Download Supplementary Data SD1: Materials and methods SD1.1 Reagents

Supplementary Data SD1: Materials and methods
SD1.1 Reagents
Human recombinant BMP-2 (rhBMP-2) was purchased from R&D Systems Inc.
(Ellisville, MO). The polyclonal antibodies used in this study were purchased from the
following companies: anti-Rac1 (BD Pharmingen, San Jose, CA); anti-FAK and
anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phosphotyrosine397-FAK
(Biosource, Camarillo, CA); anti-Cdc42 and phospho-myosin light chain 20 (Ser19)
(Cell Signaling, Beverly, MA). The monoclonal antibodies used were anti-myosin (20
kDa light-chain) (Sigma-Aldrich, St. Louis, MO); anti-actin (Millipore Bioscience
Research,
Temcula,
CA).
Blebbistatin
was
purchased
from
Sigma-Aldrich.
Rhodamine-phalloidin was purchased from Molecular Probes (Eugene, OR). Y-27632
was supplied by Mitsubishi Pharma Co. (Tokyo, Japan).
SD1.2. Cells
Mouse cell lines ST2 (RCB0224), 10T1/2 (RCB0247), C2C12 (RCB0987), and
MC3T3-E1 (RCB1126) were obtained from the Riken Cell Bank (Tsukuba, Japan), and
maintained in RPMI1640, BME, DMEM, and -MEM medium (Invitrogen, Tokyo,
Japan) supplemented with 10% fetal bovine serum (FBS; MP Biomedicals, Illkirch,
French), respectively. Cells were cultured at 37 °C in a fully humidified incubator under
a 5% CO2 atmosphere.
SD1.3. Detection of osteogenic differentiation
Alkaline phosphatase (ALP) staining and Alizarin red S staining were performed as
described previously [1]. ALP activity was determined by standard protocol using
-Nitrophenylphosphate as the substrate and the ALP activity assay kit, LabAssayTM
ALP (Wako, Osaka, Japan). Protein concentration was determined using BCA protein
assay reagents (Pierce, Rockford, IL).
SD1.4. RNA isolation, reverse transcription, and polymerase chain reaction
(PCR)
Total RNA was purified using the TRIzol reagent (Invitrogen). Total RNA (1 g) was
used as a template for reverse transcription using the High Capacity cDNA Reverse
Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer's
instructions. PCR was performed with Taq DNA Polymerase (Promega, Madison, WI)
using the indicated primer sets (forward and reverse respectively) for the following
genes:
Runx2,
5'-CGCATTCCTCATCCCAGTAT
-3'
and
5'-TAAAGGTGGCTGGGTAGTGC-3'; PTHR, 5'-AGCGAGTGCCTCAAGTTCAT -3'
and
5'-CCCTCCACCAGAATCCAGTA
5'-CTTAACCCAGCTCCCTACCC-3'
Osteocalcin,
and
-3',
5'-GCAGGCAGGTGAACTTCTTC-3';
5'-GCGCTCTGTCTCTCTGACCT-3'
GCCGGAGTCTGTTCACTACC-3';
Osterix,
and
Osteopontin,
5'5'-
TCTGATGAGACCGTCACTGC-3' and 5'-TCTCCTGGCTCTCTTTGGAA-3'; type1
collagen1a,
5'-ACTGGTACATCAGCCCGAAC-3'
and
5'-GGTGGAGGGAGTTTACACGA-3';
PPAR,5'-CCCTGGCAAAGCATTTGTAT-3'
and
5'-AATCCTTGGCCCTCTGAGAT-3'; LPL, 5'-CCTACTTCAGCTGGCCTGAC-3' and
5'-TGGGAGCAAATGATTCCTTC-3'; FABP4, 5'-CACCTGGAAGACAGCTCCTC-3'
and
5'-AATTTCCATCCAGGCCTCTT-3';
5'-AGGAAGCTGGCAGACCAGTA-3'
type10
Collagen,
and
Sox9,
5'-CCCTCTCGCTTCAGATCAAC-3';
5'-GCCAGGTCTCAATGGTCCTA-3'
and
5'-AAAAGCAGACACGGGCATAC-3';
5'-GCCAAGACCTGAAACTCTGC-3'
MyoD,
type2
and
Collagen,
5'-CTTGCCCCACTTACCAGTGT-3';
5'-AGCACGCACACTTCCCTACT-3'
and
5'-GCATCTGAGTCGCCACTGTA-3'; B2MG, 5'-ACGCCTGCAGAGTTAAGCAT-3'
and
5'-TGGGGGTGAGAATTGCTAAG-3';
GAPDH,
5'-AACTTTGGCATTGTGGAAGG-3' and 5'-CCCTGTTGCTGTAGCCGTAT-3'.
