RNA interference

CXCR4 stable knockdown in MDA-MB-231-B was obtained using shRNA containing constructs derived from Sigma MISSION library [TRCN0000004054 (or #1): 5′-CTTTGTCATCACGCTTCCC-3′ and TRCN0000004056 (or #2): 5′-GAATCACGTAAAGCTAGAA-3′]. Lentivirus virions were produced by transfecting HEK293T cells with pKLO1-puro plasmid (containing the CXCR4-targeting shRNA or a non-mammalian shRNA control), pCMV-VSV-G (envelope plasmid), pMDLg-RRE (gag and pol elements) and pRSV-REV (rev or HIV1gp6) as packaging vectors. Plasmid mix was added to cell medium together with CaCl₂ and incubated for 18-20 h. Virus-containing supernatant was collected 48 and 72 h post-transfection and virus concentration was measured using Lenti-X™ p24 Rapid Titer Kit (Clontech). For transduction, MDA-MB-231-B cells were seeded in a 24-well plate (25,000 cells/well) and lentiviruses [1-3 multiplicity of infection (MOI)] added together with polybrene overnight (O.N.). Cells were cultured with complete medium containing 1 µg/ml puromycin for four passages and samples were collected for RNA isolation.