ONLINE SUPPLEMENTARY MATERIALS AND METHODS
Histological analysis
After fixed in 4% formalin and embedded in paraffin, tissues were cut into 4-μm
sections. H&E staining were performed following the conventional protocol [1].
Immunohistochemistry (IHC) staining was performed using rabbit antibodies for DLC3
(Proteintech, Wuhan, China), MACC1 (Abnova, Taipei, China) and Ki-67 (Cell
signaling technology, Danvers, MA) and stained by Dako Envision System (Dako,
Glostrup, Denmark). The protein expressions were analyzed by a semi-quantitative
method [1]. The intensities of the protein were scored as 0 (negative), 1 (weak), 2
(medium) or 3 (intense), while the staining extent were scored as 0 (0%), 1 (1%-25%),
2 (26%-50%), 3 (51%-75%) or 4 (76%-100%) according to the stained cells in
percentage. Multiplying the staining intensity and extent scores together gets the final
protein expression score. Western blotting and Immunofluorescence
For western blotting, Goat Anti-Rabbit antibodies of DLC1 (Abcam, Cambridge, MA,
US), DLC3 (Proteintech), MACC1 (Abnova), JNK (Abcam), phosphorylated-
JNK1/2/3 at sites Y185/Y185/Y223 (Abcam), c-JUN (Cell Signaling Tech., Danvers,
MA, US), phosphorylated-c-JUN at site S63 (Cell Signaling Tech.), E-cadherin (Cell
Signaling Tech.), vimentin (Cell Signaling Tech.) and GAPDH (Cell Signaling Tech.)
were used. For immunofluorescence, antibodies of DLC3 (Proteintech) and MACC1
(Abcam), E-cadherin (Cell Signaling) and vimentin (Cell Signaling) were used.
RhoA activity assay
RhoA activity was measured using a RhoA activation assay kit (cytoskeleton, Denver,
OH). Briefly, freshly prepared cell lysates of 800 μg total protein were incubated with
50 μg rhotekin-RBD beads at 4℃ on a rotator for 1h. After centrifugation, supernatant
removal and carefully washing, the bead pellet was suspended in 2× Laemli buffer and
boiled for 2 min. Then, the prepared samples were subjected for Western blotting using
the anti-RhoA antibody included in the kit. Total RhoA expressions were also blotted
as reference.
Protein array assay
To find out DLC3 downstream signaling, protein array assay was performed using
Human Phospho-kinase array kit (R&D Systems, Minneapolis, MN) and following the
manufacturer's protocol. qRT-PCR
Trizol kit (Life science, Carlsbad, CA) for cells and tissues total RNA extraction and a
reverse transcriptase (Roche, Penzberg, Germany) were used according to the protocols
as recommended. qRT-PCR was performed using SYBR Green I Master kit (Roche) on
a LightCycler 480 system. Primer sequences involved in present study are listed in
online supplementary Table 1.
Dual-luciferase reporter assay
According to the documented functional binding site for AP-1 in the MACC1
promoter [2], mutated MACC1 reporter fragments was generated (see online
supplementary Table 2). To detect the influence of DLC3 on the MACC1 transcription,
MACC1 and its corresponding mutated reporter fragments were cloned into the
luciferase pGL3 vector. Luciferase activities of each groups was detected using Dual-
Luciferase Reporter Assay System (Promega, Madison, WI, US) according to the
manufacturer’s instructions. ROS detection and cell apoptosis assays
ROS was measured by fluorescent DCFH-DA dye (Jiancheng, Nanjing, China) and
observed under fluorescent microscope after 6 hours glucose deprivation. Cell
apoptosis was analyzed by staining Annexin V-FITC and Propidium Iodide after 12
hours glucose deprivation, and detected by flow cytometry analysis using the
FACSCanto II system (BD Biosciences, San José, CA).
Cell viability, proliferation and colony formation assays
Cells viability was measured by methyl-thiazolyl-tetrazolium (MTT) assay as
previously described [3]. Cell proliferation ability was reflected by EdU incorporation
assay using the EdU Assay Kits (Life Technologies, Carlsbad, CA, USA) according to
the manufacturer's instruction and observed under fluorescent microscope. For colony
formation, trypsinized cells were suspended in 2 mL of complete medium (Sigma, St.
Louis, MO, US) in complete medium. After 14 days culturing, cells were observed by
crystal violet. Migration, invasion and glucose chemotaxis assays
For scratch healing assay, trypsinized cells were seeded on 24-well culture plates
(5×104/well). Upon confluence, cells were scratched with a sterile 200 μL pipette tip.
