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. 2010 Sep 7;78(11):4532–4541. doi: 10.1128/IAI.00802-10

FIG. 8.

FIG. 8.

ARAP2 is needed for cytoskeletal rearrangements during InlB-mediated entry. (A) Inhibition of internalization of InlB-coated beads by siRNA-mediated depletion of ARAP2. HeLa cells treated with control or ARAP2-2 siRNA were incubated with InlB-coated beads for 10 min. Entry of beads was measured using a fluorescence microscopy-based assay (8). Panel i, entry expressed as internalization relative to that in control siRNA-treated cells. Panel ii, entry expressed as absolute values (percentage of beads internalized). Values are averages ± SD from three experiments. P = 0.0054. (B) Inhibition of F-actin cytoskeletal changes by depletion of ARAP2. HeLa cells treated with control or ARAP2-2 siRNA were incubated with InlB-coated beads for 2.5 min, followed by fixation and labeling for beads (red) or F-actin (green). Panel i, images of several cells showing labeling of both F-actin and beads or F-actin only. Arrows indicate beads encircled by F-actin. Arrowheads show cell-associated beads that do not display obvious F-actin recruitment. Scale bars indicate 5 μm. Panel ii, enlarged images of selected beads (numbered 1 to 4) and associated F-actin indicated in panel i. In the case of beads that recruit F-actin, recruitment in control siRNA-treated cells (bead 1) is typically more intense than that in cells depleted for ARAP2 (bead 3). (C) Quantification of the effect of ARAP2 depletion on cytoskeletal changes. Panel i, confocal microscopy was used to quantify the percentage of beads associated with F-actin. For examples of beads recruiting or not recruiting F-actin, see beads 1 to 4 in panel ii of panel B. HeLa cells treated with ARAP2-2 siRNA had fewer actin-associated beads than control siRNA-treated cells. P = 0.0007. Panel ii, confocal microscopy was used to quantify the degree of F-actin enrichment around beads in cells treated with control or ARAP2-2 siRNA. This analysis included only beads that displayed obvious association with F-actin. For examples, see beads 1 and 3 in panel ii of panel B. Beads bound to cells treated with ARAP2-2 siRNA displayed reduced enrichment of F-actin compared to beads adhering to control siRNA-treated cells. P = 0.0002. The data in both panel i and ii are averages ± SD from three experiments.