Liu, Y et al
Supplementary Information
Supplemental figure legend
Supplemental figures
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Liu, Y et al
Supplemental figure legend
Fig. S1 siRNA knockdown of BRMS1 promotes lung cancer cells invasion. H1299 lung
cancer cells were transfected with siRNA control or BRMS1. Post-transfection 48 hrs, the
invasion capability was quantified as described. Bar graphs show average cell counts. Data are
presented as mean ± S.D.
Fig. S2 RelA/p65 mediates TNF-induced BRMS1 methylation. (A) TNF induces BRMS1
methylation. The relative methylation of BRMS1 was analyzed by quantitative MSP in NHBE
cells stimulated with TNF at the indicated doses for 4 hrs. (B) H157 V/I cells were treated with
or without 5-Aza (5µM) for 4 days. Bisulfite sequencing PCRs were performed in three
independent experiments to detect the methylated CG dinucleotides in the BRMS1 promoter.
Each CpG dinucleotide bar from the schematic is depicted by a circle, the fill pattern of which
indicates the methylated cytosine and the blank indicated the unmethylated cytosine in CpG
dinucleotide.
Fig. S3
NF-B binds to the -B binding sites on the BRMS1 promoter. (A) EMSAs
performed using P33-labeled wild-type or mutant -B binding sites as probe incubated with
NHBE cells nuclear extracts. (B) TNF enhances NF-B binding to the NF-B sites I and II.
NHBE cells were treated with TNF (40ng/ml) for 15 min. EMSAs performed as described in (B).
The relative expression of RelA/p65 in nuclear (NE) and cytoplasmic (CE) extracts was
evaluated by Western blot. β-actin and RNA pol II were probed as controls for cytoplasmic and
nuclear protein, respectively.
Fig. S4 DNMT-3b participates in RelA/p65 mediated methylation and transcriptional
repression of the BRMS1 promoter. (A) RelA/p65 enhances DNMT-1 and -3b mediated
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Liu, Y et al
BRMS1 transcriptional repression. p65-/- MEFs cells were co-transfected with BRMS1-Luc.
reporter and expression vector encoding Flag-tagged RelA/p65, myc-tagged DNMT-1, -3a, -3b
or control. Luciferase activity was determined. (B) ChIP analysis was performed at the indicated
time points in H157 V/I cells treated with 5-Aza as described above by quantitative PCR specific
to the BRMS1 promoter, as well as GAPDH promoter. (C) RelA/p65 induces chromatinassociated DNMT-1 and -3b. 293T cells were treated with TNF (20ng/ml) and harvested at
indicated time points. ChIP analysis was performed across the BRMS1 promoter. The cIAP2 and
GAPDH promoters were examined as controls.
Fig. S5 The PTD-p65 (38-47) peptide enhances BRMS1 expression (A) Mutation of E39
abolishes The PTD-p65 (38-47) peptide binding to the BRMS1 promoter. In vitro EMSAs were
performed using indicated GST-PTD peptides incubated with
32
P-labeled -B binding site II of
BRMS1 promoter as the probe. A supershift band was produced by adding antibody against GST.
(B) The PTD-p65 (38-47) peptide increases BRMS1 protein expression in NSCLC. BRMS1
protein levels were analyzed by Western blot in H157 cells treated with the indicated PTD
peptides (5g/ml) for 24 hrs. (C) H157 cells were treated with PTD peptides at indicated doses
for 24 hrs. The mRNA levels of cIAP2, Bfl1/A1 and IB were determined by QRT-PCR. (*)
p<0.05 compared to PTD. (D) H157 cells were treated with 5-Aza as described previously.
Following 5-Aza removal, cells were treated with indicated peptides (5µg/ml) and TNF
(20ng/ml) for additional 24 hrs. ChIP analysis was performed across the BRMS1 and GAPDH
promoter.
Fig. S6 Mutating p65 (109-120) peptide failed to block the interaction of RelA/p65 and
DNMT-1. (A) HEK 293T cells were treated with PTD alone, PTD-p65 (109-120) wild type
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Liu, Y et al
peptide or mutant peptide (5µg/ml) for 24 hrs. IPs were performed using anti-DNMT-1 antibody
and the presence of RelA/p65 was detected by Western blots. (B) NSCLC cells were treated with
the PTD alone, PTD-p65 (109-120) wild type peptide or mutant peptide (5g/ml) for 24 hrs. The
mRNA of BRMS1 was analyzed by quantitative RT-PCR.
