Internalization of rEtpE-C-coated beads is dependent on DNase X.

<p>(A) Immunofluorescence labeling of rEtpE-C-coated or non-coated beads incubated with human macrophages derived from peripheral blood monocytes. At 30 min pi, cells were labeled with α-DNase X without permeabilization. rEtpE-C-coated beads cluster and colocalize with DNase X on the cell surface, but non-coated beads do not. A single <i>z-</i>plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 5 µm (see also suppl. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s007" target="_blank">Fig. S7</a> and suppl. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s011" target="_blank">Movie S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s012" target="_blank">S3</a>). (B) A selected image showing the orthogonal view of macrophage incubated with rEtpE-C-coated (left panel) or non-coated (right panel) beads in (A). The orthogonal view was obtained from the reconstituted 3-D view of serial <i>z</i>-stack images (combined z-section width of 7.2 µm). Scale bar, 5 µm. The fluorescence intensity profiles of green (DNase X) and red (beads) signals were shown. (C) Fluorescence and phase contrast merged images of rEtpE-C-coated and non-coated beads incubated with BMDMs from DNase X^−/− and wild-type mice. Cells and beads were incubated for 45 min followed by trypsin treatment to remove non-internalized beads. Scale bar, 10 µm. (D) Numbers of internalized rEtpE-C-coated beads/cell of similar experiment as (C), relative to the number of non-coated beads set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05) (see also suppl. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s008" target="_blank">Fig. S8</a>).