Supplementary Materials and Methods
Immunohistochemical and immunofluorescent staining
For immunohistochemical staining, paraffin-embedded tissue sections were first
deparaffinized and rehydrated. Antigen retrieval was performed in 0.01M sodium
citrate (pH 6.0) using a vegetable steamer. The sections were incubated with 0.3%
hydrogen peroxide, then 0.3% Triton X-100 in PBS at room temperature for 15
minutes, and blocked with 10% goat serum for 1 hour. Sections were then incubated
with anti-Shp2 antibody (Upstate, 06-118) in a 1:500 dilution for 1 hour at 37°C,
washed three times in PBS, and then incubated with HRP conjugated secondary
antibody and developed using the using DAB substrate kit according to the
manufacturer's instructions (Abcam). Hematoxylin was used for the counterstain.
Immunostaining images were captured by a Leica DM2500 microscope. The analysis
of the human prostate tumor samples for Shp2 expression was blinded to the scoring
investigator. For immunofluorecent staining of polarized MDCK cells, cells are grown
on Transwell filters for 48 hours. Transwell filters were then washed twice with PBS
buffer and the cells were fixed with BD Cytofix fixation buffer (BD bioscience) for
10 minutes at room temperature. After additional wash with PBS, cells were
permeablized by -20°C BD Phosflow Perm Buffer III (BD bioscience) for 5miniutes,
blocked with 10% gout serum for 1 hour at room temperature, and then incubated
with primary antibody at 4°C overnight. Cells were washed 3 times with PBS buffer
and incubated with a fluorochrome-conjugated secondary antibody for 1 hour at room
temperature. After washing with PBS for 3 times, the cells were stained with 1μg/ml
DAPI (Sigma) for 10 minutes at room temperature, and then mounted with Vector
H-1000 mounting medium (Vector Laboratories, Inc,#H-1200). Immunostaining
images were captured on a Leica SP5 II confocal microscope (Leica, Germany) with a
63 x Plan oil immersion objective. Z-stack x/y images were obtained with 1.5μm
interval in a 30μm total depth with LAS AF Lite software (Leica Microsystems CMS
GmbH) (Leica, Germany). More than 15 different fields were captured in each assay
for analysis.
Quantitative Real-time PCR
One microgram of total RNA was used for reversed transcription for cDNA
synthesis using PrimeScript RT reagent (Takara). SYBR green qPCR super mix
(Toyobo) was used for quantitative PCR. Quantitative real-time PCR was performed
on the Step one Plus Real-Time PCR Systems (Applied Biosystems). The primers for
real time PCR were listed in the following table.
Forward Primer 5’-3’
Reverse Primer 5’-3’
Ptpn11
GAACTGTGCAGATCCTACCTCT
TCTGGCTCTCTCGTACAAGAAA
E-cadherin
CGAGAGCTACACGTTCACGG
GGGTGTCGAGGGAAAAATAGG
N-cadherin TCAGGCGTCTGTAGAGGCTT
ATGCACATCCTTCGATAAGACTG
Vimentin
GACGCCATCAACACCGAGTT
CTTTGTCGTTGGTTAGCTGGT
Zeb1
GATGATGAATGCGAGTCAGATGC ACAGCAGTGTCTTGTTGTTGT
Snail
TCGGAAGCCTAACTACAGCGA
AGATGAGCATTGGCAGCGAG
Slug
CGAACTGGACACACATACAGTG
CTGAGGATCTCTGGTTGTGGT
Twist
GTCCGCAGTCTTACGAGGAG
GCTTGAGGGTCTGAATCTTGCT
actin
CATGTACGTTGCTATCCAGGC
CTCCTTAATGTCACGCACGAT
Immunoprecipitation and immunoblotting
For immunoprecipitation, cells were lysed in NP-40 lysis buffer (50mM Tris
(pH7.4)，150mM NaCl，1% NP-40) supplemented with the protease inhibitor cocktail,
the phosphatase inhibitor cocktail (Thermo scientific) and 5mM NaF. Cell lysates
were clarified at 12,000g for 10min at 4°C. For 293T cells, and 0.5-1mg total cell
lysates were incubated with indicated antibodies or IgG at 4°C overnight. Protein
A/G-plus Agarose beads (Roche) were then added and incubated for an additional 2
hours at 4°C. The immunoprecipitates were then washed three times with lysis buffer
and boiled for 5 minutes at 98℃ in SDS loading buffer for subsequent
immunoblotting. For the immunoprecipitation of endogenous PAR3 protein in DU145
and RWPE1 cells, 2mg of cell lysates and 2.5μg anti-PAR3 antibodies were used.
For immunoblotting, the protein concentration was determined by a BCA assay
(Bio-Rad). Proteins were separated by SDS-PAGE gels and transferred to a
nitrocellulose membrane (Amersham). Membranes were first incubated in 5% fat free
milk in TBS for one hour, and then incubated with indicated primary antibodies for 4
hours or overnight at 4˚C, followed by washing three times in TBS containing 0.8%
Tween20. The membranes were then incubated with the HRP conjugated secondary
antibody for 1 hour, and developed by the ECL reagent (Millipore).
