FIGURE 1.

DNA-PK is required for VCAM-1 expression in response to TNF treatment. A, immunoblot analysis of extracts from untreated M059K (DNA-PK-proficient) or M059J (DNA-PK-deficient) cells with antibodies to human DNA-PKcs or GAPDH. B, M059K and M059J cells were stimulated with TNF for different times, after which protein extracts were subjected to immunoblot analysis with antibodies to VCAM-1 or GAPDH. Con, control. C, M059K and M059J cells were stimulated with 2 or 10 ng/ml IL-1β for 18 h, after which protein extracts were subjected to immunoblot analysis with antibodies to VCAM-1 or GAPDH. D and E, cells were treated with TNF for 6 h, after which total RNA was prepared and subjected to cDNA generation followed by conventional (D) or quantitative (E) PCR with primers specific to human VCAM-1 or β-actin. *, different from respective untreated cells; #, different from TNF-treated M059K cells; p < 0.01. F, M059K cells were treated with different doses of DNA-PKcs siRNA; 48 h later, protein extracts were prepared and subjected to immunoblot analysis with antibodies to DNA-PKcs or GAPDH. G, M059K cells were treated with siRNA against DNA-PKcs or control siRNA; 48 h later, cells were treated with TNF for 18 h. Protein extracts were subjected to immunoblot analysis with antibodies to VCAM-1 or GAPDH. H, M059K cells were transduced with a lentiviral vector (Santa Cruz Biotechnology) expressing either control shRNA or an shRNA targeting DNA-PKcs. Forty-eight hours later, cells were treated with TNF for 18 h, and the resulting protein extracts were subjected to immunoblot analysis with antibodies to DNA-PKcs, VCAM-1, or GAPDH.