Presentation on theme: "NEGpre-221 pre-222pre-221/222 NEG DMSO 100 nM Fulvestrant for 72h Figure S1. Cell morphology of MCF7 cells after treatment with fulvestrant, with or without."— Presentation transcript:
NEGpre-221 pre-222pre-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. p27 Kip1 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. p27 Kip1 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. p27 Kip1 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 manufacturers 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-221 si-222 si-221/222 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. AB
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 NTpre-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)