Pcdh cmv lentiviral vector

NB4 [60 (link)], HL-60 [61 (link)] and COS-7 cells were cultured as described [36 (link), 62 (link), 63 (link)]. We generated NB4 cell populations silenced for PML-RAR and the RARα isoforms infecting cells with the SCRsh, PMRsh, RA1sh and RA2sh retroviral vectors according to standard protocols (System Biosciences). Following infection, NB4 cells were selected in RPMI medium containing 10% bovine serum and puromycin (1.0 μg/ml) for at least 15 days. Only NB4 cell populations characterized by >95% positivity to GFP following FACS analysis were considered. Subsequent passages of the cell populations were performed in complete RPMI medium containing puromycin (0.5 μg/ml). To isolate NB4 populations over-expressing RARα2, cells were electroporated with pCDH-CMV lentiviral vectors (System Biosciences) containing RARα2 (pRA2) using Neon Transfection (Invitrogen, Life Technologies). HL-60 cells were infected as above with the RA2sh, SCRsh or the void (Void) retroviral vector.

The source of the cell lines and the infection procedures are described in Supplementary Methods. We generated SK-BR-3, A549, HL-60, and NB4 cell populations silenced for S100A3 with lentiviral vectors (pGREENpuro, System Biosciences) containing the shRNA. To isolate NB4 populations over-expressing S100A3, cells were infected with pCDH-CMV lentiviral vectors (System Biosciences) containing the human S100A3 cDNA. To this purpose, S100A3 was inserted in the XbaI and NotI sites of pCDH-CMV.

cDNA encoding human miR-587 precursor (~300 bp) was cloned into pCDH-CMV lentiviral vector purchased from System Biosciences (SBI, Mountain View, CA, USA). PPP2R1B lentiviral expression vector was purchased from GeneCopoeia, Inc. (Rockville, MD, USA). Packaging cells (293) were co-transfected with pPackH1 packaging plasmid mix (SBI) and the lentiviral vectors using Fugene HD (Promega). Viruses were harvested 48 h later to infect target cells.