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. 2014 Jan;82(1):21–28. doi: 10.1128/IAI.01059-13

FIG 6.

FIG 6

Intracellular trafficking of T3SS-delivered ExoS does not require functional Rab5. (A) HeLa cells were mock transfected or transfected with pExoS(G−A+)-HA for 20 h and then intoxicated by PA103(ΔexoU exoT::Tc)[pUCP-ExoS(G−A−)-FLAG] at a multiplicity of infection of 16 at 37°C for 1 h. Cells were fixed and probed for HA (transfected ExoS) and FLAG (T3SS-delivered ExoS), and the merged image is shown. Red, ExoS(G−A−)-FLAG; green, ExoS(G−A+)-HA. (B) Expression of a dominant negative Rab5 construct does not block ExoS trafficking to the perinuclear region. HeLa cells mock transfected or transfected with Rab5(Ser34Asn) for 20 h were intoxicated with PA103(ΔexoU exoT::Tc)[pUCP-ExoS(G−A−)-FLAG] for 1 h. The cells were then fixed, permeabilized, and stained for the FLAG epitope. (C) Percentage of cells displaying perinuclear localization of T3SS-delivered ExoS(G−A−) in pExoS(G−A+)- or mock-transfected cells. Approximately 100 cells were counted under each transfection condition in four independent experiments. Columns were compared by unpaired Student's t test. (D) Percentage of cells displaying perinuclear localization of T3SS-delivered ExoS(G−A−) in pEGFP-Rab5(Ser34Asn)-transfected cells. Approximately 100 cells were counted under each transfection condition in four independent experiments. Columns were compared by unpaired Student's t test.