Antibodies against Shp2, HA-tag, Zeb1, N-cadherin, Slug, and Snail were all
purchased from Cell Signaling Technology (the catalog number are #3397, #2367,
#3396, #14215, #9585, #3879 respectively). Anti-E-cadherin was bought from BD
(#610181). Antibody against Vimentin was purchased from Epitomics (#2707-1).
Antibody against PAR3 and phosphotyrosine were purchased from Upstate (#07-330).
Anti-ZO-1 antibody was purchased from Life Technologies (#339100). Anti-FAK
antibody (#3285) and anti-phospho-FAK (Tyr397) (#3283) was purchased from Cell
Signaling Technology. Antibody against β-actin（Cell Signaling Technology #4970）
was used for immunoblotting internal control.
Colony, sphere and epithelial lumen forming assays
For the colony forming assay, single prostate cancer cells were plated on 6-well
plates at densities of 500, 800, or 1000 cells/well in triplicates and cultured for 7-10
days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal
violet for 1 hour. For the sphere forming assay, prostate cancer cells were plated in
96-well ultra low cluster plates (Corning) at cell densities of 10, 50, or 100 cells/well
in triplicates. After a two-week culture in DMEM:F12 medium (Gibco) supplemented
with 20ng/ml EGF (peprotech), 20ng/ml FGF (peprotech) and 2% B27(Gibco),
spheres were examined under a Nikon light microscope, and photographed. For
epithelial lumen forming assay, single MDCK or RWPE1 cells were suspended in
DMEM medium (containing 10% FBS) or KSFM medium (containing 5% FBS) with
2% Matrigel (BD Biosciences) at 1.5x104/ml. 200μL mixture was plated on Lab-Tek
II chamber slides (Thermo Fisher Scientific) which were pre-coated with 50μL of
Matrigel. The culture medium was changed every other day. The epithelial cysts were
collected, fixed and subjected to immunofluorescent staining at 10 days or 20 days
after plating for MDCK and RWPE1 cells respectively.
Small molecule inhibitor treatment
FAK inhibitor PF573228 was purchased from Medchem Express. DU145 cells
were treated with 50nm PF573228 or equivalent amount of vehicle (DMSO) 24 hours
before and during the migration assay.
Shp2 inhibitor IIB-08 was purchased from Santa Cruz Biotechnology.
To
examine the effect of IIB-08 on prostate cancer growth in vivo, one week after the
inoculation of DU145 cells, 100 mg/kg II-B08 were injected intraperitoneally each
day for 7 consecutive days into tumor bearing nude mice. Tumor size was monitored
each week for 3 weeks after the treatment. Mice were sacrificed at 4 weeks after
inoculation and then the tumors were collected and imaged.
Real time cell recording and imaging
Real time cell migration analysis was performed on Xcelligence Real-Time Cell
Analyzer DP instrument. 3x104 cells were seeded in the upper chamber and of the
CIM-16well plates (Roche Applied Science). The Kinetics of the cell migration was
recorded every 15 min.
Real time microscopic imaging was used to record cell mobility. Cells were
plated on LAB-TEK glass-bottom dishes (Thermo Fisher Scientific) at around 20%
confluence and cultured in the IX3W TOKAI HIT time-lapse system installed in an
IX83 Olympus Fluorescent microscope. Differential interference contrast images were
captured with a 10x objective lens at a 10 min interval continuously for 2 days. Then
the video was analyzed by the CellSens Dimension 1.9 software from Olympus.
Supplementary Figure 1. Expression levels of Shp2 in different prostate cancer
cell lines.
Immunoblotting result shows higher Shp2 expression levels in PC3 and DU145 cells
than LnCap cells.
Supplementary Figure 2. Shp2 knock down using a second shRNA sequence
leads to suppressed prostate cancer cell growth and metastasis.
A. Efficient knock down of Shp2 by shRNA #2 lentivirus is confirmed by
immunoblotting.
B. shShp2 #2 lentivirus transfection represses the cell cycle progression of DU145
cells. (n=3)
C. Shp2 gene silencing by shShp2 #2 suppresses xenograft tumor growth of DU145
cells in vivo. (n=10)
D. Shp2 knockdown by shShp2 #2 leads to reduced DU145 cell migration. (scale bar
is 50μm)
E. Shp2 knockdown by shShp2 #2 inhibits metastasis of PC3 cells in vivo. (n=7 in
scramble group, n=9 in shShp2 #2 group)
(****P < 0.0001, *** P < 0.001, ** P < 0.01,* P < 0.05, data are shown as means +/S.E.M.)
Supplementary Figure 3. Upregulated Shp2 promotes migration and motility of
prostate cancer cells.
A. WT-Shp2 or DA-Shp2 expression causes enhanced prostate cancer cell motility.
LnCap cells transfected with control, WT-Shp2 or DA-Shp2 retrovirus were
plated at around 20% confluence and monitored by a microscopic time-lapse
system. Cell migration distance at 48hours after the initiation of recording was
measured. At least 25 cells per each group were followed and counted.
