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Supplemental Figure 1: Chemical structures of fludioxonil and fenhexamid. FludioxonilFenhexamid.

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Presentation on theme: "Supplemental Figure 1: Chemical structures of fludioxonil and fenhexamid. FludioxonilFenhexamid."— Presentation transcript:

1 Supplemental Figure 1: Chemical structures of fludioxonil and fenhexamid. FludioxonilFenhexamid

2 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. RNU DMSO100nM Flu100nM Fen GAPDH DMSO100nM Flu100nM Fen RNU DMSO100nM Flu100nM Fen 18S DMSO100nM Flu100nM Fen CT value

3 Supplemental Figure 3: Fludioxonil and fenhexamid do not decrease ER  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 E 2, 100 nM fludioxonil (Flu), 100 nM fenhexamid (Fen), or 100 nM 4- hydroxytamoxifen (4-OHT, an ER antagonist) for 48 h. 30  g protein from whole cell lysates were separated by 10 % SDS-PAGE and immunoblotted for ER . Membranes were stripped and re-probed for  -actin as a loading control. Immunoreactive bands were quantified and the ratio of ER  -actin normalized to the DMSO control is indicated.

4 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  g protein from whole cell lysates were separated by 10 % SDS-PAGE and immunoblotted for PARP. Membranes were stripped and re-probed for  -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/ C+F *100 where C = the 85 kDa cleaved band and F = the full length 116 kDa band. DMSO E2 Flu Fen PARP 116kDa  -tubulin PARP 85kDa DMSO E2 % PARP cleavage Fen Flu

5 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 Dunnett’s Multiple Comparison Test. * P < 0.05 versus DMSO (control). # p < 0.05 versus 10 nM E 2. CCND1PGRESR1ESR2 Relative mRNA Expression * * DMSOE2FluFenE2 + FluE2 + Fen * * * * * * * * * * # ## # # # # #

6 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. 18S rRNA CT values DMSOE2100nM Flu 100nM Fen E2 + FluE2 + Fen CT

7 Supplemental Figure 7: Fludioxonil and fenhexamid do not increase cyclin D1 expression in ER  -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 > 38). Values are the average of triplicate determinations within one representative experiment. Statistical analysis used one way ANOVA followed by Dunnett’s Multiple Comparison Test. * P < 0.05 versus DMSO (control). MDA-MB-231 DMSO E2 10 nM Flu 10 nM Fen 100 nM Fen RelativeExpression 100 nM Flu CCND1 BCL2 * * * * * *


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