FIG 4 .

AnxA2 surface accessibility promotes CARDS toxin binding to A549 cells. (A) CARDS toxin binding to A549 cells was reduced by anti-AnxA2 monoclonal antibody (mAb). A549 cells were incubated with or without anti-AnxA2 monoclonal antibody or negative-control isotype-matched monoclonal antibody for 10 min followed by incubation with CARDS toxin-mCherry protein for 30 min at 4°C. Binding of CARDS toxin-mCherry protein to A549 cells was analyzed by fluorometry as described in Materials and Methods. (B) Reduced CARDS toxin binding following suppressed AnxA2 expression. CARDS toxin (1 µg) was added to A549 cells transfected with siRNAs specific to AnxA2 or control random siRNAs and incubated for 1 h at 37°C. AnxA2 expression and CARDS toxin association with A549 cells were analyzed by immunoblotting with anti-AnxA2 monoclonal antibody and anti-CARDS polyclonal antibody. Comparative GAPDH intensities were used as loading controls. (C) Quantification of CARDS toxin associated with A549 cells upon suppression of AnxA2. Immunostained bands from panel B were quantified as detailed in Materials and Methods and normalized with GAPDH. Experiments were repeated two times (experiments 1 and 2), and the relative signal differences in binding levels of CARDS toxin and expression levels of AnxA2 are presented. (D) Decreased CARDS toxin-mediated vacuolization in A549 cells following suppressed AnxA2 expression. CARDS toxin was added to A549 cells transfected with control random siRNAs (a) or siRNAs specific to AnxA2 (b) and incubated for 24 h at 37°C. Vacuole formation was analyzed microscopically. (E) Quantification of CARDS toxin-induced vacuoles in A549-AnxA2-siRNA cells at 24 h. As described above for panel C, the cells were incubated with CARDS toxin, and the numbers of vacuoles per cell were counted and compared.