ExoT/ADPRT interferes with integrin-mediated survival signaling.

<p>(A) HeLa cells were treated with PBS or infected with PA103∆<i>exoU</i> (∆U); PA103∆<i>exoU/exoT(R149K)</i> (∆U/T(G^-A⁺)); or the T3SS mutant PA103 <i>pscJ</i>::<i>Tn5</i> (<i>pscJ</i>) at MOI~10. At indicated time points after infection, cell lysates were probed for activated Akt (p-Akt^S473), Akt-mediated GSK-3β inactivation by phosphorylation (p-GSK-3β^S9), or β-catenin levels by Western blotting. The data were normalized to GAPDH and the fold changes in levels, as compared to mock, are shown underneath. (B) β-catenin transcriptional activity was assessed by transfecting HeLa cells with the TOPFlash luciferase reporter plasmid for 24 hr before infecting the cells with either ΔU, ΔU/T(G^-A⁺), <i>pscJ</i>, or PBS. At indicated time points luciferase was assessed by a luminometer using the Luciferase Assay System (see Experimental Procedures). The experiment was performed in triplicate and the luciferase readings were normalized to baseline levels. Data are shown as mean ± SEM, * <i>p</i><0.001, Student’s t-test).</p

ExoT and ADPRT disrupt focal adhesion sites.

<p>(A-D) HeLa cells were treated with PBS or infected with ∆U, ∆U/T(G^-A⁺), or <i>pscJ</i> at MOI~10. Four hours after infection, cells were fixed and stained with nuclear stain DAPI and p-FAK (A) or p-p130Cas (B). Representative images are shown in (A and B) and the tabulated results are shown in (C and D). (E-H) HeLa cells were transiently transfected with pIRES2-GFP expression vector harboring ExoT (pExoT), ExoT/ADPRT (pExoT(G^-A⁺), or inactive ExoT (pExoT(G^-A^-), all C-terminally fused to GFP, or empty vector (pGFP). 24 hr after transfection, cells were fixed and stained with nuclear stain DAPI and analyzed for p-FAK and p-p130Cas localization to the FA sites. Representative images are shown in (E and F) and the tabulated results from 3 independent experiments are shown in (G and H). (Statistical analysis was performed using one-way ANOVA. Scale bar = 25 μm). (I) HeLa cells were infected as in (A). 4hr after infection, the cell lysates were probed for total FAK and p130Cas or their phosphorylated forms (p-FAK^Y397 & p-p130Cas^Y165 respectively) by Western blotting. Expression levels were normalized to GAPDH, which was used as the loading control.</p

Crk mediates ExoT-induced apoptosis.

<p>Crk^-/- cells were transiently transfected with pCrkI, or pGFP on 3 consecutive days to obtain more transfected cells. 24 hr after the final transfection, cells were infected with either ∆U, ∆U/T(G^-A⁺), or <i>pscJ</i> at MOI~10. Cytotoxicity of transfected host cells (green) was assessed by fluorescent time-lapse microscopy, using PI uptake (red cells are dead). Cytotoxicity is expressed as a percentage of the total number of transfected cells. Video images were captured every 15 min and selected movie frames at indicated time points are shown in (A). (For clarity, phase panels were excluded from the merged images). The corresponding tabulated data are shown in (B). (C) The corresponding time to death, defined as the time of infection to the time of PI uptake (red) is expressed as the mean ± SEM. The data in B and C comprise 3 independent experiments. Statistical analysis was performed using one-way ANOVA.</p

CrkI/R38K mutant disrupts FA sites in HeLa and Crk^-/- cells, phenocopying ExoT/ADPRT adverse effect on FA sites.

<p>HeLa cells were transiently transfected with pIRES2-GFP expression vector harboring wild-type CrkI (pCrkI), SH2 DN (pCrkI/R38K), all C-terminally fused to GFP, or empty vector (pGFP). 24 hr after transfection, cells were fixed and stained with nuclear stain DAPI and analyzed for p-FAK localization to the FA sites. Representative images are shown in (A and D) and the tabulated results from 3 independent experiments are shown in (B-C and E-F). (E) Crk^-/- cells were transiently transfected with pIRES2-GFP expression vector harboring wild-type CrkI (pCrkI), SH2 DN (pCrkI/R38K), or SH2 and SH3 double mutant (pCrkI/R38K,W170K), all C-terminally fused to GFP, or empty vector (pGFP). 24 hr after transfection, cells were fixed and stained with nuclear stain DAPI and analyzed for p-FAK localization to the FA sites. Representative images and enlargements of fields indicated by a white box are shown in (G) and the tabulated results from 3 independent experiments are shown in (H-I). (Statistical analysis was performed using one-way ANOVA. Scale bar = 25 μm).</p

CrkI/R38K induces cytotoxicity in Crk^-/- cells.

<p>Crk^-/- cells were transiently transfected with pCrkI, pCrkI/R38K, pCrkI/R38K, W170K or the vector control pGFP. Video images were captured every 15 min and selected movie frames at indicated time points are shown in (A) (For clarity, phase panels were excluded from the merged images). (B) Cytotoxicity of transfected host cells (green) was assessed by fluorescent time-lapse microscopy, using PI uptake (red cells are dead). Cytotoxicity is expressed as a percentage of the total number of transfected cells. * Signifies significance with <i>p<</i>0.001 by χ² analyses analysis. Data are representative of 3 independent experiments.</p