Supplementarytable 1. Primer Sequences Used in the Experiment

SupplementaryTable 1. Primer sequences used in the experiment.

Gene / Primer sequences
SOX2 / F 5’ – CATGCACCGCTACGACG – 3’
R 5’ – CGGACTTGACCACCGAAC – 3’
PPAR-γ / F 5’ – CCTCCGGGCCCTGGCAAAAC – 3’
R 5’ – CTCCTGCACAGCCTCCACGG – 3’
C/EBP-β / F 5’ – GCGCGCTTACCTCGGCTACC – 3’
R 5’ – TGGCCTTGTCGCGGCTCTTG – 3’
COL1A1 / F 5’ – CTGGTGCTGCTGGCCGAGTC – 3’
R 5’ – GGGACCAGGGGGACCACGTT – 3’
COL1A2 / F 5’ – AACCAAGGATGCACTATGGA– 3’
R 5’ – GCTGCCAGCATTGATAGTTT– 3’
VDR / F 5’ – CGGCCGGACCAGAAGCCTTT – 3’
R 5’ – CTGGCAGTGGCGTCGGTTGT – 3’
c-MYC / F 5’ –CAGCTGCTTAGACGCTGGATTT – 3’
R 5’ –ACCGAGTCGTAGTCGAGGTCAT – 3’
SOX9 / F 5’ –TTCATGAAGATGACCGACGA – 3’
R 5’ –CACACCATGAAGGCGTTCAT – 3’
AGGRECAN / F 5’ –CCCTGGAAGTCGTGGTGAAAGG – 3’
R 5’ –AGGTGAACTTCTCTGGAGATGT – 3’
GAPDH / F 5′ – GAGTCAACGGATTTGGTCGT – 3’
R 5′ – GACAAGCTTCCCGTTCTCAG – 3’
Realtime
SOX2 / F 5’ – GTATCAGGAGTTGTCAAGGC – 3’
R 5’ – AGTCCTAGTCTTAAAGAGG – 3’
Realtime
DKK1 / F 5’ – CTTGGATGGGTATTCCAGA – 3’
R 5’ – CCTGAGGCACAGTCTGATGA – 3’
Realtime
DKK4 / F 5’ – CGCGATGAGAAGCCGTTCTGTGC – 3’
R 5’ – TCACACAGAGTGTCCCAGGGCA – 3’
Realtime
FZD1 / F 5’ – GGCCAGAACACGTCCGACAAGG – 3’
R 5’ – GCAGGAGAACTTGCCTCGCTCC – 3’
Realtime
FZD2 / F 5’ – ATCCCGCCGTGCCGCTCTATC – 3’
R 5’ – CGTCCTCGGAGTGGTTCTGGCC – 3’
Realtime
FRAT1 / F 5’ – CGCGTTGGCGGAGACTGTGG – 3’
R 5’ – AGGCCCATCAAGGTCGGCCT – 3’
Realtime
FRAT2 / F 5’ – CGCTTCGGCTGAGACGGTGG – 3’
R 5’ – GGAGGTGACCGCGTCCCTGA – 3’
Realtime
β-CATENIN / F 5’ – GAAACGGCTTTCAGTTGAGC – 3’
R 5’ – CTGGCCATATCCACCAGAGT – 3’
Realtime
LRP5 / F 5’ – GACCCAGCCCTTTGTTTTGAC – 3’
R 5’ – TGTGGACGTTGATATTGGT – 3’
Realtime
LRP6 / F 5’ – CCCATGCACCTGGTTCTACT– 3’
R 5’ – CCAAGCCACAGGGATACAGT– 3’
Realtime
GAPDH / F 5’ – CATGAGAAGTATGACAACAGCCT – 3’
R 5’ – AGTCCTTCCACGATACCAAAGT – 3’
ChIP
DKK1 promoter / F 5’ – CTTTGTTGTCTCCCTCCCAA – 3’
R 5’ – ATGACCGTCACTTTGCAAGC – 3’
Mutagenesis #1 / F 5’ – TGAAATCCCATCCCGGCATTGTTGTCTCCCTCCCA – 3’
R 5’ – TGGGAGGGAGACAACAATGCCGGGATGGGATTTCA – 3’
Mutagenesis #3 / F 5’–TTGAAATCCCATCCCGGCTTCAGTGTCTCCCTCCCAAGGGGCC–3’
R5’–GGCCCCTTGGGAGGGAGACACTGAAGCCGGGATGGGATTTCAA–3’

SupplementaryTable 2. Genes up- and down-regulated in SOX2-inhibited hUCB-MSCs by more than 4-fold.

