Slit2N inhibits HIV-1-gp120-induced migration and podosome formation of iMDDCs.

<p>(<b>A</b>) iMDDC transwell migration: cells were incubated with Slit2N, Slit2 negative control, or media alone for 2 hours, then seeded to the upper compartments of transwell chambers. Media +/− M-gp120 or Slit2N was added to the bottom compartments of the chambers. After 3 hours, iMDDCs that had migrated to the lower chambers were counted by hemocytometer. Data indicate the mean ± SD of 5 independent experiments (***p≤0.001). (<b>B</b>) iMDDC transendothelial migration: HUVECs were seeded into the upper compartment of transwell chambers and incubated at 37°C, 5% CO₂, to confluency. iMDDCs were incubated with Slit2N, Slit2 negative control, or media alone for 2 hours, then seeded to the upper compartments of transwell chambers. Media +/− M-gp120 or Slit2N was added to the bottom compartments of the chambers. After 8 hours, iMDDCs that had migrated to the lower chambers were counted by hemocytometer. Data indicate the mean ± SD of 5 independent experiments (***p≤0.001). (<b>C</b>) Vinculin expression by confocal microscopy. iMDDCs were cultured on chamber slides and left untreated or incubated with M-gp120, Slit2N then M-gp120, or Slit2N alone (Slit2N incubation: 2 hours; M-gp120 incubation: 1 hour) before fixing and staining cells. Green = Vinculin; Blue = DAPI. Scale bars = 2 µm. Arrows indicate a dense accumulation of podosomes at the cell's leading edge (“M-gp120” image), a reduced number of podosomes clustered at a weaker leading edge (“Slit2N + M-gp120” image), and a reduced number of podosomes localized around the cell's periphery (“Slit2N” image). Representative images are shown. (<b>D</b>) Percent of cell area covered by podosomes in the slides from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#pone-0048854-g001" target="_blank">Figure 1C</a>, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#s4" target="_blank">Materials and Methods</a>. (<b>E</b>) Number of podosomes per cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#s4" target="_blank">Materials and Methods</a>. Cells were treated as above. Data indicate the mean ± SD of 3 independent experiments x 50 cells per experimental condition (***p≤0.001).