B. The kinetic information of the migration of LnCap cells transfected with control,
WT-Shp2 or DA-Shp2 retrovirus was recorded using Xcelligence real-time cell
analysis. Overexpression of WT-Shp2 or DA-Shp2 leads to accelerated cell
migration (n=4). (****P < 0.0001, ** P < 0.01,* P < 0.05, data are shown as
means +/- S.E.M.)
Supplementary Figure 4. Effect of FAK activity inhibition on the migration of
scramble shRNA or shShp2 tranfected DU145 cells.
A. Impact of Shp2 knock down on the phosphorylation level of FAK at Y397.
Scramble RNA or shShp2 tranfected DU145 cells were serum starved for 8
hours and unstimulated or treated with 100ng/ml EGF for 10 minutes. Cell
lysates were analyzed by immunoblotting with anti-phospho-FAK (Y397),
anti-FAK, anti-Shp2 and anti-actin antibodies
B. Inhibition of FAK activity by PF573228 does not rescue the suppression of
DU145 cell migration caused by Shp2 silencing. DU145 cells were treated
with 50nm PF573228 or vehicle 24 hours before and during the migration
assay. (scale bars are 50μm)
(*** P < 0.001, ** P < 0.01,* P < 0.05, data are shown as means +/- S.E.M.)
Supplementary Table 1. Clinical features of prostate cancer samples in this
study.
Patient
Number
Age
Gleason
score
1
62
6
2
80
7
3
81
5
4
57
6
stage
Ⅱ
Ⅱ-Ⅲ
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
cancer
Ⅱ-Ⅲ
Ⅱ-Ⅲ
Ⅰ-Ⅱ
Ⅱ
Ⅱ-Ⅲ
5
7
Ⅱ
6
80
6
7
68
5
8
76
7
9
76
8
10
76
4
11
66
5
Ⅰ-Ⅱ
Ⅱ-Ⅲ
Ⅲ-Ⅳ
Ⅰ
Ⅱ
Ⅱ-Ⅲ
12
7
Ⅱ
13
53
6
14
80
9
15
71
5
16
55
5
17
62
7
18
72
7
19
72
5
20
55
8
Tumor
type
Ⅲ-Ⅳ
Ⅰ-Ⅱ
Ⅱ
PSA
ng/
ml
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
TNM
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
cancer
NA
NA
Ⅱ
cancer
NA
NA
Ⅱ-Ⅲ
cancer
NA
NA
Sample
type
Shp2
up-regulation
Shp2 down-regulation
or no change
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
Paraffin
embedded
√
√
Paraffin
embedded
Paraffin
embedded
√
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
Paraffin
embedded
Paraffin
embedded
Paraffin
√
√
√
√
embedded
21
74
5
22
69
6
23
75
7
24
71
4
25
68
5
26
74
5
27
67
6
28
56
7
29
65
4
Ⅱ
cancer
NA
NA
Ⅱ
cancer
NA
NA
Ⅱ
cancer
NA
NA
Ⅱ
cancer
NA
NA
Ⅰ-Ⅱ
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
adeno
carcin
oma
NA
NA
adeno
carcin
oma
5.39
adeno
carcin
oma
8.78
adeno
carcin
oma
20.9
7
adeno
carcin
oma
22.7
2
adeno
carcin
oma
9.7
adeno
carcin
oma
55.7
73
adeno
carcin
oma
NA
adeno
13.4
Ⅱ
Ⅱ
Ⅰ
Ⅰ-Ⅱ
NA
30
64
9
NA
31
51
8
NA
32
70
6
NA
33
65
6
NA
34
63
9
NA
35
68
8
NA
36
68
7
NA
37
55
6
38
65
6
NA
NA
NA
NA
NA
6.8
NA
NA
NA
NA
NA
T2bn0
m0
T2cn0
m0
T2cn0
m0
NA
T3bn1
m0
T2an0
m0
NA
T2bn0
Paraffin
embedded
Paraffin
embedded
Paraffin
embedded
Paraffin
embedded
Paraffin
embedded
√
√
√
√
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Paraffin
embedded
√
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
√
√
√
√
√
√
√
√
√
carcin
oma
NA
39
74
6
NA
40
56
8
NA
41
70
9
NA
42
65
6
NA
43
total
73
6
m0
adeno
carcin
oma
13.6
2
adeno
carcin
oma
17.5
2
adeno
carcin
oma
NA
adeno
carcin
oma
4.16
adeno
carcin
oma
8.62
T2cn0
m0
NA
NA
T1cn0
m0
T2an0
m0
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
Fresh
sample
from
surgical
removal
√
√
√
√
√
26
17
(No.1-29 patient samples were used for the IHC staining. No.30-43 patient samples were used for
the immunobloting analysis.)
Supplementary Video 1. Time-lapse recording video of LnCap cells transfected
with control retrovirus.
Supplementary Video 2. Time-lapse recording video of LnCap cells transfected
with WT-Shp2 retrovirus.
Supplementary Video 3. Time-lapse recording video of LnCap cells transfected
with DA-Shp2 retrovirus.