Plk1 Overexpression and miR-509 Rescue

Plk1 cDNA was cut off from pcDNA3 3xFlag-Plk1^{7 (link)} and subcloned to pLenti-puro (Addgene #39481) at XhoI and ApaI sites. pLenti-puro has 3′UTR and poly A of BGH, which does not have miR-509 targeting sequence, and is resistant to miR-509. To produce viruses, 293FT cells were transfected with pLenti-puro encoding Plk1 plus packaging vector pCMV and envelop vector pVSV-G. Viruses were collected 48 h after transfection. For infection, smooth muscle cells were incubated with viruses 12 h. They were then cultured in the F12 growth medium for 3 days. Positive clones were selected by puromycin. Stable Plk1 expressing cells were treated with miR-509 to generate Plk1 rescue cells.