Wound closure was observed at indicated time point. For Boyden chamber transwell
assay, cells were planted in serum free medium in the upper chamber, which consisted
of 8 μm membrane filter inserts (5×104/ml, 200 μL/well) with Matrigel. The lower
chamber was complete medium with serum as chemoattractant. After 48 hours culturing,
cells at upper chamber were fixed by fixed in 4% paraformaldehyde and stained by
crystal violet. Cells were photographed under microscope. For glucose chemotaxis
assay, cells were seeded on the upper chamber coated with Matrigel (BD Biosciences)
and incubated with RPMI 1640 glucose free medium, while the lower chamber was
supplemented with RPMI 1640 high glucose medium (2 g/L) as attractant. Cells were
cultured for 48 hours before crystal violets staining.
Glucose uptake and pH evaluation
Cells were seeded on 6-well plates (5×105/well) and cultured in 2 ml complete medium.
The 24-hour conditioned medium and the original medium (baseline control) were
collected. Glucose uptake was determined using a glucose assay kit by conventional
enzymatic methods (Randox, Antrim, UK). Total protein of cells was detected by
conventional BCA method (Beyotime, Haimen, China). The pH value was detected by
a pH meter.
Reference 1. Wang L, Wu Y, Lin L, Liu P, Huang H, Liao W, et al. Metastasis-associated in colon cancer-1 upregulation predicts a poor prognosis of gastric cancer, and promotes tumor cell proliferation and invasion. International Journal of Cancer. 2013; 133: 1419-30. 2. Juneja M, Ilm K, Schlag PM, Stein U. Promoter identification and transcriptional regulation of the metastasis gene MACC1 in colorectal cancer. Mol Oncol. 2013; 7: 929-43. 3. Wang L, Lin L, Chen X, Sun L, Liao Y, Huang N, et al. Metastasis-associated in colon cancer-1 promotes vasculogenic mimicry in gastric cancer by upregulating TWIST1/2. Oncotarget. 2015; 6: 11492-506.
OnlineSupplementary
Figures and Tables
Supplementary Figure S1 DLC3 is associated with perfusion status in tumor microenvironment, andDLC3 low expression indicates poor cancer prognosis.(A) Quantified of the RhoA activity changes upon glucose deprivation by three independent repeats. (B)Using TCGA GC tissue data, the mRNA expressions of DLC family members in correlation with CD31, andthree representative GEF members (TRIO, ARHGEF1 and MCF2L) were analyzed. Angiogenic factorVEGFA in correlation with DLC3 was also analyzed. (C and D) Using online Kaplan-Meier plotter forbioinformatic analysis, DLC3 downregulation was associated with poor survival in (C) breast cancer(GSE42568) and (D) lung cancer (CaArray) respectively.
0 40 80 1200
20
40
60
80
100 DLC3 lowDLC3 high
Time (month)
Recu
rren
ce F
ree
(%)
n=52
n=52
0 40 80 1200
20
40
60
80
100 DLC3 lowDLC3 high
Time (month)
Ove
rall
Surv
ival
(%)
n=52
n=52
Breast Cancer (GSE42568)
0 60 120 180 2400
20
40
60
80
100 DLC3 lowDLC3 high
n=246
n=122
Time (month)Free
of p
rimar
y pr
ogre
ssio
n (%
)
0 60 120 180 2400
20
40
60
80
100 DLC3 lowDLC3 high
n=302
n=302
Time (month)
Ove
rall S
urviv
al (%
)Lung Cancer (CaArray)
C D
0 1 2 3 4 50
2
4
6
8 r2=0.4387p<0.001
CD31
DLC
1
0 1 2 3 4 50
2
4
6
8
10 r2=0.1796p<0.001
CD31
DLC
2
0 1 2 3 4 50
1
2
3
4
5 r2=0.6203p<0.001
CD31
DLC
3
A
0 1 2 3 4 50
2
4
6 r2=0.0333p<0.001
CD31
TRIO
0 1 2 3 4 50
2
4
6 r2=0.0044p=0.199
CD31
ARHG
EF1
0 1 2 3 4 50
2
4
6 r2<0.0001p=0.907
CD31
MC
F2L
0 1 2 3 4 50
2
4
6
8 r2=0.0013p=0.4933
DLC3
VEG
FAB
+ -0
1
2
3
*
Glucose
Activ
e Rh
oA/T
otal
Rho
A
Supplementary Figure S2 Constructing DLC3-overexpression and -silenced GC cell models.
DLC3 was successfully (A) overexpressed and (B) silenced in MKN45 cells. (C) Correspondingly, RhoAactivities were altered by DLC3. (D) Glucose deprivation did not influence DLC3 expression inoeDLC3 cells.
