mice). Note the nuclear translocation after 4 weeks of tamoxifen administration, best assessed in the high power images inserted in each panel. De Frutos, C.A. ...
Snail1-ER - TAM
+ TAM
trabecular bone
B
cortical bone
wt Snail1
hER
Perichondrium
A
Supplementary Fig. 1. (A) Endogenous Snail1 expression in the perichondrial area (white star) and in the growth plate (black star; see de Frutos et al., 2007) of the long bones. (B) Subcellular localization of Snail1-ER is regulated by Tamoxifen in transgenic bones. Immunostaining of the Snail1-ER protein in adult bones (12 week-old mice). Note the nuclear translocation after 4 weeks of tamoxifen administration, best assessed in the high power images inserted in each panel. De Frutos, C.A., Vega, S., Manzanares, M., Flores, J.M., Huertas, H., Martinez-Frías, M.L. and Nieto M.A. (2007). Snail1 is a transcriptional effector of FGFR3 signaling during chondrogenesis and achondroplasias. Dev. Cell 13, 872-883.
wt -TAM
+TAM
B B
C
D
E
F
TRAP
Calcein
Von Kossa
A A
Supplementary Fig. 2. Bone mineralization, cortical thickness, osteoblasts activity and osteoclasts differentiation are not affected by Tamoxifen administration in wild type mice. (A-B) Von Kossa staining in sections of long bones from 16 week-old mice. (C-D) Osteoblasts activity measured by in vivo calcein incorporation into trabecular bone. (E, F) TRAP staining of osteoclasts. Scale bars, 1 mm (A, B) and 100 µm (E, F).
wt -TAM
+TAM
B
C
D
Von Kossa
A
Snail1-ER - TAM + TAM
Histomorphometry
E 20
50
5
350
10
25
2.5
175
0
B.V (mm3)
0
Tb.Th. (mm)
wt + oil wt + tam
0
Tb.N. (mm-3)
0
Tb.Sp. (mm2)
tg + oil tg + tam
Supplementary Fig. 3. (A-D) Von Kossa staining of vertebrae sections from 16-week-old wild type and Snail1-ER mice (black staining). (E) Histomorphometric analysis (n=5 per condition). Bone volume (B.V.); Trabecular thicknes (Tb. Th.); trabeculae number (Tb.N.) and trabecular spacing (Tb. Sp.). Scale bar, 1mm.
3
A
int
ERK-2
Snai1-ER
bone
wt -TAM
Snail1-ER - TAM + TAM
+TAM
C
D
E
F
G
H
I
J
K
L
M
VDR
Intestine epithelium
H/E
hER
B
Supplementary Fig. 4. (A) Western blot and (B-E) immunohistochemical analysis confirms that no transgenic protein (hER) is present in the intestine. Thus, tamoxifen administration does not have any impact on tissue morphology (F-I) or VDR expression (J-M). The inset in (J) shows a negative control for the VDR antibody. Int, intestine
4
+ 4-OH-TAM
D
E
F
G
H
I
J
K
L
M
N
O
P
day 2
day 4
day 6
DAPI
C
PH3
B
DAPI
A
PH3
- 4-OH-TAM
Snail1-ER
day 8
tg - 4-OHT
20
tg + 4-OHT
0 0
2
4
relative mRNA levels
R
p21
1,6
0,8
0
0
2
4
6
8
6
relative mRNA levels
PH3 positive cells (% total cells)
Q
8 tg - 4-OHT tg + 4-OHT
p27
1,6
0,8
0
0
2
4
6
8
Supplementary Fig. 5. Snail1 activation does not affect the proliferation in osteoblasts in culture. (A-D and I-L) Phospho-histone 3 immunofluorescence (PH3) shows the mitotic transgenic osteoblasts during in vitro differentiation in the presence or in the absence of 4-OH-TAM. (E-H and M-P) Cells were counterstained with DAPI to reveal their nuclei. (Q) Quantification of PH3-positive cells. (R) Real time PCR shows no variations in the mRNA levels of p21 and p27, indicating that contrary to its action in chondrocytes, Snail1 does not activate p21 in osteoblasts.
5
B wt
50
Relative mRNA levels
Relative mRNA levels
A
25
0 days 0
2
4
6
8
50
wt+Snail1 siRNA
25
0 days 0
10
wt
2
4
6
8
proliferation maturation mineralization
D Snail1-ER
Snail1-ER + 4-OHT
Relative mRNA levels
Relative mRNA levels
C 50
25
0 days 0
2
4
6
8
50
4-OHT wash
25
0 days 0
10
+ 4-OHT
2
4
6
8
10
Realtive mRNA levels
E Differentiating wt osteoblasts 2
Atf4
Dmp1 Phex
1
0 days
0
2
4
Mock
6
4
6
Snail1 CR
4
6
si-RNA
Supplementary Fig. 6. Snail1 is necessary for osteoblast differentiation in culture. (A, B) Relative mRNA levels of Dmp1 and Phex in wild type and transgenic mesenchymal cells during their differentiation to osteoblasts. (C, D) Snail1 activation inhibits Dmp1 and Phex expression in cultured osteoblasts. This effect is reversible, as assessed by the onset of their expression when 4OHT is washed out. Dmp1 (light brown) and Phex (light green). (E) the expression of Atf4 is not affected by Snail1 activation.
