V3lhs 364015

The p53 wild-type A549 and H460 cells were purchased from ATCC (Manassas, VA). The p53 deficient A549-p53shR and H460-p53shR cells were established by stable transduction with two different pBABE-puro shRNA retroviral vectors against p53 in A549 and H460 cells, respectively, as previously described [37 (link)]. The p53 shRNA sequences are as follows: p53-shR-1 (5'-GACTCCAGTGGTAATCTAC-3') and p53-shR-2 (5'-GTCCAGATGAAGCTCCCAGAA-3'). Control cells (A549-con-shR and H460-con-shR) cells were stably transduced with a control pBABE vector. The lentiviral shRNA vectors against human RRAD (V3LHS_364015 and V3LHS_409093) were purchased from Open Biosystems (Huntsville, AL). The siRNA oligos against human GLUT1 (5'- CGAACTATGAACTACAAAGCTTCTA-3', and 5'-TCAAAGTTCCTGAGACTAAAGGCCG- 3') were purchased form Integrated DNA Technologies. siRNA oligos were transfected into cells using Lipofectamine 2000 (Invitrogen). pLPCX-RRAD-Flag vectors were constructed by inserting full length human RRAD cDNA with Flag tag at C-terminus into the pLPCX vectors. For cells with stable ectopic RRAD overexpression, cells were transduced with a pLPCX-RRAD-Flag retroviral vector and selected by puromycin. For hypoxia treatment, cells were treated with hypoxia (0.1% O₂) in a hypoxia chamber.

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).