FIG 2 .

EspH induces focal adhesion disassembly in a caspase-independent manner. (A) Immunofluorescence microscopy of uninfected HeLa cells or cells infected with WT EPEC, ΔespH EPEC, or ΔespH EPEC complemented with EspH (ΔespH/pEspH) for 1 h, then incubated with gentamicin for 3 h. Bacteria were visualized with anti-intimin antibody (green), active caspase-3 was detected with anti-caspase-3 (cleaved) antibody (magenta), actin was stained with TRITC-phalloidin (red) and cell nuclei were stained with DAPI (cyan). The ΔespH mutant induced reduced levels of caspase-3 activation compared to WT EPEC. Complementation with EspH resulted in a marked increase in caspase-3 activation when compared to ΔespH alone. Bar = 20 µm. (B) Quantification of caspase-3 activation of infected cells from (A). STS treatment of HeLa cells for 5 h was used as a positive control. 100 infected cells were counted in triplicates. Results are means ± SD of three independent experiments. *, P < 0.05), ***, P < 0.001). (C) Immunofluorescence microscopy of HeLa cells transfected with Myc-GFP or Myc-EspH. Myc-tagged proteins were visualized with anti-Myc antibody (green), active caspase-3 was detected with anti-caspase-3 (cleaved) antibody (magenta), actin was stained with TRITC-phalloidin (red) and nuclei were stained with DAPI (cyan). Ectopic expression of EspH, but not GFP, induced caspase-3 activation. Bar = 20 µm. (D) Quantification of caspase-3 activation of transfected cells from (C). STS treatment of HeLa cells for 5 h was used as a positive control. 100 transfected cells were counted in triplicates. Results are means ± SD of three independent experiments. **, P < 0.01). (E) Quantification of detachment levels of HeLa cells, with or without zVAD-fmk treatment, infected with ΔespH EPEC complemented with EspH (ΔespH/pEspH) for 1 h, then incubated with gentamicin for 3 h. STS treatment of HeLa cells for 5 h was used as a positive control. Cell detachment was expressed as a percentage of uninfected cells. Treatment with zVAD-fmk inhibited STS-induced cell detachment, but not cell detachment following infection with ΔespH/pEspH EPEC. Results are means ± SD of three independent experiments. (F) Immunofluorescence microscopy of HeLa cells infected with ΔespH EPEC complemented with EspH (ΔespH/pEspH) for 180 min, with or without zVAD-fmk treatment. Bacteria were visualized with anti-intimin antibody (magenta), vinculin was detected with anti-vinculin antibody (green), and actin was stained with TRITC-phalloidin (red). Treatment with zVAD-fmk did not prevent focal adhesion disassembly. Bar = 10 µm.