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Supplemental Materials
Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment
Jianjian Shi, Xiangbing Wu, Michelle Surma, Sasidhar Vemula, Lumin Zhang, Yu Yang,
Reuben Kapur and Lei Wei*
*To
whom correspondence should be addressed. E-mail: lewei@iupui.edu (L.W.)
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sFigure 1.
A
5.8 kb
B
X Bg
Ex 2 X
Ex 3
Bg
Ex 2 B
X
ROCK2 locus
(WT allele: +)
B
Ex 3
B
Ex 3
B
Neo
B
Ex 2 B
X Bg
X
Neo
Targeting
vector
Targeted allele
(flox)
4.5 kb
B
X
X Bg
Ex 3
B
Knockout allele
Neo
Probe
B
C
WT
ROCK2+/-
ROCK2
WT allele (5.8 kb)
KO allele (4.5 kb)
Tail DNA Xba I (X) digestion
ROCK1
GAPDH
Heart homogenate
sFigure 1. Generation of ROCK2+/- mice. A. Schematic illustration of the strategy used for
disruption of ROCK2 gene. A targeting vector was constructed containing loxP sites (arrows)
flanking exon 2 of ROCK2 and Frt sites (diamonds) flanking the PGK-Neo cassette. Restriction
enzymes sites are shown for BamH I (B), Bgl II (Bg), Xba I (X) and genomic probe. We first
obtained germline transmission of the ROCK2flox allele, which contains the loxP-flanked exon 2
and neo cassette. The ROCK2 knockout allele was generated by crossing male ROCK2+/flox
mice with female Tie2-Cre mice to generate female ROCK2+/flox/Tie2-Cre mice. Female Tie2-Cre
mice express Cre in the female germ cells prior to the expression in endothelial lineage,
allowing germline deletion from floxed allele.
B. Southern blot analysis of genomic DNA
obtained from mouse tail. C. Western blot analysis of ROCK1 and ROCK2 levels in the heart of
WT and ROCK2 heterozygous knockout mice showing 50% reduction in ROCK2 expression
observed in ROCK2+/- heart homogenate. An anti-GAPDH was used to confirm equal loading.
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sFigure 2.
A
500
WT
400
* *
ROCK1-/-
300
*
*
200
*
*
100
0
Day
1
2
3
4
Attached cells (% of day 1)
Attached cells (% of day 1)
B
400
WT
* *
ROCK2-/-
300
*
*
200
*
*
100
0
Day
1
2
3
4
sFigure 2. ROCK1 or ROCK2 deletion in mouse embryonic fibroblasts (MEFs) does not
affect cell proliferation. Proliferation assays of wild type (WT) versus ROCK1-/- cells (A) and
WT versus ROCK2-/- cells (B) were performed at a density of 1 x 106/plate in 10%FBS DMEM
supplemented with 10%FBS. Cells were counted every 24 h. Cell viability was estimated by
using trypan bleu staining. Assays were performed in triplicate. Attached viable cell number was
expressed as percentage of attached cells at day 1. Error bars represent standard deviation * P
< 0.05 vs. control of the same genotype. # P < 0.05 vs. WT under the same condition.
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sFigure 3.
siRNA: Control
ROCK1
ROCK1
ROCK2
GAPDH
ROCK2
siRNA: Control
ROCK1
ROCK1
ROCK2
GAPDH
ROCK2
GAPDH
ROCK1&2
DOX
Phalloidin
p-MLC2
Merge
Phalloidin
p-MLC2
Merge
siROCK1
siROCK1&2
siROCK2
siROCK1
siROCK1&2 siROCK2
Control
siRNA
B
siRNA: Control
Control
siRNA
A
sFigure 3. siRNA-mediated knockdown of ROCK1 or ROCK2 mimics the effects of
ROCK1 or ROCK2 deletion. A. Representative image of Western blot of ROCK1 and ROCK2
performed with cell lysates from WT MEFs transfected with scrambled siRNA, ROCK1 siRNA
and ROCK2 siRNA. GAPDH was used to confirm equal loading. B. Representative images of
rhodamine-phalloidin staining for F-actin (red), p-MLC staining (green) and DAPI staining (blue)
of siRNA transfected cells with or without 3 M doxorubicin treatment for 16 h. Actomyosin
contractile rings are indicated with white arrows and folded periphery membranes with white
arrowheads. Bar, 50 m.
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sFigure 4.
Attached cells
A
0.88%
Floating cells
B
5.37%
0.31%
14.15%
WT
WT
23.29%
62.25%
0.23%
2.11%
0.13%
3.40%
ROCK1-/-
96.81%
0.85%
Annexin V-FITC
7-AAD
1.69%
7-AAD
92.06%
ROCK1-/-
22.57%
73.90%
Annexin V-FITC
sFigure 4. Representative scatter plots of apoptosis quantified by FACS analysis after staining
with Annexin V and 7-amino-actomyocin D (7-AAD) in attached (A) and floating (B) WT and
ROCK1-/- cells collected after treatment for 16 h with 3 M doxorubicin. Viable cells are Annexin
V−/7-AAD−. Annexin V+/7-AAD− cells are in early apoptosis, whereas Annexin V+/7-AAD+ cells
are in late apoptosis, and have lost cell membrane integrity and taken up 7-AAD. Necrotic cells
are Annexin V−/7-AAD+.
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sFigure 5
50
40
30
20
10
0
CytoD 0.2 M
DOX 1 M
C
*¶
*
*#
*
-
+
-
+
WT
+
+
-
#
*
*
+
-
+
ROCK1-/-
+
+
¶
ROCK1-/(0.2 M CytoD)
ROCK1-/-
Attached cells
(% of control)
Floating cells
(% of total cells)
B
WT
(0.2 M CytoD)
WT
A
120
100
80
60
40
20
0
CytoD 0.2 M
DOX 1 M
*
*
-
+
-
*
*¶
+
+
+
WT
*#
*
-
+
-
+
#
¶
+
+
ROCK1-/-
sFigure 5. Disruption of stress fibers by cytochalasin D facilitates doxorubicin-induced
cell detachment. A. Representative images of rhodamine-phalloidin staining for F-actin of WT
and ROCK1 deficient cells treated with 0.2 M cytochalasin D for 4 h. Bar, 50 m. B and C.
Floating cells and attached cells were separately collected and counted after treatment with 1
M doxorubicin and/or 0.2 M cytochalasin D for 16 h. * P < 0.05 vs. control of the same
genotype.
#
P < 0.05 vs. WT under the same treatment condition. ¶ P < 0.05 vs. the same
genotype under doxorubicin only condition.