MTT and dimethyl sulphoxide (DMSO) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Transwell chambers were purchased from Corning Inc. (Corning, NY, USA). Anti-TAZ (catalog no. 4883) primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-CTGF (catalog no. SC-365970), anti-Cyr61 (catalog no. SC-374129), anti-mothers against decapentaplegic homolog (Smad) 3 (catalog no. SC-101154) and anti-Smad4 (catalog no. SC-7966) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and anti-GAPDH primary antibody was purchased from Sigma-Aldrich (catalog no. G8795; Sigma-Aldrich; Merck KGaA).

hDPSCs were isolated from healthy third molars or premolars of healthy human subjects (male; age, 16–30 years; n=12). Individual dental pulps were minced into small pieces (1 mm3) and digested using type I collagenase (3.0 mg/ml) and dispase (4.0 mg/ml; Sigma-Aldrich; Merck KGaA) for 45 min at 37°C. The solution was filtered through a 70-mm cell strainer. Single-cell suspensions were obtained and seeded into 35 mm culture dishes at a density of 1×105/ml. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 2.0 mM/l glutamine (Invitrogen; Thermo Fisher Scientific, Inc.), and 100 IU/ml penicillin and streptomycin, and maintained at 37°C in a humidified 5% CO₂ atmosphere. When confluent, cells were collected by trypsinization (0.2% trypsin and 0.02% EDTA) and subcultured with DMEM supplemented with 10% FBS. The medium was replaced every 2–3 days. Cells between 3 and 5 passages were used in experiments.

The present study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (Shijiazhuang, China). Written informed consent was obtained from all human subjects prior to enrollment in the present study.

A total of 1×105/ml hDPSCs were seeded onto uncoated coverslips and were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Fixed samples were permeabilized using 0.3% Triton X-100 (Sigma-Aldrich; Merck KGaA) in PBS for 15 min at room temperature. Following blocking with 10% normal goat serum diluted with 1% immunoglobulin G (IgG)-free bovine serum albumin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and 0.3% Triton X-100 in PBS at 37°C for 2 h, the samples were incubated with rabbit anti-TAZ polyclonal antibody (1:200) overnight at 4°C, and with DyLight™ 488-conjugated goat anti-rabbit IgG (catalog no. 611-141-002; 1:500; Jackson ImmunoResearch Laboratories, Inc.) for 2 h at room temperature. A total of 5 µg/ml Hoechst 33342 was used to stain the nuclei at 37°C for 15 min. Fluorescently-labeled cells were observed under an upright fluorescence microscope (magnification, ×20; Olympus Corporation, Tokyo, Japan).

siRNA duplex oligonucleotides targeting TAZ mRNA (siTAZ) and non-targeting duplex oligonucleotides used as negative controls (siCON) were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Sequences of siTAZ and siCON were as follows: siTAZ sense, 5′-GGCCAGAGAUAUUUCCUUATT-3′; anti-sense, 5′-UAAGGAAAUAUCUCUGGCCTT−3′; and siCON sense, 5′-UUCACCGAACGUGUCACGUTT-3′; anti-sense: 5′-ACGUGACACGUUCGGAGAATT-3′;. Following incubation for 24 h, hDPSCs were transfected with 1 µg/ml of siRNAs, using RNAi-Mate transfection reagent (Shanghai GenePharma Co., Ltd., Shanghai, China), according to the manufacturer's protocol, and cultured in DMEM. Cells were subjected to a second round of transfection prior to subsequent experiments (18).

Following treatment, cells in different groups were harvested and total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA at 42°C for 1 h using Moloney murine leukemia virus reverse transcriptase (GeneCopoeia, Inc., Rockville, MD, USA). qPCR was performed on cDNA using the All-in-One™ qPCR mix (GeneCopoeia, Inc.) and an ABI7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primers were obtained from Invitrogen (Thermo Fisher Scientific, Inc.): TAZ: sense, 5′-ATGTTGACCTCCGGACTTTGG−3′; anti-sense: 5′-GAGGAAGGGCTCGCTTTTGT-3′; Cyr61: sense, 5′-GGCAGACCTTGTGAATATA-3′; anti-sense: 5′-GTATTAGGCTTTATTTACCA-3′; CTGF: sense, 5′-CCCAGACCCAAATATGATT-3′; anti-sense: 5′-CAATGTACGATAGTGCAGT-3′; and GAPDH sense: 5′-AGTCCAACGGCACAGTCAAGG-3′; anti-sense: 5′-AGCACCAGCATCACCCAT-3′;. Amplification conditions were as follows: Initial denaturation at 95°C for 1 min; followed by 40 cycles of 95°C for 30 sec, 60°C for 20 sec and 72°C for 20 sec. The relative expression levels of each gene were normalized to GAPDH expression with 2-^ΔΔCt method (19).

