Fig. 3. The cellular level of miR-451 affects growth and colony formation.

A) Growth of Tam^R cells in the presence of Tam or Veh was followed for times up to 6 days after transfection of pri-miR-451 (+miR-451) or Ctrl vector (Ctrl) or pri-miR-451 and 14-3-3ζ (+miR-451 + 14-3-3ζ). The growth index was assessed at day 0, 3, and 6. B) Effect of antagomiR-451transfection (denoted miR-451 KD) on miR-451 and 14-3-3ζ expression levels. C) Effect of miR-451 KD on viability of Tam^R cells. Cell viability was assessed at day 0, 3, and 6 of Tam or Veh treatment. D) After 14-3-3ζ knockdown by siRNA, growth of Tam^R cells in the presence of Tam or Veh was evaluated at day 6. E) Sensitivity to SERMs was assessed after 72 h treatment with control vehicle, or 1μM Tam, Raloxifene or ICI in cells with control plasmid (Ctrl) or with miR-451 overexpression. F) miR-451 overexpression or miR-451 downregulation has no impact on ERα mRNA or protein level. G) Tam^R cells were assayed for soft agar colony formation after overexpression of miR-451 or control plasmid and exposure to Tam or Veh. Number of colonies and colony size were assessed and photography of colonies is also presented.