Figure 2. LSP1 contributes to M-gp120-induced migration and podosome formation of iMDDCs.
(A) LSP1 expression by confocal microscopy. iMDDCs were cultured on chamber slides and left untreated or incubated with M-gp120 for 1 hour before fixing and staining cells. Red = LSP1; Blue = DAPI. Scale bars = 2 µm. Arrow indicates the cell's leading edge. Representative images are shown. (B) Representative Western blot analysis of LSP1 expression in untreated iMDDCs and in iMDDCs transfected with NT-siRNAs or LSP1-specific siRNAs. β-actin used as loading control. (C) Quantitative analysis of LSP1 expression in untreated iMDDCs and in iMDDCs transfected with NT-siRNAs or LSP1-specific siRNAs. The band intensity in each lane of 3 independent Western blots (as in Figure 2B) was determined by densitometry. Percent expression was calculated relative to the untreated control cells, whose LSP1 levels were established as 100%. Data represent the mean ± SD of 3 independent experiments (**p≤0.01). (D) LSP1 and vinculin expression by confocal microscopy. iMDDCs transfected with NT-siRNAs or with LSP1-specific siRNAs were cultured on chamber slides and incubated with M-gp120 for 1 hour before fixing and staining cells. Green = Vinculin; Red = LSP1; Blue = DAPI. Scale bars = 2 µm. Arrows in the upper left panel and lower left panel indicate a dense accumulation of podosomes at the cells' leading edge; while arrows in the upper right panel and lower right panel indicate fewer podosomes, more diffusely distributed throughout cells without a distinct leading edge. Representative images are shown. (E) Transwell Migration: iMDDCs and iMDDCs transfected with NT-siRNAs or with LSP1-specific siRNAs were seeded to the upper compartments of transwell chambers. Media +/− M-gp120 was added to the bottom compartments of the chambers. After 2 hours, cells that had migrated to the lower chambers were counted by hemocytometer. Data indicate the mean ± SD of 4 independent experiments (**p≤0.01).