Supplemental Figure 1: Chemical structures of fludioxonil and fenhexamid.

1
Fludioxonil
Fenhexamid
Supplemental Figure 1 Chemical structures of
fludioxonil and fenhexamid.
2
23
23
RNU38
RNU48
22
22
21
21
CT value
CT value
20
20
19
19
18
18
17
17
DMSO
100nM Flu
100nM Fen
DMSO
100nM Flu
100nM Fen
22
17
GAPDH
18S
21
16
15
20
CT value
CT value
14
19
13
18
12
17
11
16
DMSO
100nM Flu
100nM Fen
DMSO
100nM Flu
100nM Fen
Supplemental Figure 2 Fludioxonil and
fenhexamid do not affect control genes used in
Q-PCR. MCF-7 cells were serum-starved for 72 h
prior to treatment with DMSO (vehicle control),
fludioxonil (Flu) or fenhexamid (Fen), as
indicated for 6 h. A) RNU38 and B) RNU48 are used
as controls in miRNA Q-PCR. C) GAPDH and D) 18S
rRNA are used as controls in mRNA Q-PCR. Values
are the mean SEM of 9 experiments.
3
Supplemental Figure 3 Fludioxonil and
fenhexamid do not decrease ERa protein in MCF-7
cells. MCF-7 cells were seeded in 6-well plates
and were serum starved for 72 h prior to
treatment with DMSO (vehicle control), 10 nM E2,
100 nM fludioxonil (Flu), 100 nM fenhexamid
(Fen), or 100 nM 4-hydroxytamoxifen (4-OHT, an ER
antagonist) for 48 h. 30 mg protein from whole
cell lysates were separated by 10 SDS-PAGE and
immunoblotted for ERa. Membranes were stripped
and re-probed for b-actin as a loading control.
Immunoreactive bands were quantified and the
ratio of ERa/b-actin normalized to the DMSO
control is indicated.
4
DMSO E2 Flu Fen
150-
PARP 116kDa
PARP 85kDa
75-
a-tubulin
7
6
5
PARP cleavage
4
3
2
1
0
Fen
E2
Flu
DMSO
Supplemental Figure 4 Fludioxonil and
fenhexamid do not stimulate apoptosis in MCF-7
cells. MCF-7 cells were seeded in 6-well plates
and cultured in phenol red-free IMEM 5 DCC-FBS
for 48 h prior to treatment with DMSO (vehicle
control), 10 nM E2, 100 nM fludioxonil (Flu), 100
nM fenhexamid (Fen) for 96 h. 30 mg protein from
whole cell lysates were separated by 10
SDS-PAGE and immunoblotted for PARP. Membranes
were stripped and re-probed for a-tubulin.
Intact (116 kDa) and cleaved (85 kDa) PARP were
measured and the percentage of PARP cleavage was
calculated from the formula PARP cleavage C/
CF 100 where C the 85 kDa cleaved band and F
the full length 116 kDa band.
5
2.0
CCND1
PGR
ESR1
ESR2



1.5



Relative mRNA Expression
1.0














0.5
0.0
DMSO
E2
Flu
Fen
E2 Flu
E2 Fen
Supplemental Figure 5 Fludioxonil and fenhexamid
have antiestrogenic activity in MCF-7 cells.
MCF-7 cells were serum-starved for 48 h and then
treated with DMSO, 10 nM E2, 100 nM fludioxonil,
or 100 nM fenhexamid, alone or in combination, as
indicated for 6 h. CCND1 (cyclin D1), PGR
(progesterone receptor, PR), ESR1 (ER?), and ESR2
(ER?) expression was determined by Q-PCR. Values
are the average of triplicate determinations
within one representative experiment.
Statistical analysis used one way ANOVA followed
by Dunnetts Multiple Comparison Test. P lt 0.05
versus DMSO (control). p lt 0.05 versus 10 nM
E2.
6
16
18S rRNA CT values
15
CT
14
13
12
DMSO
E2
100nM Flu
100nM Fen
E2 Flu
E2 Fen
Supplemental Figure 6 Fludioxonil and
fenhexamid do not affect 18S rRNA control gene
expression in MCF-7 cells. MCF-7 cells were
serum-starved for 72 h prior to treatment with
DMSO (vehicle control), 10 nM E2, or 100 nM
fludioxonil or 100 nM fenhexamid, as indicated
for 24 h. 18S rRNA was used as a control for
RT-Q-PCR for Figure 6. Values are the mean /-
SEM of triplicate determinations.
7
CCND1
2.5
BCL2
MDA-MB-231
2.0



1.5
RelativeExpression
1.0



0.5
0.0
100 nM Flu
10 nM Flu
10 nM Fen
100 nM Fen
DMSO
E2
Supplemental Figure 7 Fludioxonil and fenhexamid
do not increase cyclin D1 expression in
ERa-negative MDA-MB-231 cells. MDA-MB-231 cells
were serum-starved for 48 h and then treated with
DMSO, 10 nM E2, 10 or 100 nM fludioxonil or
fenhexamid, as indicated for 6 h. CCND1 (cyclin
D1), BCL2, PGR (progesterone receptor, PR)
expression was determined by Q-PCR. PGR was not
expressed in MDA-MB-231 (CT values gt 38). Values
are the average of triplicate determinations
within one representative experiment.
Statistical analysis used one way ANOVA followed
by Dunnetts Multiple Comparison Test. P lt 0.05
versus DMSO (control).