H. pylori and PaCS Figure 7. H. pylori products/virulence factors induce PaCS formation in vitro. (A) Confocal microscopy of HeLa cells incubated for 24 h with Cy5-labeled purified VacA for the simultaneous detection of proteasome (green), ubiquitinated proteins (FK2; red), and VacA (blue). The enlargements show in a1 colocalization (purple) between ubiquitinated proteins and VacA, and in a2 colocalization (light blue/white) of proteasome, ubiquitinated proteins, and VacA. The graphs on the right represent the respective colocalization charts showing the intensity profile for each of the three fluorescences taken along the dotted lines. Pictures are from one single confocal section. Bar: 10 mm. (B) Confocal microscopy of HeLa cells (nuclei in blue) incubated for 24 h without (control) or with H. pylori BCF shows the presence in the cytoplasm of BCF-treated cells of spots with the yellow component representing the colocalization of ubiquitinated proteins (FK2; red) with proteasome (green) (top line) or of ubiquitinated proteins (red) with VacA (green) (bottom line), respectively. These cytoplasmic colocalizations in either control or treated cells are quantitatively analyzed in the histograms (right) showing the percentage of FK2-positive spots colocalizing with proteasome or VacA, respectively. Pictures are from one single confocal section. Bar: 10 mm. (C) Time-course of HeLa cells (nuclei in blue) incubated with H. pylori BCF shows a time-dependent increase in the cytoplasmic spots of ubiquitinated proteins (FK2; red). The histogram (right) shows the number (means 6 SEM) of FK2-positive cytoplasmic spots per cell. Bar: 10 mm. (D) Confocal microscopy of HeLa cells (nuclei in blue) incubated for 24 h without (control) or with H. pylori BCF shows the presence in the cytoplasm of BCF-treated cells of yellow spots representing the colocalization of ubiquitinated proteins (FK2; red) with NOD1 (green). Each individual labeling is shown in the enlarged areas of the boxed regions. Pictures are from one single confocal section. Bar: 10 mm. doi:10.1371/journal.pone.0009716.g007 PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9716