SD1.5. Immunofluorescence and phase contrast microscopy
Immunofluorescent staining analysis was performed as described previously [2].
Fluorescence and phase contrast images were obtained using a CCD camera VB-7010
(Keyence, Osaka, Japan) and a BX60 (Olympus, Tokyo, Japan) microscope with a
UplanApo 40/1.00 Oil Iris Ph3 lens. Images were analyzed with ImageJ (NIH
Software, Bethesda, MD). Cells were defined as having a motile phenotype if it had an
elongated shape with strong cell protrusion. Using these criteria, more than 100 cells per
sample were independently counted by five individuals and the mean number of cells
with a motile phenotype during osteogenic differentiation was calculated.
SD1.6. Cell migration assays
A modified Boyden chamber migration assay was performed as described previously
[3]. In this experiment, we carefully adjusted cell number to avoid an effect of cell
density on cell migration ability. For the wound healing migration assay, ST2 cells and
BMDCs were cultured in a plastic culture dish. A confluent monolayer of these cells
was then wounded with a 200 l yellow pipette tip and the media was replaced with
fresh medium. Treatment of reagents was continued through the entire experiment after
1 h pretreatment. Closure of the wound was monitored by time-lapse photography using
the Cool Snap Cf camera (Roper Scientific, Ottobrunn, Germany) and an IX70 inverted
microscope (Olympus). Supplemental movies were made using iMovie software with a
Macintosh computer (Apple Inc., Tokyo, Japan).
SD1.7. Cell adhesion assay
The cell adhesion assay was performed as described previously [4]. Briefly, the cells
(2105 cells/well) were plated in 12-well culture plates and incubated for 30 min.
Non-attached cells were removed by three PBS washes and attached cells were counted
(n=6).
SD1.8. Culture and expansion of bone marrow derived cells (BMDCs)
C57BL/6 mice (SLC, Shizuoka, Japan) were sacrificed under diethyl ether anesthesia.
The bone marrow was flushed out from the tibia and femur and suspended in -MEM
medium supplemented with 20% FBS, 100 units/ml penicillin, 100 g/ml streptomycin,
and 55 M 2-mercaptoethanol. Non-adherent cells were discarded and adherent cells
were cultured in a culture dish. These cells were passaged using trypsin treatment when
the cells reached 80% confluency. Osteogenic differentiation was induced by treatment
with induction medium. Adipogenic induction was induced by treatment with 2 M
troglitazone. Oil red O staining was performed using a staining kit (Diagnostic
Biosystems, Pleasanton, CA) according to the manufacturer’s protocol. Chondrogenic
induction of ST2 cells was performed as described previously [5].
SD1.9. Small-GTPase family pull-down assays
Levels of activated GTP-RhoA, GTP-Rac1 and GTP-Cdc42 were measured using a
pull-down assay [6]. Briefly, prepared cells were cultured in a type I-collagen coated
dish (BD Falcon) for 24 h. Cell lysates were clarified by centrifugation at 13,000 × g at
4 °C for 10 min. Lysates containing an equal volume of protein were mixed with
glutathione-Sepharose beads (Amersham Pharmacia Biotec., Buckinghamshire, UK)
that were coupled with bacterially expressed GST-rhotekin fusion protein for the
GTP-RhoA assay, or bacterially expressed GST-p21 activated kinase (PAK) fusion
protein for the GTP-Rac1 and GTP-Cdc42 assays, and were then incubated for 2 h at
4 °C. Activated GTPases bound to beads and the total protein level of the GTPase in
cell extracts were detected by immunoblotting. Specific GTPase activities were
calculated by normalization of the amount of the bead-bound GTPase against the
amount of the same GTPase in whole cell lysates (n=4).
SD1.10. Immunoblotting
Immunoblotting was performed as described previously [7]. Band intensity was
quantified using ImageJ.
