FIG 3 .

Identification of shRNAs that result in defective YopB/D function. (A) YopB/D function correlates with membrane permeability. 293T cells were challenged with Y. pseudotuberculosis YPIII ΔyopB, YPIII ΔyopHMOJE, or YPIII yopE⁺ ΔyopHMOJ at an MOI of 25 for 2 h at 37°C. The cells were then stained using the FITC LIVE/DEAD cell viability kit (Invitrogen) for 30 min at room temperature (RT) and analyzed by flow cytometry. Representative histograms depicting relative FITC fluorescence after bacterial challenge are shown. (B) Depletion of host cell factors inhibit YopB/D function. 293T cells were transfected with individual shRNA plasmids coexpressing mCherry. After 72 h of knockdown, cells were incubated with YPIII ΔyopHMOJE for 2 h at an MOI of 25 followed by staining and flow cytometry analysis as described above for panel A. Representative histograms are overlays of mCherry⁺ cells from each labeled shRNA (gray) compared to vector control (white).