Cheng X et al: MKL1 potentiates lung cancer cell migration and invasion by epigenetically
activating MMP9 transcription
Online supplementary material
Supplementary Figures: 6
No treatment
Hypoxia
No treatment
Hypoxia
TGF-
MKL1/DAPI
A
TGF-
MKL1/DAPI
B
Fig.S1: (A) A549 cells were exposed to 1% O2 or TGF- (2ng/ml) treatment for 24 hours.
Nuclear localization of MKL1 was assessed by immunofluorescence staining. Scale bar, 50m
(B) H1299 cells were exposed to 1% O2 or TGF- (2ng/ml) for 24 hours. Nuclear localization
of MKL1 was assessed by immunofluorescence staining. Scale bar, 20m
0.8
0.6
0.4
0.2







H1299
1.2
1
siMKL1#2
1
0
SCR
siMKL1#1
siMKL1#2
1.4
SCR
A549
Relative MKL1 RNA expression
Relative MKL1 RNA expression
1.2
siMKL1#1
A
←MKL1 (145kd)
0.8
A549
0.6
←-actin (42kd)
0.4
←MKL1 (145kd)
0.2
0
SCR
siMKL1#1
siMKL1#2
H1299
←-actin (42kd)







B
0.06
0.15
Weight of nodules (g)
WT
KO
*
Volume of nodules (cm3)
*
0.04
0.02
0.10
0.05
0.00
0.00
WT (N=10)
KO (N=10)
WT (N=10)
KO (N=10)
C
Relative Mkl1 RNA expression
1.2
1
0.8
←Mkl1 (145kd)
0.6
←-actin (42kd)
0.4
SCR shMkl1
0.2
0
SCR shMkl1
D
SCR
shMkl1
Fig.S2: (A) A549 and H1299 cells were transfected with indicated siRNAs. Expression levels of
MKL1 were examined by qPCR and Western. (B) LLC cells were inoculated into wild type or
MKL1 deficient mice as described under Methods. Tumors were surgically removed after
three weeks and weighed. (C) LLC cells were infected with lentivirus carrying indicated
shRNAs. Expression levels of MKL1 were examined by qPCR and Western. (D) In vivo
metastasis assay was performed as described under Methods. H&E staining of lung sections
showing metastasized tumor nodules.
RLU
RLU
1.5
1
A549
H1299
2.5
2.5
2
2
2
1.5
1.5
1.5
*
1
1
*
1
*
0.5
0
1% O2
MKL1 DN
B
H1299
2.5
RLU
A549
2
RLU
A
0.5





0
1% O2
MKL1 DN
0.5





0
TGF-
MKL1 DN


*
0.5



0
TGF-
MKL1 DN





Fig.S3: (A, B) A549 and H1299 cells were transfected with or without dominant negative
MKL1 followed by exposure to 1% O2 (A) or treatment with TGF- (B) for 24 hours. Data are
expressed as relative luciferase unit (RLU).
A
MLL1
7
3
2
Relative enrichment
4
2
1.5
1
0
1% O2 (h)
0
12
24
4
3
2
1
0.5
1
MLL3
5
2.5
5
Relative enrichment
Relative enrichment
6
6
MLL2
3
0
1% O2 (h)
0
12
24
0
1% O2 (h)
0
12
24
B
MLL1
3.5
MLL2
3
3
3
2.5
2.5
2
1.5
1
0.5
0
12
24
Relative enrichment
2
Relative enrichment
Relative enrichment
2.5
0
TGF- (h)
MLL3
3.5
1.5
1
0.5
0
TGF- (h)
0
12
24
2
1.5
1
0.5
0
TGF- (h)
0
12
24
C
MLL1
4
3
2
1




2
1.5
1
0.5
0
SCR
siMKL1
3.5
Relative enrichment
5
0
SCR
siMKL1
MLL2
2.5
6
Relative enrichment
Relative enrichment
7
MLL3
3
2.5
2
1.5
1
0.5




Hypoxia
0
SCR
siMKL1
Hypoxia




Hypoxia
D
3
MLL1
Relative enrichment
Relative enrichment
6
5
4
3
2
2
1.5
1
0.5
1
0
SCR
siMKL1
MLL2
2.5