Genes / Fold Changes
Up-regulated / KRT15 / 5.524460
AXIN1 / 5.190355
GJB2 / 5.178377
Down-regulated / PARD6A / 0.240232
CDH1 / 0.233124
MYC / 0.185030

KRT15, Keratin 15; AXIN1, Axin 1; GJB2, Gap junction protein, beta 2, 26kDa ; PARD6A, Par-6 partitioning defective 6 homolog alpha (C. elegans); CDH1, Cadherin 1, type 1, E-cadherin (epithelial); MYC, V-myc myelocytomatosis viral oncogene homolog (avian).

Supplementary Figure Legends

Supplementary Figure1. Localization of SOX2 and the expression of pluripotent markers.

(a) SOX2 was expressed in the nucleus of tera-1 cells and hUCB-MSCs. The scale bar represents 10 m.

(b) SOX2, OCT4 and c-MYC expression in hMSCs. LAMIN A and -actin were used as loading controls.

Supplementary Figure2. Cell proliferation after SOX2 inhibition by siRNA.

(a) The population of hUCB-MSCs was reducedin si-SOX2compared to the si-control.The scale bar represents 100 m.

(b) By real-timePCR, SOX2 expression in si-SOX2 cells decreased to 20% of the si-control. ***, p<0.001.

(c) FACS analysis showed that the number of cells in S phase decreased, and the number in G0/G1 phase increased in cells treated with si-SOX2 compared to the si-control. ***, p<0.001.

(d) Cell proliferation, measured by the MTTassays, was significantly decreased by si-SOX2 treatment. ***, p<0.001.

Supplementary Figure3. Differentiation analysis after SOX2 inhibition by siRNA.

(a-c) Adipogenic differentiation was analyzed using siRNA. Oil red O droplets and adipogenic marker expression was reduced after SOX2 inhibition but were not severe.The scale bar represents 100 m.

(d-f) Osteogenic differentiation after SOX2 inhibition by siRNA. Enhanced osteogenesis after SOX2 inhibition was observedby cell morphology, alizarin red S elution and molecular marker gene expression.The scale bar represents 100 m. **, p<0.01.

(g-i) Chondrogenic differentiation after SOX2 knockdown with siRNA. The maximum diameter of si-SOX2treated cells was larger than that of the si-control, and the expression of chondrogenic markers was increased after SOX2 inhibition. The scale bar represents 100 m.

Supplementary Figure4. SOX2 inhibition using siRNAalters WNT signaling through DKK1.

(a,b) DKK1expression was significantly reduced in SOX2-inhibited hUCB-MSCs. The expression of p--CATENIN decreased, but the expression of total--CATENIN increased after SOX2 inhibition. *, p<0.05; and ***, p<0.001. Protein levels were normalized to -ACTIN using Image J analysis software.

(c) The TOP/FOP Flash assay showed increased WNT signaling in si-SOX2 treated.

Supplementary Figure 5. ChIP and luciferase assays after SOX2 inhibition with siRNA.

(a) ChIP assay around the -76 bps region of DKK1 showed SOX2-specific binding.

(b) Luciferase activity aftersi-SOX2treatment was significantly decreased compared toafter thesi-control treatment. ***, p<0.001.

Supplementary Figure 6. Ectopic expression of DKK1 and WNT ligands cannot recover the proliferation defects in SOX2-inhibited hUCB-MSCs.

(a, b) Cell proliferation after treatment with SOX2 siRNAdid not increase in response to DKK1 treatment, as confirmed by MTT assays and FACS analysis. ***, p<0.001.

(c, d) Treatment with WNT3A and WNT5A could not recover the proliferation defect in SOX2 inhibited hUCB-MSCs. *,p<0.05;**, p<0.01;***, p<0.001.

(e) Treatment with WNT3A increase S phase instantly in sh-SOX2 cells.

Supplementary Figure 7.The expressions of c-MYC and SOX2 were decreased after SOX2 inhibition. LAMIN A and -ACTIN were used as loading controls.