celastrol inhibits ezrin-mediated migration of ...10.1038... · a. the coomassie brilliant blue...
TRANSCRIPT
Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells
Shihao Du1, 2, 5, Xiaoyu Song3, 5, Yuan Li2, 5, Yalei Cao1,5, Fuhao Chu1, Olanrewaju Ayodeji Durojaye3, Zeqi Su1, Xiaoguang Shi2, Jing Wang2, Juan Cheng2, Tangshun Wang2, Xiang Gao2, Yan Chen1,2, Wuzhekai Zeng3, Fengsong Wang4, DongMei Wang3, Xing Liu3, Xia Ding1,2,*
1 School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China 100029; 2 Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China 100700; 3 MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics & Hefei National Center for Physical Sciences at the Microscale, University of Science & Technology of China, Hefei, China 230027; 4 School of Life Science, Anhui Medical University, Hefei, China 230032.
5 Equal contribution
*Correspondence: [email protected]
Supplemental Figure 1
B
A
Figure S1A. The inhibitory effect of several TCM monomers (1μM) on MHCC97H cell motility were tested by wound
healing assay. The wound healing ratio (%) of 8 hours were calculated. B. MHCC97H cells were treated with 2‰ DMSO and different concentration of celastrol (5 nM, 10 nM, 50
nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM) for 24h before MTS assay, the cell viability was calculated by dividing the OD of each group by OD of the control group.
C. HepG2 cell were treated in the same way as above. The error bars represent S.E.; n = 3 preparations, *P<0.05, **P < 0.01.
C
60
100
20
60
100
20 Cel
l via
bilit
y (%
) C
ell v
iabi
lity
(%)
10 100 1000
Celastrol, nM10000
60
100
20
10 100 1000
Celastrol, nM10000
Du et al., W
ound
hea
ling
(%)
Supplemental Figure 2
B
A
Figure S2A. Wound healing area of Huh7 cell. Images were collected before and 8 hours after 20% fetal calf
serum addition. 2‰ DMSO were added to the Control group and 500 nM celastrol were added to the treatment group. Bar = 100 μm.
B. Wound healing area of HepG2 cell.
0 h 8 h
DM
SOC
elas
trol
Huh7
HepG2
DM
SOC
elas
trol
0 h 8 h
Du et al.,
Supplemental Figure 3
B
A
Figure S3A. The flowchart shows the synthesis process of celastrol-biotin. B. The mass spectrometry identified the celastrol-biotin (peak 719.4204).
Celastrol-biotin
HOBT/EDCI/DIPEADry DMF
0.6
0.2
1.0
×106
Counts vs. Mass-to-Charge (m/z)
Du et al.,
Supplemental Figure 4
BA
Figure S4A. The coomassie brilliant blue (CCB) stain result shows the protein pulled down by celastrol-biotin.
The MHCC97H cell lysates were treated with 10 μM celastrol-biotin and equal volume of DMSO (-), and then incubated with avidin-beads.
B. Western-blotting result of the co-immunoprecipitation assays. HEK293T cells transfected with Flag-ROCK2 and GFP-ezrin were lysed and incubated with FLAG M2 beads, followed by the western blotting with anti-GFP and anti-FLAG antibodies. The transfected cells were treated with 500 nM celastrol for 6 hours, while the control group were treat with equal volume of DMSO. The cells transfected with Flag-ROCK2 and GFP vector were negative control. Full-length blots are presented in Supplementary Figure S8.
CBBStaining
Mr(K)
1 2
+ - Celastrol-Biotin
Immuno-Precipitation:
FLAG Antibody
1 2 3 4 5 6
GFP blotting
GFP-ezrin
IgG (HC)
FLAG-ROCK2
Input IP
GFP
GFP-ezrin +CelastrolGFP-ezrin
FLAG-ROCK2
+ - - + - - - + - - + - - - + - - + + + + + + +
Input:GFP-ezrin
Mr(K)
116-97-
66-
44-
29-
Du et al.,
130-180-
70-
55-
40-
35-
100-
25-
200-
Residues Distance (A0)
MET-172ASP-218ASP-232
2.45.62.8
A
B
Supplemental Figure 5
Figure S5A. Cartoon representation of Y27632 (green) docked onto the ROCK2 model. Potential area of Y27632
interacted with the residues of ROCK2 were shown in yellow dotted lines. The numbers represent the distance between each residue and Y27632.
B. The amino acid residues of ROCK2 that formed polar bonds with Y27632 and the distance between each residue and Y27632.
Du et al.,
A
B
Supplemental Figure 6
Figure S6A. Western blotting analyses of ezrin pT567, ezrin and Tubulin, after treating Huh7 cells with different
concentration of celastrol for 6h. Full-length blots are presented in Supplementary Figure S8.B. Western blotting analyses of ezrin pT567, ezrin and Tubulin, after treating HepG2 cells with different
concentration of celastrol for 6h. Full-length blots are presented in Supplementary Figure S8.
Du et al.,
Mr(K)
ezrin
Tubulin
Celastrol, nM 0 5 50 500 5000Mr(K)
1 2 3 4 5
80- ezrin pT567
80-
50-
ezrin
Tubulin
Celastrol, nM 0 5 50 500 5000
1 2 3 4 5
80- ezrin pT567
50-
80-
Supplemental Figure 7
Figu
re S
7O
ncop
rint v
isua
lizat
ion
of c
linic
al a
nd g
enom
ic fe
atur
es o
f 442
TC
GA
hepa
toce
llula
r car
cino
ma.
Clin
ical
info
rmat
ion
for t
his
exam
ple
incl
udes
mut
atio
n sp
ectru
m, o
vera
ll su
rviv
al a
nd s
ex. R
OC
K2 g
enom
ic fe
atur
es a
re v
isua
lized
, inc
ludi
ng m
utat
ions
, cop
y nu
mbe
r am
plifi
catio
ns a
nd m
RN
A. In
divi
dual
col
umns
are
for i
ndiv
idua
l pat
ient
s. P
atie
nts
with
the
indi
cate
d al
tera
tions
are
sho
wn.
Du et al.,
Supplemental Figure 8Figure 2 B Figure 3 A
80- ezrin
Du et al.,
50-
Figure 4 E
180-
Mr(K)
180- ROCK2
50- Tubulin
80-
Mr(K)
ezrin pT567
Figure S6B
ezrin pT567Mr(K)
Mr(K)
50-
Tubulin
Figure S4B
116-
200-
66-
29-
97-
44-
Mr(K)FLAG-ROCK2
50- Tubulin
ezrin pT567Mr(K)
80-
Figure 3 B
130-
180-
70-
100-
80-
80-
ezrin
Figure 2 E
50-
180-
Tubulin
Mr(K)
Flag-ROCK2
Figure S6A
Tubulin
ezrin pT56780-
80- ezrin
** non-specialMr(K)
80- ezrin
Figure 4 A
Mr(K)
80-
200-
116-97-
66-
44-
29-
ezrin pT567
Flag-ROCK2
His-ezrin
Figure 4 B
Mr(K)
80-ezrin pT567
29-
44-
66-
97-116-
200- Flag-ROCK2
His-ezrin
Tubulin
ROCK2 pS1366
50- Tubulin
ROCK2Mr(K)