Siport amine reagent

The HeLa cells were transfected in 12-well plates with the Renilla luciferase-based hTERT 3′UTR reporter constructs using FuGENE HD transfection reagent (Promega, Madison, WI). Transfection efficiency was controlled by co-transfection with a firefly luciferase-based control reporter pCI-firefly [90] (link) and the pEGFP-N1 fluorescent reporter (Clontech Laboratories, Mountain View, CA). After 24 hours the cells were super-transfected with miRNA mimics at 60 nM final concentration using siPORT amine reagent (Ambion). Cells were harvested 72 hours after reporter transfection and analyzed using a Dual-Luciferase Reporter Assay System (Promega). The reporter assay employing constructs with TCF7, MSI1, and PAX5 3′UTR were performed similarly with the following modifications: the HeLa cells were co-transfected in 24-well plates with the Renilla luciferase-based UTR reporter constructs, pCI-firefly, and pEGFP-N1 reporters, and 60 nM miRNA mimics using Metafectene Easy transfection reagent (Biontex-USA). Cells were harvested 48 hours after transfection and analyzed using a Dual-Luciferase Reporter Assay System (Promega). The Renilla luciferase signal of each sample was normalized for differences in transfection efficiency using the activity of co-transfected pCI-firefly reporter as control.

The Jurkat, HeLa, DLD-1, MCF-7 and Caco-2 cells were the gifts from P. Tucker, C. Sullivan, and J. Dudley (University of Texas, Austin). DLD-1 and Caco-2 colon carcinoma cell lines were described previously [88] (link), [89] (link). The cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Atlanta Biologicals, Norcross, GA). HeLa, DLD-1, Caco-2, or MCF-7 cells were plated in 6-well (8×10⁴ cells per well) or 12-well plates (4×10⁴ cells per well) and transfected after 24 hours using siPORT amine reagent (Ambion, Austin, TX) or Metafectene SI (Biontex-USA, San Diego, CA) with miRNA mimics (Ambion) at a final concentration of 60 nM (Table S1). All cells (including floating cells) were harvested after 48 hours, counted using a hemocytometer, and cell extracts were prepared for Western blot analysis. Trypan blue staining was used to evaluate the number of dead cells. For TRAP analysis cells were harvested 4 hours after transfection. miRNA inhibitors (Ambion) were electroporated into Jurkat cells under 200 V, 1 µF in siPORT electroporation buffer (Ambion) using Gene Pulser II electroporation apparatus (BioRad).