FIG 1.

Three levels of screening for novel regulators of NF-κB regulation. (A) Primary screen where siRNA oligonucleotides (4 for every gene and 1 gene per well) were prearrayed into 384-well plates. THP-1 macrophages with the NF-κB reporter were reverse transfected for 72 h before being treated with TNF-α or LPS for 6 h and assayed for luciferase activity. Each whole-genome library was assayed in duplicate for each of the two stimuli. (B) Secondary screen where the top hits from panel A were screened again using TNF-α and LPS, with a test for cell viability to rule out essential genes from the final hit list (n = 3 replicates). (C) Tertiary screen where hits from panel B were validated using an endogenous NF-κB reporting system in response to TNF-α (COX2 protein readout, measured via Western blotting and subsequent ImageJ analysis) in U87 cells (n = 2 replicates).