BCtrl oeDLC3
GAPDH
DLC3
A
120kDa
36kDa
C
GAPDH
DLC3shN -#1 -#2
shDLC3
120kDa
36kDa
RhoA-GTP 21kDa
Total RhoA 21kDa
shN -#1 -#2shDLC3
D
RhoA-GTP 21kDa
Total RhoA 21kDa
Ctrl oeDLC3
0 1 3 6GAPDH 36kDa
DLC3 120kDa
Glucose deprivation time (h)12
shDLC3■-#1 ■-#2■shN
BGC
823
0
100
200
300
400 +
#
Col
ony
num
ber
0
50
100
150
+
Col
ony
num
ber
□oeDLC3■Ctrl
BGC
823
BGC823
■Ctrl
□oeD
LC3
EdU Hoechst Merge
0
10
20
30
40
+
EdU/
Hoec
hst (
%)
A B
C
0.0
0.4
0.8
1.2
+
Cel
l via
bility
(fol
d)
■Ctrl□oeDLC3
BGC823
0
500
1000
1500 +
#
Inva
ded
cells
BGC823
shDLC3■-#1 ■-#2■shN
0
500
1000
1500
*
Inva
ded
cells
□oeDLC3
BGC823
■Ctrl
D
shDLC3
shN
BGC823
0h 6h 12h 24h
-#1
-#2
BGC823
0h 6h 12h 24h
Ctrl
oeDLC3
E
Supplementary Figure S3 DLC3 suppressed proliferation and migration in BGC823 cells.
(A) The viability and (B) DNA replication of BGC823 were suppressed by DLC3 overexpression. (C)DLC3 influenced colony formation of BGC823. (D) The migration and (E) invasiveness was inhibitedby DLC3 overexpression and enhanced by its silencing. *P<0.05, #P<0.01, +P<0.001.
Supplementary Figure S4 DLC3 inhibited MACC1 expression via RhoA/JNK/AP-1 signaling inMKN45 cells.(A) OeMACC1 did not influence DLC3 expression, but oeDLC3 significantly downregulated MACC1expression. OeMACC1 reversed the epithelial mesenchymal transition marker alterations inducedby oeDLC3 in MKN45 cells. Right panel: semi-quantified MACC1 relative gray values (n=3). (B and C)Glucose deprivation upregulated (B) MACC1 protein and (C) mRNA expressions via DLC3 in MKN45cells. (B) Right panel: semi-quantified MACC1 relative gray values (n=3). (D) In protein array assay,shDLC3 enhanced the phosphorylation of JNK and several other protein kinases. (E) Semi-quantifiedMACC1 relative gray values of shDLC3 and corresponding control (n=3). (F and G) RhoA, ROCK orJNK inhibitors suppressed shDLC3-promoted MKN45 (F) colony formation and (G) invasion.Quantified results related to Figure 6G and H.
A
B
JNK1/2/3EGFR
TORSrcFynHck
p53AMPKα1
HSP27Yes
MSK1/2
Lyn
shN
shDLC3-#2
oeDLC3 - - + +oeMACC1 - + - +
DLC3MACC1
E-cadherinvimentinGAPDH
120kDa
36kDa
97kDa135kDa57kDa
Glucose + - + -oeDLC3 - - + +
DLC3
MACC1GAPDH
120kDa
36kDa
97kDa
D
0
1
2
3 *
GlucoseoeDLC3
+ - + -- - + +
*
*
MAC
C1
mRN
A(fo
ld to
Ctrl
)
0
12
34
5#
#
oeDLC3oeMACC1
- - + +- + - +
*ns
MAC
C1
gray
val
ue
0.0
0.5
1.0
1.5
2.0 *
GlucoseoeDLC3
+ - + -- - + +
*
*
MAC
C1
gray
val
ue
F
G
E
0100
200
300
400
500
shDLC3RhoAi
- + - +- - + +
- +- -
ROCKi - - - - + +
P valuesCtrl vs.. shDLC3Ctrl vs. RhoAiCtrl vs. ROCKiCtrl vs. JNKiCtrl vs. shDLC3+RhoAiCtrl vs. shDLC3+ROCKiCtrl vs. shDLC3+JNKishDLC3 vs. RhoAishDLC3 vs. shDLC3+RhoAishDLC3 vs. ROCKishDLC3 vs. shDLC3+ROCKishDLC3 vs. JNKishDLC3 vs. shDLC3+JNKiRhoAi vs. shDLC3+RhoAiROCKi vs. shDLC3+ROCKiJNKi vs. shDLC3+JNKi
<0.01<0.001<0.001<0.001<0.001<0.01<0.01<0.001<0.001<0.001<0.001<0.001<0.001<0.05<0.05<0.05ROCKi - - - - - -