Total protein was extracted from hDPSCs using RIPA lysis buffer (C1053; Applygen Technologies, Inc., Beijing, China), according to the manufacturer's protocol. Protein concentrations were determined using a bicinchonic acid protein assay kit (Thermo Fisher Scientific, Inc.). A total of 50 µg extracted protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk with TBS containing Tween-20 for 2 h at 37°C and then incubated with anti-TAZ (1:1,000), anti-CTGF (1:200), anti-Cyr61 (1:200), anti-Smad3 (1:200), anti-Smad4B (1:200) and anti-GAPDH (1:3,000) primary antibodies at 4°C overnight. Membranes were subsequently incubated with IRDye800^®-conjugated secondary antibody (catalog no. 608-445-002; 1:20,000; Rockland Immunochemicals, Inc., Limerick, PA, USA) for 1 h at 37°C, followed by scanning with the Odyssey^® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Data were normalized to GAPDH levels and analyzed with Image-Pro Plus software, version 7.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Cellular proliferation was evaluated using the MTT assay. hDPSCs were seeded in 96-well plates, at a density of 3×10³ cells/well, and incubated at 37°C for 24 h. Subsequently, 20 µl MTT solution (5 mg/ml) was added and cells were incubated at 37°C for 4 h. The culture medium was replaced with DMSO (150 µl/well) and the plates were agitated at room temperature for 10 min to dissolve the crystals. The optical density at 570 nm for each well was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cellular proliferation was also assessed via the detection of 5-bromo-2′-deoxyuridine (BrdU)-labeled DNA using the Cell Proliferation ELISA, BrdU kit (Roche Applied Science, Penzberg, Germany). Briefly, hDPSCs were seeded in 96-well plates (3×10⁴ cells/well) and were incubated for 24 h at 37°C in the presence of siTAZ or siCON. Subsequently, cells were labeled with BrdU at 37°C for 1 h. BrdU-labeled DNA was quantified using ELISA.

Cellular migration was assessed using a wound healing assay. hDPSCs were seeded in 6-well plates (~5×10⁴ cells/well) and cultured to form a monolayer. The cell monolayer was scratched with a 200-µl pipette tip. Cells were cultured for 24 h and wound healing was observed at 0 and 24 h post-wound infliction under a phase-contrast microscope (Olympus Corporation). Healing was assessed using Scion Image Software, version 4.03 (Scion Corporation, Frederick, Maryland, USA). Cellular invasion was also assessed using Transwell inserts coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Following treatment, hDPSCs were seeded into the upper chambers at a density of 5×104 cells in 200 µl serum-free DMEM; culture medium supplemented with 10% FBS was added to the lower chambers. Following incubation for 24 h at 37°C, the cells on the upper surface of the membrane were removed. The invaded cells on the lower membrane were fixed for 10 min with 4% paraformaldehyde at 4°C, stained at 37°C for 10 min with 5 µg/ml Hoechst 33342 and counted under an upright fluorescence microscope (Olympus Corporation).

hDPSCs were seeded into 6-well plates at a density of 2×10⁵ cells/well and cultured for 24 h. Subsequently, cells were incubated at 37°C for 10 min with r-hTHBS1, a TGF-β activator (200 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA). Cells were harvested following treatment and used for further analysis.

The statistical significance of the difference between groups was assessed by the Student's t-test for pair-wise comparisons or a one-way analysis of variance, followed by a post hoc Student-Newman-Keuls test for multiple comparisons. Data are expressed as the mean ± standard deviation. The experiment was repeated three times. P<0.05 was considered to indicate a statistically significant difference. The analysis was performed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA).

hDPSCs were cultured for 48 h, fixed and processed for immunofluorescence. TAZ was revealed to be expressed in hDPSCs (Fig. 1).

Successful transfection with siTAZ in hDPSCs was confirmed using RT-qPCR and western blot analysis. The present results suggested that TAZ mRNA and protein expression levels were significantly decreased following transfection with siTAZ compared with control cells and cells transfected with siCON (Fig. 2).

hDPSCs were transfected with siTAZ to silence TAZ expression. Results of the MTT assay demonstrated that TAZ silencing significantly inhibited hDPSC proliferation compared with control cells and cells transfected with siCON (Fig. 3A). Similarly, the results of the BrdU assay revealed that transfection with siTAZ decreased hDPSC proliferation in vitro (Fig. 3B).

A wound-healing assay demonstrated that the migration distance of hDPSCs transfected with siTAZ following a scratch wound was significantly reduced compared with control cells and cells transfected with siCON (Fig. 3C). These results suggested that TAZ silencing significantly impaired the migratory capabilities of hDPSCs. Similarly, the results of the Transwell invasion assay revealed that transfection with siTAZ suppressed hDPSC invasion in vitro (Fig. 3D).

To explore the molecular mechanism underlying the inhibitory effects of TAZ silencing on hDPSC proliferation and migration, putative downstream targets of TAZ were investigated. CTGF and Cyr61 have been suggested to be downstream targets of TAZ (20), which may be implicated in the regulation of cellular proliferation, migration and apoptosis. Treatment of hDPSCs with siTAZ for 24 h significantly decreased the mRNA and protein expression levels of CTGF and Cyr61, compared with control cells and cells transfected with siCON (Fig. 4). These results indicated that siTAZ inhibited hDPSC proliferation and migration through the modulation of CTGF and Cyr61 expression.

To further explore the molecular mechanisms underlying the implication of TAZ in the regulation of CTGF and Cyr61 expression, the TGF-β pathway was investigated. Following treatment with r-hTHBS1, the protein expression levels of Smad3, Smad4, CTGF and Cyr61 in hDPSCs were significantly upregulated compared with the control (Fig. 5A-D). These results suggested that TAZ may be involved in the regulation of CTGF and Cyr61 expression via a TGF-β-mediated pathway. Therefore, the expression of Smad3/4 in hDPSCs was investigated following silencing of TAZ expression. Western blot analysis demonstrated that Smad3/4 protein expression levels were significantly downregu lated in TAZ-depleted cells compared with in control cells and cells transfected with siCON (Fig. 5E and F). These results suggested that TAZ may be implicated in TGF-β-dependent pathways that may modulate the expression of CTGF and Cyr61.