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Figure 2


Fig. 2. IRSp53 is required for generation of CSF-1-induced F-actin rich membrane protrusions and cell migration. (A) The level of IRSp53 and WAVE2 present in IRSp53-shRNA-treated (IRSp53 sh) RAW/LR5 cells was analyzed by western blotting with the indicated antibodies and compared with mock-shRNA-treated cells. A representative blot and quantification of IRSp53:β-actin signal-intensity ratios are shown; n=3; *, P<0.05 compared with mock-shRNA-treated cells. (B) The level of IRSp53 present in IRSp53-shRNA-treated (IRSp53 sh) RAW/LR5 cells was analyzed by immunofluorescence with the indicated antibodies and compared with mock-shRNA-treated cells. Representative images and quantification of the IRSp53 signal intensity are shown; n=3; *, P<0.05 compared with mock-shRNA-treated cells. (C) Mock or IRSp53-shRNA-treated RAW/LR5 cells were treated with, or without, CSF-1 for 5 minutes, and F-actin-rich protrusions were visualized by Alexa-568-phalloidin staining. Bar, 10 µm. (D) The number of CSF-1-elicited protrusions in mock- (white bar) or IRSp53-shRNA-treated RAW/LR5 cells from C was quantified and expressed as a percentage of the CSF-1 stimulation observed in mock-shRNA-treated cells; n=3; *, P<0.05 compared with mock-shRNA-treated cells. The dotted line represents basal ruffling. (E) Mock- (white bar) or IRSp53-shRNA-treated (gray bar) RAW/LR5 cells were fixed after treatment with, or without, CSF-1, and the total F-actin content, normalized to the cell number, was quantitatively measured as described in Materials and Methods and compared with unstimulated control cells; n=3; *, P<0.05 compared with mock-shRNA-treated cells. (F) Chemotaxis and chemokinesis in response to CSF-1 in mock- (white bars) or IRSp53-shRNA-treated (gray bars) RAW/LR5 cells were evaluated using a transmigration chamber assay as described in Materials and Methods. The CSF-1-stimulated migration of each cell population was compared with the corresponding unstimulated condition and expressed as a fold induction. n=3; *, P<0.05 compared with mock-shRNA-treated cells.





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