RRAD Overexpression and Knockdown in Lung Cancer

Human lung epithelial cancer H1299 and H460 cells were purchased from ATCC (Manassas, VA). For cells with stable ectopic RRAD overexpression, cells were transduced with a pLPCX-RRAD-Flag retroviral vector and selected by puromycin [16 (link)]. The pCMV-RRAD-Flag WT and deletion mutations were constructed by PCR amplification. pCMV-p65-HA vector was constructed by using DNA fragment from pCMV4-p65 (Addgene). The lentiviral shRNA vectors against human RRAD (V3LHS_364015 and V3LHS_409093) were obtained from Open Biosystems (Huntsville, AL). To avoid off-target effects, two different siRNA oligos against each gene were employed for all knockdown experiments. The siRNA oligos against human GLUT1 (5′- CGAACTATGAACTACAAAGCTTCTA-3′ and 5′-TCAAAGTTCCTGAGACTAAA GGCCG-3′) and human p65 (5′-GGAGTACCCTGAGGCTATAACTCGC-3′ and 5′- AGCACAGATACCACCAAGACCCACC-3′) were obtained from Integrated DNA Technologies. Vectors and siRNA oligos were transfected into cells using Lipofectamine 2000 (Invitrogen).