression was markedly decreased when rASCs were cultured at a higher density (Fig. 1B). In addition, the effects of the actin cytoskeleton on YAP subcellular distribution were investigated. The results demonstrated that under normal conditions, YAP was mainly localized in the nuclei of rASCs (Fig. 2A); however, disruption of the actin cytoskeleton with 1 µg/ml LatB for 30 min induced marked YAP cytoplasmic translocation (Fig. 2B).

RT-qPCR was conducted to analyze the expression levels of the YAP target genes, CTGF and Ankrd1. The results indicated that the mRNA expression levels of CTGF and Ankrd1 were significantly decreased in the high density group compared with in the low density group, which was consistent with decreased YAP nuclear localization (Fig. 3A). Furthermore, CTGF and Ankrd1 mRNA expression was markedly inhibited by LatB treatment compared with in the control group, thus suggesting that the integrity of the actin cytoskeleton may be closely associated with YAP activity (Fig. 3B).

It has previously been reported that LPA activates YAP expression in epithelial cells, such as MCF10A cells (14). However, to the best of our knowledge, the effects of LPA on YAP protein expression in rASCs have yet to be determined. The present study demonstrated that SS of rASCs for 24 h markedly decreased YAP protein expression (Fig. 4). However, when cells were treated with LPA (10 µmol/l) for 1 h after 24 h SS, YAP protein expression was significantly increased (Fig. 4). When cells were treated with the actin cytoskeleton disruption agent, LatB, for 1 h after 24 h SS treatment, YAP protein expression was further decreased compared with in the SS group (Fig. 4). Notably, the LPA-induced upregulation of YAP protein expression in rASCs was inhibited in the SS + LPA + LatB group compared with in the SS + LPA group (Fig. 4). These results suggested that LPA may promote YAP protein expression in rASCs, and that integrity of the actin cytoskeleton is critical for the regulation of YAP protein by LPA.

rASCs at passage three were infected with lentiviruses carrying shYAP-GFP or shControl-GFP. Fluorescence microscopy was used to detect GFP expression in transduced rASCs. GFP-expressing rASCs were observed by fluorescence microscopy 24 h post-transduction (Fig. 5A–C). To verify that the lentiviral system had been successfully infected into rASCs, the mRNA expression levels of YAP were measured by RT-qPCR 5 days post-transduction. The results revealed that YAP mRNA expression in the shYAP#1 and shYAP#2 groups was significantly lower than in the shControl group (Fig. 5D). Furthermore, YAP protein expression in the shYAP#1 and shYAP#2 groups was markedly lower compared with in the shControl group 7 days post-transduction (Fig. 5E). The shYAP#1 lentiviral system was used for subsequent experiments.

YAP is a transcriptional coactivator that promotes the expression of various downstream genes, including CTGF and Ankrd1 (14). The present results demonstrated that CTGF, Ankrd1 and YAP mRNA expression levels were significantly increased when rASCs were treated with LPA (10 µmol/l) or S1P (10 µmol/l) for 1 h compared with in the SS control group (Fig. 6A–C). However, rASCs transduced with shYAP lentiviruses exhibited no increase in CTGF, Ankrd1 and YAP mRNA expression when treated with LPA or S1P (Fig. 6A–C). Furthermore, the expression levels of PCNA in rASCs were evaluated following treatment with LPA and S1P for 3 days. The results demonstrated that LPA and S1P significantly increased PCNA mRNA expression in rASCs (Fig. 6D). However, when rASCs were transduced with a shYAP lentivirus, the effects of LPA and S1P on PCNA expression were abrogated (Fig. 6D).

rASC proliferation was investigated using CCK-8 cell proliferation assays and flow cytometric analysis. CCK-8 results revealed that LPA and S1P significantly increased rASC proliferation compared with in the SS control group 2 and 3 days after treatment (Fig. 6E and F). Infection with a shYAP lentivirus significantly inhibited LPA and S1P-induced cell proliferation (Fig. 6E and F). Cell cycle distribution of rASCs was also investigated using flow cytometry, and the representative flow cytometric profile is presented. Flow cytometric analysis revealed that treating rASCs with LPA and S1P for 3 days significantly increased the percentage of rASCs in S and G₂/M phases compared with in the SS control group (Fig. 6G and H). However, when rASCs were infected with a shYAP lentivirus, no significant difference was observed in the percentage of rASCs in the S and G2/M phases compared with in the SS control group (Fig. 6G and H). In conclusion, these results suggested that LPA and S1P may induce rASC proliferation by activating YAP expression.

The protein expression levels of YAP were investigated 7 days after osteogenic and adipogenic differentiation of rASCs. The results revealed that YAP protein expression was increased during osteogenic differentiation of rASCs (Fig. 7A and B). Conversely, the protein expression of YAP was significantly decreased during adipogenic differentiation (Fig. 7A and B).

The present study also investigated the effects of YAP depletion on osteogenic and adipogenic differentiation of rASCs. YAP knockdown following transduction with shYAP led to a marked increase in osteogenic differentiation, as demonstrated by increased ALP staining in the Osteo + shYAP group compared with in the Osteo and Osteo + shControl groups 5 days after cell culture (Fig. 8A). In accordance with this, the expression levels of the osteogenic differentiation-related gene RUNX2 were also increased in the Osteo + shYAP group compared with in the Osteo and Osteo + shControl groups 5 days after cell culture (Fig. 8B). The effects of YAP knockdown on adipogenic differentiation of rASCs were also investigated. The results suggested that adipogenic differentiation of rASCs was reduced following YAP inhibition, as adipocyte formation was markedly decreased 5 days after cell culture in the Adipo + shYAP group compared with in the Adipo and Adipo + shControl groups (Fig. 8C). The adipogenic differentiation-related gene PPARγ was also decreased by YAP knockdown 5 days after cell culture compared with in the control groups (Fig. 8D). In conclusion, these results suggested that YAP may serve an important role in rASCs osteogenic and adipogenic differentiation.