Figure 2

Estradiol (E2) increased anchorage-dependent colony formation and repressed miRNA (miR)-34b expression. (A) Anchorage-dependent colony formation assay results. MCF-7 cells were seeded into six-well plates and treated with different concentrations of E2 (0 to 10 nM) for 14 days. Colonies were fixed and stained with crystal violet dye. Representative results are shown in the upper panel. The number of colonies are shown in the bottom panel (n = 3). (B) miR-34b expression of MCF-7 cells treated with E2 (10 nM) for different time periods (n = 3) or with different concentrations of E2 for 24 hours (n = 3). The results were normalized to internal U6 snRNA expression. (C) Western blots of cyclin D1 in MCF-7 cells treated with E2 (10 nM) for different time periods (n = 3) or with different concentrations of E2 for 24 hours (n = 3). β-actin was probed as an internal control. The results are expressed as means ± SD. *P < 0.05. (D) Stable clones of pTRE-miR-34b/Tet-On were induced by doxycycline (DOX) (2 μM) for 48 hours, and miR-34b expression was analyzed by performing quantitative PCR. (E) Cell viability of pTRE-miR-34b/Tet-On in the presence or absence of DOX (2 μg/ml) was detected by using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (n = 3). (F) Western blot showing cyclin D1 after miR-34b induction. β-actin was probed as an internal control.