0
SCR
siMKL1
3.5
Relative enrichment
7
2.5
2
1.5
1
0.5




TGF-
0
SCR
siMKL1
TGF-
TGF-
Hypoxia
control
TGF-
Hypoxia
control
E
←ASH2 (80kd)
←MKL1 (145kd)
input
MLL3
3
IP: -MKL1




TGF-
Fig.S4: (A, B) H1299 cells were exposed to 1% O2 (A) or treated with TGF- (B) and harvested
at indicated time points. ChIP assays were performed with indicated antibodies. (C, D) H1299
cells were transfected with indicated siRNAs followed by exposure to 1% O2 (C) or treatment
with TGF- (D) for 24 hours. ChIP assays were performed with indicated antibodies. (E)
H1299 cells were exposed to 1% O2 or treated with TGF- for 24 hours. Co-IP assays were
performed with anti-MKL1.
A
1.2
←-actin (42kd)
0.2
B
Relative RNA expression
1.2
A549
1.2
1
*
0.8
0.6
*
*
0.4
0.2
0
SCR
siMLL1
siMLL2
siMLL3













←-actin (42kd)
0.2
0
SCR siASH2
C
H1299
1
0.8
*
0.6
*
0.4
0.2
0
SCR
siMLL1
siMLL2
siMLL3
Hypoxia
*













A549
H1299
1.2
1.2
1
1
*
0.8
0.6
*
*
0.4
0.2
0
SCR
siMLL1
siMLL2
siMLL3
Hypoxia
D













0.8
0.6
*
*
*
0.4
0.2
0
SCR
siMLL1
siMLL2
siMLL3













TGF-
TGF-
E
3
*
1.5
1
0.5
0
SCR
siASH2
2.5




Hypoxia
*
1
0.5
0
SCR
siASH2
3.5
H3K4Me3
2
2
1.5
H3K4Me2
3
2.5
2
Relative enrichment
2.5
H3K4Me3
1.5
*
1
0.5
Relative enrichment
H3K4Me2
Relative enrichment
3
Relative enrichment
←ASH2 (80kd)
0.4
SCR siASH2
Relative RNA expression
0
0.6
Relative RNA expression
←ASH2 (80kd)
0.4
0.8
siASH2
0.6
H1299
1
SCR
SCR
0.8
siASH2
1
Relative RNA expression
A549
Relative ASH2 RNA expression
Relative ASH2 RNA expression
1.2
2.5
2
1.5
*
1
0.5




Hypoxia
0
SCR
siASH2




TGF-
0
SCR
siASH2




TGF-
Fig.S5: (A) A549 and H1299 cells were transfected with indicated siRNAs. Expression of ASH2
was measured by qPCR and Western. (B, C) A549 and H1299 cells were transfected with
indicated siRNAs followed by exposure to 1% O2 (B) or treatment with TGF- (C) for 24 hours.
Expression levels of MMP9 were measured by qPCR. (D, E) H1299 cells were transfected with
indicated siRNAs followed by exposure to 1% O2 (D) or treatment with TGF- (E) for 24 hours.
ChIP assays were performed with indicated antibodies.
A
Relative Ash2 RNA expression
1.4
1.2
1
0.8
←Ash2 (80kd)
0.6
←-actin (42kd)
0.4
SCR
shAsh2
0.2
0
SCR shAsh2
B
SCR
shAsh2
C
D
8
MMP-9 mRNA
Relative RNA expression
p=.0215
SCR
siAsh2
MMP9 (92kd)
-actin (42kd)
6
4
2
0
SCR (N=6)
siAsh2 (N=6)
Fig.S6: (A) LC cells were infected with lentivirus carrying indicated shRNAs. Expression levels
of ASH2 were examined by qPCR and Western. (B) In vivo metastasis assay was performed as
described under Methods. H&E staining of lung sections showing metastasized tumor
nodules. (C, D) Heterotopic xenographt assay was performed as described under Methods.
Tumors were dissected and MMP9 expression was examined by qPCR (C) and Western (D).