Supplemental Figure 1 - jbc.org€¦ · Supplemental Figure 5. Expression of epiprofin/Sp6...

7
dental pulp SP MP Oil-Red-O control adipogenic cond. A B dental pulp SP MP control neurogenic cond. C - + - + - + dental pulp SP MP PPARγ Hprt D Relative expression 40 0 30 10 20 50 PPARγ/GAPDH - + - + - + dental pulp SP MP Relative expression 100 0 75 25 50 125 Neurofilament-M/β-actin - + - + - + dental pulp SP MP * * Supplemental Figure 1

Transcript of Supplemental Figure 1 - jbc.org€¦ · Supplemental Figure 5. Expression of epiprofin/Sp6...

Page 1: Supplemental Figure 1 - jbc.org€¦ · Supplemental Figure 5. Expression of epiprofin/Sp6 transcription factor in iPS cells co-cultured with SF2-24 cells. Time course analyses of

dental pulp SP MP

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PPARγ Hprt

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50 PPARγ/GAPDH

- + - + - + dental pulp SP MP

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*

*

Supplemental Figure 1

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rat dental epithelium (SF2 cells)

pEF6/GFP-PDGFtm-myc-HA

SF2/GFP cells （expressing GFP-myc-HA tag）

selection

co-cultured with SP cells

GFP

phase (SF2)

SF2/GFP

SP

Supplemental Figure 2

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A B Ambn

CK14

Klf4

Sox2

Oct3/4

Nanog

GAPDH

SF2-24 iPS

GAPDH

Ambn 7 12 13 17 19 24 Clone No.

Rat dental epithelium (SF2 cell)

Supplemental Figure 3

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A B

MEF iPS tooth germ

mAmbn GAPDH

m total-RNA

GAPDH

rAmbn

0 1 10 100 120 150 200 (ng)

200 150 120 100 10 0 1 (ng) r total-RNA

mAmbn

Supplemental Figure 4

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Supplemental Figure 5

Ambn

GAPDH

Epiprofin/Sp6

1 7 10 (days)

SF2-24+iPS

Page 6: Supplemental Figure 1 - jbc.org€¦ · Supplemental Figure 5. Expression of epiprofin/Sp6 transcription factor in iPS cells co-cultured with SF2-24 cells. Time course analyses of

SUPPLEMENTAL INFORMATION Supplemental Figure 1. Adipogenic and neurogenic induction of SP cells. (A) Adipocyte-specific Oil-Red-O staining of dental pulp, SP, and MP cells in regular or adipogenic induction culture (adipogenic cond.) medium. (B) RT-PCR and real-time PCR analyses of the adipocyte marker gene PPARγ in dental pulp, SP, and MP cells in adipogenic induction culture medium. (C) Phase images of dental pulp, SP and MP cells cultured in regular or neurogenic induction culture (neurogenic cond.) medium. (D) Western blotting of dental pulp, SP, and MP cells cultured in regular (-) or neurogenic induction culture (+) medium was performed using the anti-Neurofilament-M antibody. The relative intensities of bands obtained with western blots were scanned and quantified using an LAS UV4000 system (Fujifilm-GE Healthcare, Tokyo, Japan). Supplemental Figure 2. Schematic diagram of establishment of SF2 cells expressing PDGFtm-GFP-myc-HA. Rat-derived dental epithelial SF2 cells were transfected with a GFP-PDGFtm-myc-HA expression vector and selected with antibiotics. GFP in the SF2 cells was visualized using fluorescence microscopy. SF2/GFP cells were co-cultured with SP cells (bottom) and separated with the anti-HA antibody. Supplemental Figure 3. Gene expression of subcloned SF2 cell lines and iPS cells. (A) Ambn expression in dental epithelial cells subcloned from SF2 cells. Clone SF2-24 expressed Ambn at the highest level and was used for co-culturing with iPS cells. (B) SF2-24 cells expressed the ameloblast marker genes CK14 and Ambn, but not stem cell markers. In contrast, stem cell markers were observed in iPS cells, but not ameloblast markers. Supplemental Figure 4. Design of mouse-specific Ambn primer. Mouse and rat Ambn mRNA showed a high degree of homology (94.2%), thus regular Ambn primers for RT-PCR are able to precisely distinguish between them. To resolve this problem, we employed a mouse Ambn LNA primer. The accuracy of the mouse-specific Ambn LNA primer was confirmed by PCR with gradient cDNA mixtures containing serial dilution ratios of mouse and/or rat cDNAs. (A) The expected size of the amplicon using the mouse LNA primer was observed only with mouse cDNA, while the intensity of the band increased in a cDNA dose-dependent manner. (B) Undifferentiated iPS cells and MEF feeder cells did not express Ambn mRNA. A mouse tooth germ sample served as a positive control for the mouse LNA primer. Supplemental Figure 5. Expression of epiprofin/Sp6 transcription factor in iPS cells co-cultured with SF2-24 cells. Time course analyses of Ambn and epiprofin/Sp6 gene expressions in iPS cells co-cultured with SF2-24 cells for 1 (1D), 7 (7D) and 10 days (10D) were analyzed by RT-PCR.

