NEG pre-221

pre-222 pre-221/222

NEG

DMSO 100 nM Fulvestrant for 72h

Figure S1. Cell morphology of MCF7 cells after treatment with fulvestrant, with or without pre-221 and/or -222 transfection (Magnification 20× objective).

Figure S2. p27Kip1 and ERα protein level in MCF7 cells. 24 hours after transfection with pre-miR-221 (pre-221) and/or pre-miR-222 (pre-222), cells were treated with 10 nM fulvestrant for 2 days and subjected to immunoblotting analysis.

GAPDH

p27

ERα

- + - + + + +

- - + + - - -

- - - - + - +

- - - - - + +

10nM Fulvestrant

Scramble

pre-221

pre-222

GAPDH

p27

MCF7 MCF7-F NEG si-221/222

MCF7-F

Figure S3. p27Kip1 protein level in MCF7, MCF7-F cells, and MCF7-F cells transfected with negative control (NEG) or antagomirs (si-221/222) for 72 hours.

Figure S4. Expression level of miR-221/222 in fulvestrant-resistant MCF7-F cells after knockdown of miR-221 (left panel) or miR-222 (right panel) using 2’-O-Me-antagomirs. (mean±SE, n=3). **P < 0.01.

** **

**

Figure S5. Clonogenic activity of MCF7 and fulvestrant-resistant MCF7-F cells. (mean±SE, n=3). **P < 0.01.

**

**

**

GAPDH

p27

NEG si-221 si-222

Figure S6. p27Kip1 mRNA (left) and protein level (right l) in MCF7-F cells transfected with si-221 or si-222 for 72 hours. (mean±SE, n=2, two independent experiments). **P < 0.01.

Downregulated by si-221

(688)

Downregulated by si-222

(685)

224

Upregulated by si-221

(919)

Upregulated by si-222

(601)428

Figure S7. Venn diagrams showing the number of probes regulated by miR-221 and miR-222 in fulvestrant-resistant MCF7-F cells. A. Probes upregulated by si-221 or si-222; B. Probes downregulated by si-221 or si-222.

A

B

Figure S8. Fold-change of pathway activities in fulvestrant-resistant MCF7-F cells compared to MCF7 cells. Cignal Finder™ Cancer Pathway Reporter assay (SABiosciences Corporation, Frederick, MD) was performed according to the manufacturer’s instruction. (mean±SE, n=4). **P < 0.01.

**

******

****

**

**

**

Figure S9. qPCR results showing the time-course gene expression after transfection of MCF7 cells with miR-221/222 (solid lines), compared with scramble (dotted lines).

β-catenin

GAPDH

Scramble

si-22

1si-

222

si-22

1/222

MCF7-F

Figure S10. Immunoblotting result of β-catenin in MCF7-F cells after antagomiR treatment.

**

Figure S11. Cell proliferation assay of MCF7 cells. A. Cells were cultured in normal growth medium for 4 days. B. Cells were cultured in estrogen-free medium for 7 days. . (mean±SE, n=6). **P < 0.01.

A B

Figure S12. Dose dependent growth inhibition of MCF7 (black bars) and MCF7-F cells (gray bars) by TGF-β1 treatment. Cell numbers were determined using MTT assay and normalized to vehicle-treated cells.

PTEN

MCF7

ERα

p27

GAPDH

NT pre-scramble

pre-221

pre-222

pre-221/222

PTEN

GAPDH

MCF7-F

NT NEG

si-221

si-222

si-221/222

p27

Figure S13. Immunoblotting results of PTEN protein level in MCF7 and fulvestrant-resistant MCF7-F (PTEN antibody obtained from Cell Signaling Technology, Inc., Danvers, MA)