- +- -- -+ +
Col
ony
num
ber
0
100
200300
400
500
shDLC3RhoAi
- + - +- - + +
- +- -
ROCKi - - - - + +
P valuesCtrl vs.. shDLC3Ctrl vs. RhoAiCtrl vs. ROCKiCtrl vs. JNKiCtrl vs. shDLC3+RhoAiCtrl vs. shDLC3+ROCKiCtrl vs. shDLC3+JNKishDLC3 vs. RhoAishDLC3 vs. shDLC3+RhoAishDLC3 vs. ROCKishDLC3 vs. shDLC3+ROCKishDLC3 vs. JNKishDLC3 vs. shDLC3+JNKiRhoAi vs. shDLC3+RhoAiROCKi vs. shDLC3+ROCKiJNKi vs. shDLC3+JNKi
<0.001<0.001<0.001<0.001<0.01<0.001<0.05<0.001<0.001<0.001<0.001<0.001<0.001<0.05<0.05<0.01ROCKi - - - - - -
- +- -- -+ +
Inva
ded
cells
C
shN shDLC30.0
0.5
1.0
1.5
2.0 #
MAC
C1
gray
val
ue
Supplementary Figure S5 The DLC3/MACC1 axis modulated glycolysis remodeling.(A) Indicated by EdU staining, siMACC1 significantly reversed shDLC3 accelerated DNA-replication inMKN45 cells. (B) The DLC3/MACC1 axis regulated cellular 18F-FDG uptake in culturing plates. (C)Silencing MACC1 reduced glucose uptake in shDLC3 MKN45 cells. (D) The DLC3/MACC1 axisinfluenced acidosis in the conditioned medium. (E) SiMACC1 further increased glucose deprivation-promoted ROS generation. #P<0.01, +P<0.001.
- +shDLC3
siM
ACC
1 -
+
EdU/Hoechst0
10
20
30
40
50+
shDLC3siMACC1
- + - +- - + +
+ +E
dU/H
oech
st (%
)
A
7.0
7.2
7.4
7.6
pH v
alue
shDLC3 +- - +siMACC1 -- + +
siMACC1shDLC3 - -+ +
- +- +
Low High
B
C D E
0
100
200
300
400 +
shDLC3siMACC1
- + - +- - + +
+
+
Glu
cose
/Pro
tein
(mm
ol/g
)
- +siMACC1
Glu
cose
+
-
DCFH-DA/Bright field
Supplementary Figure S6 The DLC3/MACC1 axis modulated GC cell invasion and glucosechemotaxis.(A and B) The DLC3/MACC1 axis modulation on MKN45 cell motility was confirmed by (A) scratchhealing migration assay and (B) transwell invasion assays. (C) DLC3/MACC1 axis modulation onepithelial and mesenchymal marker expressions were confirmed by immunofluorescence. (D and E)Silencing MACC1 (D) reversed the glucose-deprivation-induced cytoskeleton changes and (E)inhibited glucose chemotaxis in MKN45 cells. *P<0.05, #P<0.01, +P<0.001.
- +shDLC3
siM
ACC
1 -
+
siMACC1shDLC3 - - + +
- + - +
E-ca
dher
inD
LC3
MAC
C1
vim
entin
50μm
050
100150
200
250 ++
shDLC3siMACC1
- + - +- - + +
+
+
Inva
ded
cells
A
siMACC1shDLC3
0h
++1
-+1
+-1
--1
48h
0.660.730.440.58
B
D
E
C
0 100 200 300 400
#
++
GlucosesiMACC1
+ -
- -
+ +
- +
Invaded cells
Ctrl Glc(-)Glc(+)
Ctrl Glc(+)Glc(+)
siMACC1
siMACC1
+
-
-siMACC1
+
20μm
Glu
cose
CTTCAGCTCTGAATCACCGAAAGAGAATCTCTTCAGCTCTGAATTGCCGAAAGAGAATCT
MACC1mutated
AP-1
Supplementary Table S1 The qRT-PCR primer sequences used in this study
Target SequenceMACC1 F GAGTTAGTCGCACGTCTCATCC
R AGTGAGCACTCCAGGTATACAGDLC3 F GACGGAGCAGTCCCTCCT
R CTTTTGGCCTCGGCTTCTβ-actin F TGGCACCCAGCACAATGAA
R CTAAGTCATAGTCCGCCTAGAAGCA
Supplementary Table S2 The functional binding site for AP-1 in MACC1 promoter
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