FIG 6 .

AnxA2 expression enhances CARDS toxin binding and vacuolization in transfected HepG2 cells. (A) Expression of AnxA2 in stably transfected HepG2 cells. HepG2 cells were transfected with pCDNA plasmid carrying the AnxA2 gene, and G418-resistant stable clones were isolated and screened for expression of AnxA2 (lanes 2 to 4) and compared to normal HepG2 cells (lane 1). The clone expressing the highest level of HepG2-AnxA2 (lane 4) was selected for further studies. (B) Increased binding of CARDS toxin-mCherry to HepG2-AnxA2 cells. HepG2 and HepG2-AnxA2 cells were incubated with CARDS toxin-mCherry protein for 30 min at 4°C, and binding of CARDS toxin-mCherry protein to cells was analyzed by fluorometry as described in Materials and Methods. (C) AnxA2 expression enhances CARDS toxin-mediated vacuolization. HepG2 and HepG2-AnxA2 cells were incubated with CARDS toxin (50 µg/ml) for 4 h at 37°C, and vacuole formation was analyzed microscopically. (a to d) HepG2 cells alone (a) and with CARDS toxin (b) and HepG2-AnxA2 cells alone (c) and with CARDS toxin (d) are shown. (D) Increased numbers of CARDS toxin-induced vacuoles in HepG2-AnxA2 cells at 4 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.