FIG 3.

Caspase-3/7 activation in A549 cells and primary lung epithelia following infection with S. maltophilia wild-type and virB10 mutant strains. Host cell monolayers were uninfected (UI) or infected with either parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+) for 24 h. (A and C) The levels of caspase-3/7 cleavage in A549 cells (A) and human bronchial/tracheal epithelial cells (ATCC PCS-300-010) (C) were determined by CellEvent caspase-3/7 green detection reagent that fluoresces upon binding the active caspase cleavage products. The raw data appear in the left panels, and data are normalized to the value for WT cells in the right panels. Asterisks indicate significant differences in the activation induced by the strains (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each). (B) Cell lysates were collected and analyzed by SDS-PAGE and then immunoblotted using an anti-caspase-7 antibody. (Left) Immunoblot from a representative experiment, with the migration of molecular mass standards (in kDa) indicated to the left of the gel image and the migration of caspase-7 and its active 20- and 10-kDa cleaved products to the right of the gel image. Immunoblot analysis using anti-GAPDH antibodies was performed to confirm equal loading. (Right) Graph showing the levels of the active cleaved products relative to GAPDH pooled from three independent experiments. Asterisks indicate significant differences between strains (**, P < 0.01). Whereas the 20-kDa species was seen in all trials, the 10-kDa cleaved product was only visible in two of three immunoblots.