Figure 4. Treatment of gastric epithelial cells with the specific SKF inhibitor PP2 impairs the arrival of VacA into endosomes.

(A and B) VacA intracellular trafficking in AGS (A) or MKN 28 (B) cells treated or not with PP2. Cells were preincubated for 30 min at 37°C in the presence or absence of 10 µM PP2 and, after a VacA binding step of 1 h at 4°C, immediately fixed (0 min) or allowed to internalize the toxin into early (30 min) or late (120 min) endocytic compartments (top left panels). VacA in green. For the 120 min time point, also LAMP1 (red; a marker of late endosomes) and its colocalization with VacA are shown (top right panels). All the pictures represent single confocal sections. Scale bar: 10 µm. The graphs (bottom panels): effect of PP2 treatment on either the percentage of cells exhibiting VacA confined in the GEECs (left) or the percentage of late endosomes (i.e., LAMP1-positive vesicles) containing VacA (right) after 2 h internalization. Mean±SEM by extensive confocal microscopy evaluation of slides from 3 independent experiments. *: P<0.05 versus PP2-untreated cells. (C) Apoptosis (shown as fold increase over the respective PP2-untreated control cells) induced by either VacA or etoposide in MKN 28 cells treated or not with PP2. Cells treated or not with PP2 but not exposed to VacA or etoposide served as controls. Mean±SEM of three different experiments. *: P<0.05 versus paired control. °: P<0.05 versus the same condition in the absence of PP2. The high cytotoxicity induced by PP2 on AGS cells after the incubation time used to measure either VacA- or etoposide-induced apoptosis did not enable us to repeat the same set of experiments in this latter cell line.