Establishing FAK-Deficient Cell Lines

Normal murine NMuMG and metastatic 4T1 cells were obtained from ATCC (Manassas, VA) and cultured as described previously [18 (link)]. 4T1 cells were engineered to express stably firefly luciferase by their transfection with pNifty-CMV-luciferase [20 (link)] and selection with Zeocin (500 μg/ml; Invitrogen, Carlsbad, CA). NMuMG cells expressing WT or the nonfunctional mutant D119A-β₃ integrin were constructed by retroviral transduction, as described previously [19 (link)]. The MCF10A cell derivates T1k, Ca1h, and Ca1a were cultured in DMEM/F12 supplemented with 5% horse serum. The construction of NMuMG and 4T1 cells lacking FAK was accomplished by their lentiviral-mediated transduction with a scrambled (i.e., nonsilencing shRNA) or verified FAK-specific shRNA sequence encoded in pLentilox3.7-puro [15 (link)]. In brief, human 293T cells were transiently transfected with lentiviral packaging vectors (i.e., pMD2.G, pRRE, pRSV, and pLentiLox 3.7) according to standard protocols [21 (link)], and 48 h after transfection, the resulting conditioned medium was collected, filtered, and mixed with polybrene (8 μg/ml). Target cells were incubated in viral-containing supernatants for 48 h, and cells expressing nonsilencing or FAK-specific shRNAs were isolated by puromycin selection (5 μg/ml) for 14 days. Afterward, puromycin-resistant NMuMG and 4T1 cells were assayed for FAK-deficiency by immunoblotting with anti-FAK antibodies, as described later.