Figure 7. siRNA Targeting of FRL1 Reduces Macrophage Adhesion and Podosome Number.

A. Western blots of lysates from differentiated macrophages, THP-1 cells, and RAW 264.7 cells treated with control siRNA, s2227 siRNA, s2228, or s4093 siRNA as described in Methods. Lysates were separated on SDS-PAGE gels and blotted for FRL1 with mouse monoclonal anti-FRL1 antibody followed by anti-mouse HRP tagged antibody or for Dia1 with goat polyclonal anti-Dia1 antibody followed by anti-goat HRP tagged antibody. Lysates were also blotted for either transaldolase or actin as controls. B. Fixed cell microscopy of cell treated with siRNA targeting formins as described for A. Cells were fixed, permeabilized, and stained with rhodamine phalloidin to visualize actin. Images are 40μm by 40μm. C. Adhesion of macrophages following FRL1 reduction by siRNA. Graph depicting average cell numbers in one square centimeter in 24 well plates after treatment with siRNA to reduce expression of formins as described for A. Macrophage counts are shown as solid bars, THP-1 cell counts are shown as open bars, and RAW 264.7 cell counts are shown as cross-hatched bars. Results are reported as mean +/− SEM. D. Podosome number and area in macrophages following FRL1 reduction by siRNA. Graph depicting average podosome counts and average individual podosome area in primary macrophages treated with siRNA. Podosome count data is shown as solid bars while average podosome size data is shown as open bars. Results are reported as mean +/− SEM. E. Actin Intensity in Podosomes. Graph depicting average actin intensity per podosome in primary macrophages treated with siRNA. Mean Actin Intensity is shown as solid bars. Results are reported as mean +/− SEM.