Cell Culture and Identification. Healthy human tooth samples were collected from 10 donors, aged from 16 to 18 years, who were undergoing routine premolar or third molar extractions for orthodontic reasons. The study was approved by the hospital ethics committee, and informed consent was obtained from the patients. Primary human periodontal ligament stem cells (PDLSCs) were cultured as described previously 8, 20, 23 . In our experiment,
PDLSCs at 2-4 passages were used and each experiment was repeated at least three times.
To assess colony forming efficiency, single cell suspensions (1x10 3 cells) were seeded in
10 cm diameter culture dishes. Day 14 cultures were fixed in 4% paraformaldehyde and stained with 0.1% toluidine blue. Cells were harvested at indicated time points and fixed in
2% paraformaldehyde for immunofluorescence detection of STRO-1 (MAB1038, R&D
Systems, Inc., Minneapolis, MN, USA), CD-146 (R&D Systems, Inc.), two early mesenchymal progenitor markers, and the mesenchymal cells marker vimentin (R&D
Systems, Inc.). Then, fl uorescence-activated cell sorting (FACS) analysis was used for analyzing STRO-1, CD146, CD29, CD90, CD105, CD31, CD34 and CD45 (R&D Systems,
Inc.).
Osteogenic Differentiation and Mineralization Assay. A total 3×10 5 PDLSCs were plated into each well of a 6-well plate and cultured. After the cells reached 70~80% confluence, they were cultured in osteogenic differentiation medium ( α -MEM supplemented with 10% FBS,
100 nM dexamethasone, 50 μ g/ml of ascorbic acid and 5 mM β -glycerophosphate) for 7-21
1

days. The cells were washed twice in phosphate-buffered saline (PBS) after fixation in 4% paraformaldehyde for 30 minutes. Calcium accumulation was detected by 2% alizarin red staining (pH 4.2), and calcium levels were measured using the Calcium Colorimetric Assay
Kit (BioVision Co., USA). ALP staining and activity were determined with the BCIP/NBT
Alkaline Phosphatase Color Development Kit (Beyotime Co., Shanghai, China) and Alkaline
Phosphatase (AKP/ALP) Detection Kit (Zhong Sheng Co., Beijing, China).
Adipogenic Differentiation.
To induce adipogenic differentiation, at 100% con fl uence,
PDLSCs were cultured in α -MEM supplemented with 10% FBS, 2 μ m insulin, 0.5 mM isobutylmethylxanthine, and 10 nM dexamethasone for 21 days. Intracellular lipid accumulation was detected by staining with oil red O solution.
Real-time PCR Analysis. Total RNAs, including miRNAs, were isolated from cultured cells using the mirVana miRNA Isolation Kit (Ambion, TX, USA). The conversion of miRNAs and mRNAs into cDNA and the detection of the miRNAs were carried out according to the manufacturer’s instructions using the miScript Reverse Transcription Kit and miScript SYBR
Green PCR Kit (Takara Bio Inc., Japan), respectively. Sequences were determined with the
ABI Prism 7500 HT Sequence Detection System (Applied Biosystems, Darmstadt, Germany).
The optimized miRNA-speci fi c primers for has-miR-17 and the endogenous control U6, were commercially obtained (RiboBio Co., Guangzhou, China). The expression levels of Runx2,
ALP, OCN, OPN, LPL, PPAR γ , TCF3, TCF4, LEF1, Smurf1 (Takara Bio Inc., Japan) were examined.
Western Blot Analysis. PDLSCs were harvested in RIPA lysis buffer (Beyotime Co.,
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Shanghai, China). Whole-cell protein extracts were quanti fi ed by the BCA assay, separated on SDS-PAGE 8%-12%, and then transferred to PVDF membranes (Millipore, Billerica, MA) for Western blots. Antibodies used in this study included the mouse β -actin antibody (1:5,000,
Abcam, Cambridge, England), rabbit α -tubulin antibody (1:2,000, Cell Signaling Technology,
Boston, MA), rabbit TCF3 antibody (1:500, Abcam, Cambridge, England), mouse TCF4 antibody (1:500, Abcam, Cambridge, England), rabbit LEF1 antibody (1:1,000, Cell Signaling
Technology, Boston, MA), mouse Runx2 antibody (1:500, Abcam, Cambridge, England), rabbit β -catenin antibody (1:800, Abcam, Cambridge, England), rabbit ALP antibody (1:800,
Abcam, Cambridge, England), mouse Smurf1 (1:500, Abcam, Cambridge, England). In addition, stripped membranes were reprobed with β -actin or α -tubulin antibodies for a loading control. The signals were detected using the ECL Kit after incubation with an anti-rabbit or anti-mouse IgG secondary antibody (1:5,000, CoWin Bioscience Co., Beijing, China). The relative band intensities in the scanned images were analyzed with Image J software
(National Institutes of Health, Maryland, USA).
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Primers for Real-time PCR.
Primer name Sequence (5’ to 3’)
U6 hsa-miR-17
β -actin
Runx2
OCN
OPN
ALP
Col1
Col3
LPL
PPAR γ
Smurf1
F:5’GCTTCGGCAGCACATATACTAAAAT3’
R:5’CGCTTCACGAATTTGCGTGTCAT3’
5’CAAAGUGCUUACAGUGCAGGUAG3’
F:5’TGGCACCCAGCACAATGAA3’
R:5’CTAAGTCATAGTCCGCCTAGAAGCA 3’
F:5’CACTGGCGCTGCAACAAGA3’
R:5’CATTCCGGAGCTCAGCAGAATAA3’
F:5’CCCAGGCGCTACCTGTATCAA3’
R:5’ GGTCAGCCAACTCGTCACAGTC 3’
F:5’GCCGACCAAGGAAAACTCACT 3’
R:5’GGCACAGGTGATGCCTAGGA3’
F:5’ GGAC CATT CCCA CGTC TTCA C 3’
R:5’ CCTT GTAG CCAG GCCC ATTG 3’
F:5’ TCCACATACCTTTATTCCAGGAATC 3’
R:5’ CCCGGGTTTAGAGACAACTTC 3’
F:5’ CTGGTCAGCAGGGTGCAATC 3’
R: 5’ TGGTTCCATCTTTGCCAGGAG 3’
F:5’ AAGACTTTGTAGGGCATCTGAGAACGA 3’
R:5’ TGAGAAAGGGCTCTGCTTGAGTTGTAG 3’
F:5’ GCACTGCCTATGAGCACTTCACA 3’
R:5’ GGGAGTGGTCATCCATCACAGA 3’
F:5’ GCCGCCTTGTCATCGGTTGTG 3’
R:5’ CCATTTGCTTTGCCATGTTGAGTTCT 3’
4
Temperature
( ℃ )
Length
(bp)
60
60
89
23
60
60
60
60
60
60
60
60
60
60
188
127
112
73
137
126
91
122
122
116

