Shclnd nm 001785

Stable cell lines were generated via lentiviral infection using a standard protocol⁴² with 2^nd generation packaging plasmids (pCMV-VSVG, pCMV-dR8.9, a generous gift from Bruno Amati, IIT, Milan). CDA knock-down was achieved by infecting MDA-MB-231 and SN12C cell lines with pLKO.1 vectors containing 5 different shRNA constructs (SHCLND-NM_001785, Sigma-Aldrich) and a control pLKO.1 containing shRNA silencing Luciferase (a gift from Xin Lu, Oxford Ludwig Cancer Research). Infected cells were selected by incubation with 1.5 μg/ml puromycin (Sigma) for 60 hrs. Two cell lines with the lowest CDA mRNA (shRNA TRCN0000051290 and TRCN0000051288) levels were further assessed by immunoblotting and used for experiments. Lentivirus for CDA overexpression was generated with pLenti-puro (39481, Addgene, Ie-Ming Shih laboratory) expressing dsRed-IRES-CDA. H1299 and MCF-7 were infected as above. Infected cells were selected with puromycin at 2 μg/ml for 60 hrs.