6
-8000
IX
VIII VII
VI
V IV III II
-7105 -2905
-1005 -606
+4-OHT
-7055
-7105 -7965 -7055
- 4-OHT
-7965
-7105 -7965 -7055
-74
Snail1-box E-box conserved E-box
-7105 -7965
Input
M -7055
I
-667 -420
IX VIII VII VI V
IV
III
II
I
M
Supplementary Fig. 7. Snail1 does not bind to its perfect match E-boxes (GCAGGTG; Cano et al., 2000) located from 7 to 8Kb upstream of the Runx2 gene coding region. These Snail1 E-boxes are not conserved in the human promoter. The arrows on the right indicate the positions of the predicted amplified fragments. Input material was tested for each primer set (boxes I to IX). Cano, A., Pérez, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., Del Barrio, M. G., Portillo, F. and Nieto, M. A. (2000). The transcription factor Snail controls epithelial-mesenchymal transitions by repressing Ecadherin expression. Nature Cell Biol. 2, 76-83
7
Supplementray Table I. Oligonucleotides GENE
SEQUENCE (5' to 3')
mice genotyping
ACCGAGATGATGTAGCCAGCAG RT-PCR
Gapdh
Runx2 E-box I
CCTCCTCCCTTTCCTTCATTAT CCAAGCTTAGGAAGACAAGCAA
Runx2 E-box V
CGTGGCGGCTCTTACAATAAA
TCAACTGAGTGTGTGGCGTT
TCACTGTCCACGCTGATGAAA
112
100
101
TGGCAGAAAGGAAAAGCCTTA AGAGAACGGCTCCCTGCAGCG
VDR E-box III
104
TAGGAGAGAGGACGCAACTCC CGCTGCAGGGAGCCGTTCTCT
VDR E-box II
120
CATGACTACAACTCATTCTTGCCTC AGCCAGTAAATGAATACATGTGTCTG
VDR E-box I
100
ATCTGCATGATTGGTTTGAAC CCATTAAGTTCCATCATTCTAGAATG
Runx2 E-box IX
105
CATTCTAGAATGATCCAACTTAATGG TTACAGGAATAGATTGGGTTAGAATTAG
Runx2 E-box VIII
103
GCATTTGCTTACTATCCTATAGCAAC CTGTGCCAGTGTGATCTTTATC
Runx2 E-box VII
126
CTTGCAAGTGATACAATCCCAA
Runx2 E-box IV
Runx2 E-box VI
150
TGGCAGAAAGGAAAAGCCTTA GCCTTCCTGGCATTCAGAA
Runx2 E-box III
102
AAAGAGGGAGGGAAGAGAGCAA CGAATGAAGCATTCACACAATCC
Runx2 E-box II
102
CAAGGACAACCGGCGACACT TTACGCTGCACCTCCTCATCTG
ChIP assay
101
TGATTTGCTTTTGCCTGTTTGG AGGATCTGGGTGCAGGCTGTA
Vdr
100
CCACACTGGTGAGAAGCCATTC TCTTCACATCCGAGTGGGTTTG
Spp1/Osteopontin
102
AAACACACGGACTGCAGCACAT CACTTTTGCGTGGCTTCTCTGT
Snail 1
100
CAACATTTGCTTTCGGCATCAT AACTTGGGATTTTGATGCTGGTTT
Tnfrsf11b/Osteoprotegerin
102
GATTTCCTGCTGGAGAGCTG TGTAAATTCATGGCCCACAA
Tnfsf11/Rankl
100
AGAACTAACCCGGGACTTGG CCAGGGGCTTATGATTCTGA
Phex
102
AGGAGCCAGGCCAAGATGGT GCTTTGACACCCACGGTATTCA
p27
104
ACCACACCCCAAGCACAGACT TCGTCGCTTTCCTTCACTTTTG
p21
100
CTGAGCAAGAGAGGCCCTATCC CTCCCTAGGCCCCTCCTGTT
Ibsp/Sialoprotein
102
CCACAGACACCACACCGTCC TGTCTGCCTCATCCTCACTG
Gapdh
101
TACCTGAGCCAGGTGCTCTT GGCTGTCTTCCGAATGGTTA
Dmp1
101
GGAGAGAGCATGACCGATGGA GGTGGACATTAGGCGAGGAA
Cyp27b1
100
GGCCGGGAATGATGAGAACTAC CGCTCCGGCCCACAAAT
Collagen1a1
102
GGCTGGAAGACCGCCTACAAA CCCGGAGAGCCAAAG
Cbfa1/Runx2
391
GAGAGGTCCAGGCAACTTCCA GGAATAAAGGCAGAGCCAGGAAT
Bglap1/Osteocalcin
709
AGCTGGCCAGGCTCTCGGTG TAGCAGGGTCAGCGAGGGCC
Alkaline Phosphatase
450
CAAAGTGGAGATTGTTGCCATC CACCACCTTCTTGATGTCATC
Snail 1
Q-RT-PCR
fragment size (bp)
ACGATAAGCTCGAGCCATCTGC
100
GATCCGTGATGTAGCCACCAC GTGCAGTGGTTGATTCCAAGT
100
8