TCF3
TCF4
LEF1
F:5’ GCAGTTCCTGGGAACCACAATTA 3’
R:5' CAGCCAGCTGTGTTGACACGTA 3'
F:5’GTCTGGGCTCAGGGTATGGAAC 3’
R:5'GGAAGCTGTGGAACCGGAAC 3'
F:5’ TTATCCAGGCTGGTCTGCAAGAG3’
R:5'GCAGCTGTCATTCTTGGACCTGTA3'
60
60
60
118
139
107
5

Primers for lentiviral construct transduction.
Prime name sh-homo-TCF3-99 sh-homo-TCF3-299 sh-homo-TCF3-1076 sh-homo-TCF3-1201
Negative control
Target Sequence
Sense (5’ to 3’)
GGACTTCAGCATGATGTTCCC
GCCTCTCTTCATCCACATTCC
GCAATAACTTCTCGTCCAGCC
GGCCTGCAGAGTAAGATAGAA
TTCTCCGAACGTGTCACGT sh: Lentiviral construct.
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Supplementary Figure 1 Isolation and identification of PDLSCs. ( a) PDLSCs were capable of forming a single-colony cluster stained with 0.1% toluidine blue. Immunocytochemical staining showed that STRO-1, CD146, and vimentin were expressed in the hMSCs. ( b) Flow cytometric analysis of mesenchymal stem cell surface markers. ( c) Osteoblastic and adipogenic differentiation were assessed by alizarin red staining and oil red O staining. ( d)
The expression of osteoblast-related genes and adipogenic-specific genes was investigated by real-time PCR. The expression levels were normalized to β -actin. The scale bar in the micrographs represents 50 μ m or 100 μ m. The data are shown as mean±SD. * P < 0.05, n =3.
Abbreviations: Con: control; 1: control group; 2: induction group; OS: osteogenic induction;
AD: adipogenic induction.
Supplementary Figure 2 The different dose of Wnt3a on activating canonical Wnt pathway.
PDLSCs were treated with human recombined Wnt3a 25ng/ml or 50ng/ml for 3 days. The expression level of β -catenin in nucleus was measured by Western blot. The protein levels were normalized to α tubulin. The data are shown as mean±SD. Abbreviations: Con: control;
Wnt3a 25: recombinant human Wnt3a 25 ng/ml; Wnt3a 50: recombinant human Wnt3a 50 ng/ml.
Supplementary Figure 3 The expression level of Smurf1 was not regulated by canonical
Wnt signaling. PDLSCs were cultured in fully culture medium or osteogenic differentiation medium with 50ng/ml recombined Wnt3a for 7 days. The expression level of Smurf1 was measured by real-time PCR (a) and western blot (b) . The expression levels of mRNA and
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protein were normalized to β -actin.
The data are shown as mean±SD. *
P < 0.05, n =3.
Abbreviations: Con: control; OS: osteogenic induction. Wnt3a: human recombined Wnt3a
50ng/ml.
Supplementary Figure 4 The efficiency of lentiviral vector transduction. ( a) The effect of lentiviral construct on overexpressing TCF3 was confirmed by real-time PCR and Western blot analyses. ( b) The effects of lentiviral constructs transduction for down-regulating TCF3 were veri fi ed by real-time PCR and Western blot analyses. The expression levels were normalized to β actin. The data are shown as mean±SD. * P < 0.05, n =3. sh-Con: virus produced from the two packaging vectors; sh-TCF3-99, sh-TCF3-299, sh-TCF3-1076, sh-TCF3-1201: four target lentiviral constructs; sh-NC: lentivirus negative control; plenti-Con: virus produced from the two packaging vectors; plenti-TCF3: lentiviral construct for up-regulating TCF3.
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