Secondary RNAi assays for validation of host factors required for Yersinia -mediated suppression of NF-κB signaling. RE-luc2P-HEK293 cells (20,000/well) were transfected with a 10 nM siRNA pool of four sequences per target gene in a 96-well plate and cultured for 72 h prior to (A) Y. enterocolitica WA or (B) Y. pestis Ind195 infection at MOI 2 and 10, respectively. Infection was stopped 1 h post-bacteria exposure by addition of 170 μg/ml chloramphenicol in the culture media. Cells were stimulated 5 h post-infection with 10 ng/ml TNF-α and were further cultured for 18 h. Cells were lysed and luciferase activity was measured to determine the percentage recovery of NF-κB regulated gene expression relative to that of uninfected cells. NF-κB-regulated luciferase activity was normalized to the RE-luc2P-HEK293 cell titer for each sample to obtain relative luciferase units. Numbers above the column data represent the ratio of luciferase activity in bacteria-infected versus uninfected cells. A “*” denotes significant (p≤0.05) recovery of reporter activity for targeting siRNAs compared to the control non-targeting siRNA (CTL)-treated cells infected with bacteria. Data was obtained from four independent experiments performed in duplicate.