Fig. S7 The interaction of RelA/p65 and DNMT-1 is HDAC independent. H157 cells were
transfected shRNA HDAC-1, -2, -3 or a scramble shRNA as control. Immunoprecipitations were
performed using antibody against DNMT-1 and the presence of RelA/p65 was detected by
Western blot.
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Liu, Y et al
siRNA
Cont.
BRMS1
Cell number (x100)
SD Fig. 1
10
8
6
4
2
0
p=0.002
siRNA
BRMS1
β-actin
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Liu, Y et al
SD Fig. 2
Relative methylation
(Fold over TNF 0ng/ml)
A
10
8
6
4
2
0
0 20 40
TNF (ng/ml)
B
H157V
H157I
Cont.
5-Aza
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Liu, Y et al
SD Fig. 3
A
NF-B
BRMS1-B
I
II
III
B
NF-B
BRMS1-B
I
II
III
TNF
CE NE CE NE
RelA/p65
β-Actin
RNA polII
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Liu, Y et al
SD Fig.4
BRMS1-promoter activity
(RLUX10000)
A
3.0
CMV
p65
2.5
2.0
1.5
1.0
0.5
0
DNMT1
DNMT3a
DNMT3b
B
Percent of Input
BRMS1 promoter
50
40
30
20
10
0
DNMT3a
DNMT3b
50
40
30
20
10
0
0 12 24 36 48
0 12 24 36 48
Percent of Input
Percent of Input
Removal of 5-Aza (hrs)
50
40
30
20
10
0
50
40
30
20
10
0
GAPDH promoter
RelA/p65
50
40
30
20
10
0
Removal of 5-Aza (hrs)
p50
50
40
30
20
10
0
0 12 24 36 48
Removal of 5-Aza (hrs)
DNMT3a
50
40
30
20
10
0
0 12 24 36 48
Removal of 5-Aza (hrs)
DNMT3b
0 12 24 36 48
Removal of 5-Aza (hrs)
C
I
V
DNMT3a
DNMT3b
SR-IkB
-tubulin
BRMS1
TNF (min) 0 15 30 60120
RelA/p65
Meth-H3 K9
DNMT1
50
40
30
20
10
0
0 12 24 36 48
Removal of 5-Aza (hrs)
Ac-H3
Meth-H3K9
0 12 24 36 48
Removal of 5-Aza (hrs)
GAPDH
0 15 30 60120
0 15 30 60120
IgG
8
DNMT1
0 12 24 36 48
0 12 24 36 48
Removal of 5-Aza (hrs) Removal of 5-Aza (hrs)
cIAP2
DNMT3b
Input
50
40
30
20
10
0
Liu, Y et al
SD Fig.5
GST-PTD-p65
A
B
BRMS1
GST-PTD
-tubulin
5 2.5 0.5 +Ab
GST-PTD peptides
C
Relative mRNA
(Fold of induction)
1.5
cIAP2
1.0
2.0
*
*
0.5
Bfl1/A1
2.0
1.5
1.5
1.0
1.0
*
0.5
0
1
2
5
PTD peptides(ug)
PTD
PTD/p65(38-47)
*
0.5
0
0
0
IB
0
1 2
PTD peptides(ug)
0
5
1
2
5
PTD peptides(ug)
D
BRMS1 promoter
Percent of Input
50
40
30
20
10
DNMT3b
No add
TNF
TNF
GST-PTD
-tubulin
0
PTD
0
0
38-47
12
12
Removal of 5-Aza (hrs)
Removal of 5-Aza (hrs)
GAPDH promoter
Percent of Input
RelA/p65
DNMT1
DNMT3b
Meth-H3K9
GST
50
40
30
20
10
50
40
30
20
10
50
40
30
20
10
50
40
30
20
10
50
40
30
20
10
0
0
0
0
0
0
12
0
12
0
12
0
12
Removal of 5-Aza (hrs) Removal of 5-Aza (hrs) Removal of 5-Aza (hrs) Removal of 5-Aza (hrs)
9
0
12
Removal of 5-Aza (hrs)
Liu, Y et al
SD Fig.6
B
H157
PTD-p65
(109-120)
IP:DNMT1
PTD WT Mut.
H1299
PTD-p65
(109-120)
PTD WT Mut.
RelA/p65
Input
DNMT1
RelA/p65
Relative BRMS1 mRNA
A
25
20
PTD
p65 (109-120)
p65 (109-120) Mut.
15
10
5
0
H157 H1299
10
Liu, Y et al
SD Fig. 7
siRNA
Cont.
1
HDAC
2
3
p65
IP:DNMT1
Input
shRNA
HDAC1
p65
DNMT1
-actin
HDAC2
p65
DNMT1
-actin
11
HDAC3
p65
DNMT1
-actin