Supplementary Figure S1
Fold Enrichment (Lep vs Mock)
0
2
4
6
8
B
A
Mock
**
732
G9a
Negative regulation of
epithelial-mesenchymaltransition
473
Leptin
*
STAT3 47
Positive regulation
of cell differentiation
Cell junction organization
Cell developmental process
STAT3 253 G9a
1804
Cell morphogenesis
611
MiR-200c
C
HEI193.fcs
STAT3
15
HEI193-MDM1.fcs
0
35 0
18
256
G9a
512
768
PE-A
0
0
256
512
H3K9Me2
768
Count Count
Count Count
HEI193.fcs
Mock
29
Leptin
11
40
6
256
512
768
1024
40
PE-A
8
0
0
1024
256
512
0
768
102430
PE-A
PE-A
ch12:
0
HEI193-MDM1.fcs
0
16
10240
7072862
3kb
0
7072862
3kb
E
Fold change of
mRNA level
1.5
Mock
Leptin
1
0.5
**
*
*
*
*
*
Leptin+shSTAT3
Leptin+shG9a
0
miR-200c NUMB GATA3 FOXA2 miR-34a HOXA5
Supplementary Figure S1 Leptin induces STAT3-G9a interaction and their cooccupancy on targeted gene promoters. (A) Venn diagram showing overlap of STAT3 and
G9a bound genes. (B) Gene ontology functional annotation of STAT3 and G 9a bound genes
showing enrichment in specific biological processes. ChIP-seq results for (C) STAT3-G9a cooccupied MiR-200c with enhanced H3K9Me2 (bar positions denote the TSS) and (D) STAT3
and G9a peak distribution around TSS. (E) Fold change of miRNA/mRNA expression in
MCF7 cells that expressed shSTAT3 or shG9a under leptin treatment (n=3, asterisk indicates
P<0.05, double asterisk indicates P<0.01). Error bars denote ±SD.
Supplementary Figure 2
B
A
Con-Vec
3
*
*
*
miR-34a-5p
miR-20b-5p
Let-7f-5p
miR-210-3p
miR-200c-3p
miR-200c
*
miR-125a-5p
*
**
miR-21-5p
*
2
1
D
Con-Vec
Leptin: +
Percentage of CD24CD44+ cells (%)
C
miR-200c
-
+
E-cad
N-cad
Actin
F
Con-Vec
miR-200c
-
+
200
Number of sphere per
1000 cells
E
Leptin:
+
*
miR-124-3p
Fold change of
miRNA
Mock
Leptin
4
0
-
Leptin:
5
150
Mock
40
Leptin
*
30
20
10
0
Con-Vec miR-200c
Mock
Leptin
*
100
*
50
0
Con-Vec miR-200c Con-Vec miR-200c
MCF12A
MCF7
Supplementary Figure S2 Leptin induces EMT and stem cell-like traits via down-regulation
of miR-200c. (A) Genome-wide human miRNA array analysis showing miRNAs with significant
expression changes in MCF12A cells treated with 50 ng/mL leptin for 12 hours (> 2 fold, n=3,
asterisk indicates P<0.05, double asterisk indicates P<0.01). (B) Cell morphological change, (C)
EMT protein expression (E-cadherin and N-cadherin), and (D) the percentage of CD24-CD44+
population in MCF12A cells that stably expressed miR-200c and treated with 50 ng/mL leptin for
3 days (scale bar: 20 mm, n=3, asterisk indicates P<0.05). (E) Representative images of spheres
generated from MCF12A cells, and (F) the number of spheres generated from MCF12A and
MCF7 cells that stably expressed miR-200c and treated with 50 ng/mL leptin for 7 days (scale
bar: 100 mm, n=3, asterisk indicates P<0.05). Error bars denote ±SD.
Supplementary Figure S3
A
B
Leptin:
sh-Vec sh-STAT3
+
+
OBR
sh-Vec sh-G9a
+
Leptin: +
OBR
p-STAT3
G9a
STAT3
Actin
Actin
Supplementary Figure S3 STAT3 and G9a expression is required for
OBR induction by leptin. OBR protein expression levels in MCF7 cells
that stably expressed (A) sh-STAT3 or (B) sh-G9a or the control sh-Vec
under leptin treatment.
Supplementary Figure S4
A
B
Obese
Lean
Lean
Supplementary Figure S4 Distinct tumor phenotypes in Western obese animals
compared to lean animals. (A) Animals in the highest tertile of body fat (determined
by DEXA scan) were placed in the Western-obese group and those in the lowest tertile
placed in the Western-lean group. Rats were then injected with 50 mg/kg MNU. Animals
were palpated by 8 weeks after MNU injection and tumor incidences were monitored.
(B) H&E staining showing aggressive adenocarcinoma (with tumor epithelial cells
invading adjacent muscle tissue, black arrow) is the predominant tumor type in Westernobese animals, while Western-lean animals manifest benign adenoma (with low cuboidal
epithelial cells forming duct-like structures, black arrow). Error bars denote ±SD.
Supplementary Figure S5
A Western-Obese Western-Lean Regular Chow
Western-Obese
Western-Lean
B
Regular Chow
Fold change of
miRNA/mRNA expression
LEP
OBR
p-STAT3
*
*
miR-200c
C
D
DMSO
S3I-201or DMSO treatment
-
d3 d6 d9 d12
S3I-201
d3 d6
d9 d12
Lysate collection
p-STAT3
d1
STAT3
d6
d9
Relative expression
of P-STAT3
d4 d7 d10 d13
d3
E
OBR
1.2
Actin
d12
DMSO
P< 0.01
1
0.8
0.6
0.4
S3I-201
0.2
0
Treatment:
-
d3 d6 d9 d12
d3 d6 d9 d12
Supplementary Figure S5 STAT3 activation and OBR overexpression in diet-induced
obesity rats. (A) Representative leptin (LEP), p-STAT3, and OBR staining, and (B) fold change
of miRNA/mRNA expression in mammary tissues of the obese and lean rats with Western diet,
and the control rats with regular diet (scale bar=100um, n=5 animals/group, asterisk indicates
P<0.05). (C, D) Immunoblots showing p-STAT3 levels in the tumor lysates collected on the
indicated days (in between the days of treatment) from the MNU-treated obese rats with
Western diet treated with S3I-201 or DMSO. (E) Quantification of p-STAT3 expression in (D) by
ImageJ densitometry analysis (relative p-STAT3 level is normalized with total STAT3 and Actin,
n=5 animals/group). Error bars denote ±SD.