Nuclear factor-kappa B (NF-κB)-dependent gene induction in HeLa 57A cells is NF-κB-inducing kinase (NIK) dependent. HeLa 57A cells were infected with Adv.GFP, Adv.IκBα, or Adv.NIK-kd as indicated (moi 50) and left for 24 hours to allow transgene expression. Cells were stimulated for 4 hours with vehicle (no shading), with 25 ng/ml tumor necrosis factor (TNF)-α (solid shading), or with 10 ng/ml IL-1α (hashed shading). Cell lysates were then prepared and luciferase activities assessed (n = 3, ± SEM). (b, c) NIK is not required for lipopolysaccharide (LPS)-induced or TNF-α-induced cytokine expression in primary human macrophages. Primary human macrophage colony stimulating factor-differentiated macrophages were infected with Adv.GFP or Adv.NIK-kd as indicated (moi 100) and left for 24 hours to allow transgene expression. Cells were then stimulated for a further 24 hours with vehicle (no shading), with 10 ng/ml LPS (hashed shading), or with 25 ng/ml TNF-α (solid shading). Cell supernatants were collected and secreted IL-6 and TNF-α levels were determined by enzyme-linked immunopsorbent assay (n = 3, ± SEM).