Page 7: Supplemental Figure 1 - jbc.org€¦ · Supplemental Figure 5. Expression of epiprofin/Sp6 transcription factor in iPS cells co-cultured with SF2-24 cells. Time course analyses of

Supplemental Table1

　Gene name forward primer reverse primer Size（bp）

mouse/rat　Ambn 5‘-GCGTTTCCAAGAGCCCTGATAAC-3’ 5‘-AAGAAGCAGTGTCACATTTCCTGG-3’ 366/368

mouse/rat　CK14 5‘-GAGAAGAACCGCAAGGATGC-3’ 5‘-AGGTTATTCTCCAGGGATGCTTTC-3’ 191

mouse/rat　GAPDH 5‘-GGAGCGAGACCCCACTAACATC-3’ 5‘-CTCGTGGTTCACACCCATCAC-3’ 181

mouse　Ambn 5‘-GCAGGTGGCACCATCCGALA-3’ 5‘-AAGAAGCAGTGTCACATTTCCTGG-3’ 1104

rat Ambn 5‘-GCTACCCTTCCACAGGGAAGGTLA-3’ 5‘-AAGAAGCGGTGTCACATTTCCTGG-3’ 267

mouse　Enam 5‘-GGGGACCACCAACAGCGTTTGGACGGCCA-3’ 5‘-CCCGGAGGTAGGAGAGGGAGGTTTACC-3’ 614

rat Enam 5‘-CAGGACCACCAACAGGGTTCGGACGA-3’ 5‘-TCTTGAGGTAGGAGGGGAAGGTTTGT-3’ 626

mouse CK14 5‘-GGCTGCCGATGACTTCCGGAC-3’ 5‘-CAGCAGTATCTGCGTCCACGCA-3’ 909

rat CK14 5‘-AGACTACAGCCCCTACTTCAAGACC-3’ 5‘-AGGTTATTCTCCAGGGATGCTTTC-3’ 597

mouse p63 5‘-GGCATTTCAGCACTATTTAGGTGG-3’ 5‘-CACTGGTGTGAGGAGACAAACTGTG-3’ 110

mouse Epiprofin 5‘-TCAGCCTGCTTTGGGAGGATAC-3’ 5‘-ATGCTTCTTCTTGCCCCCATCG-3’ 313

Klf4 5‘-CACCATGGACCCGGGCGTGGCTGCCAGAAA-3’ 5‘-TTAGGCTGTTCTTTTCCGGGGCCACGA-3’ 739

Sox2 5‘-GGTTACCTCTTCCTCCCACTCCAG-3’ 5‘-TCACATGTGCGACAGGGGCAG-3’ 193

Oct3/4 5‘-TCTTTCCACCAGGCCCCCGGCTC-3’ 5‘-TGCGGGCGGACATGGGGAGATCC-3’ 224

Nanog 5‘-CAGGTGTTTGAGGGTAGCTC-3’ 5‘-CGGTTCATCATGGTACAGTC-3’ 223

Fgf4 5‘-CGTGGTGAGCATCTTCGGAGTGG-3’ 5‘-CCTTCTTGGTCCGCCCGTTCTTA-3’ 197

Gdf3 5‘-GTTCCAACCTGTGCCTCGCGTCTT-3’ 5‘-AGCGAGGCATGGAGAGAGCGGAGCAG-3’ 570

Cdx2 5‘-GGCGAAACCTGTGCGAGTGGATGCGGAA-3’ 5‘-GATTGCTGTGCCGCCGCCGCTTCAGACC-3’ 493/490

Gata6 5‘-ACCTTATGGCGTAGAAATGCTGAGGGTG-3’ 5‘-CTGAATACTTGAGGTCACTGTTCTCGGG-3’ 334/328

Brachyury 5‘-ATGCCAAAGAAAGAAACGAC-3’ 5‘-AGAGGCTGTAGAACATGATT-3’ 835

BMP2 5‘-GGGACCCGCTGTCTTCTAGT-3’ 5‘-TCAACTCAAATTCGCTGAGGAC-3’ 154

BMP4 5‘-ACTGCCGCAGCTTCTCTGAG-3’ 5‘-TTCTCCAGATGTTCTTCGTG-3’ 486

Osteocalcin 5’-CCTCTTGAAAGAGTGGGCTG-3 5’-CCTCGGGAGACAAACAACAT-3’ 268

Osteonectin 5’-GTCTCACTGGCTGTGTTGGA-3’ 5’-AAGACTTGCCATGTGGGTTC-3’ 264

Runx2 5’-GACGTTCCCAAGCATTTCAT-3’ 5’-ACTCTGGCTTTGGGAAGAG-3’ 181

Dspp 5'-GGAACTGCAGCACAGAATGA-3' 5'-CAGTGTTCCCCTGTTCGTTT-3' 199/193

Bcrp1 5'- GGTGAATCTCAGAACCATTGGGC-3’ 5'-GCTGTTGTCCGTTACATTGAATCC -3' 139

PPARγ 5’-CTGATGCACTGCCTATGAGC-3’ 5’-CAGACTCGGCACTCAATGGC-3’ 373

Hprt 5’-GTTAAGCAGTACAGCCCCAAA-3’ 5’-AGGGCATATCCAACAACAAACTT-3’ 131

Gapdh (Real-time) 5’-CCATCACCATCTTCCAGGAG-3’ 5’-GCATGGACTGTGGTCATGAG-3’ 322

(mouse/rat)


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