SD1.11. Mouse model of ectopic bone formation
The mouse model of ectopic bone formation was described previously [7]. Briefly, a
rhBMP-2-collagen1 composite containing 5 g of rhBMP-2 (Astellas Pharma Inc.,
Tokyo, Japan) and 3 mg of atelocollagen (Nitta Gelatin, Osaka, Japan) were implanted
into a dorsal subfascial pocket of twelve 5-week-old male ICR mice. Six mice were
sacrificed at 4 days, and six at 1 week, after implantation of the composite. Y-27632
was supplied to the mice using Alzet osmotic pumps (Alza Corp., Palo Alto, CA).
Recovered ossicles were examined radiographically using a soft X-ray apparatus
(Softex Type SM, Osaka, Japan), as well as histologically. Tissue preparation and in situ
hybridization were carried out as previously described [8, 9]. Six sections, each 4 m
thick, were hybridized with digoxigenin-labeled antisense and sense cRNA probes for
mouse osteopontin [9]. Hybridized probes were detected using a nucleic acid detection
kit (Roche Diagnostic, Japan) according to the manufacturer’s instructions. Sections
hybridized with sense probes showed no positive signals.
SD1.12. Statistical analysis
Results are expressed as means ± SD. Statistical differences between samples were
determined using a two-sided Student’s t-test for biological assays. Values of less than
0.05 were considered to be statistically significant.
Supplementary Data SD2: Supplemental figure legends
Fig. S1. Four mesenchymal cell lines in different stages showed difference
in motility and ST2 cells was undifferentiated mesenchymal cell.
(A) Schematic presentation of differentiation stages among four mesenchymal cell lines.
(B) RT-PCR analysis of differentiation related gene expression of osteogenic,
adipogenic, chondrogenic, and myogenic lineages in four cell lines. OPN, osteopontin;
Oc, osteocalcin; LPL, lipoprotein lipase; Col, collagen; B2MG, beta-2-microglobulin (as
a control). Boyden chamber migration assay for (C) ST2, (D) 10T1/2, (E) C2C12, and
(F) MC3T3-E1 cells using different concentration of FBS as a chemoattractant. (G) ST2
was differentiated into osteogenic, adipogenic, and chondrogenic lineages confirmed by
ALP, Oil red O, and Alcian blue staining, respectively.
Fig. S2. rhBMP-2 did not affect cell migration of undifferentiated (0 day)
and differentiated (3 day) ST2 cells as a chemoattractant.
(A) Chemotaxis analysis of undifferentiated (upper panel) and differentiated (lower
panel) ST2 cells in response to rhBMP-2. rhBMP-2 was added to the upper chamber,
lower chamber or both chambers at the indicated concentrations. (B) ALP activity of
ST2 cells after treatment with rhBMP-2 (50 ng/ml) for 4 hours (n=4).
Fig. S3. ST2 cells synthesized certain amount of murine BMP-4.
RT-PCR analysis of the mRNA expression of murine BMP-4 during osteogenic
differentiation using rhBMP-2 (50 ng/ml) (A) and by treatment with Y-27632 (0 ~ 20
M) (B) for 1 day. The upper numbers indicated relative ratio of mBMP-4 expression to
control (normalized to GAPDH).
Fig. S4. Y-27632 treatment showed additive effect for ALP activity in ST2
cells.
ALP activity of ST2 cells after treatment with Y-27632 and rhBMP-2 at the indicated
concentrations for 1 day (n=3).
Fig. S5. Inhibition of myosin ATPase with blebbistatin did not affect
osteogenic differentiation.
(A) ALP activity of ST2 cells after treatment with rhBMP-2 (BMP, 50 ng/ml), Y-27632
(Y, 10 M), and blebbistatin (Ble, 10 M) (n=3). (B) Western blotting analysis of the
phosphorylation level of MLC20 (pMLC20, Ser 19) over time following treatment with
Y-27632 or blebbistatin. Total MLC levels were also assayed.
Supplementary data SD3: Supplemental movies 1-4
A wound healing assay was performed using ST2 cells. The four-second Quicktime
movie represents 4 h of recording. The scale bar indicates 30 m. (1) Control, (2)
rhBMP-2 (50 ng/ml) treatment, (3) Y-27632 (10 M) treatment, (4) combined rhBMP-2
(50 ng/ml) and Y-27632 (10 M) treatment of ST2 cells.
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