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Fig. 3. Interfering with WAVE2 function inhibits CSF-1-induced F-actin rich membrane protrusions. (A) RAW/LR5 cells, transiently transfected with the indicated constructs, were treated with 20 ng/ml CSF-1 for 5 minutes and F-actin-rich protrusions were visualized by Alexa Fluor 568 phalloidin staining. Cells expressing the constructs were identified by epitope staining (FLAG for WAVE1/2 and Myc for WASP) and the number of CSF-1-elicited protrusions was quantified as described in Materials and Methods and expressed as a percentage of the CSF-1 stimulation observed in non transfected cells on the same coverslip; n=3, *P<0.05 compared to non transfected cells (represented by the dotted line in A-D). (B) RAW/LR5 cells were transiently transfected with the same constructs as in A, and their ability to undergo Fc{gamma}-R-mediated phagocytosis was determined as described in Materials and Methods and expressed as a percentage of phagocytosis observed in non transfected cells on the same cover slip; n=3, *P<0.05 compared to non transfected cells. (C) WAVE1 or WAVE2 antibodies or control IgG were introduced into BMM cells by transient permeabilization as described in Materials and Methods. The ability of cells to form F-actin rich protrusions in response to CSF-1 was analyzed as in A; n=3, *P<0.05 compared to non permeabilized cells on the same coverslip. (D) WAVE2 antibodies or control IgG were introduced into RAW/LR5 cells by transient permeabilization, as in C, and CSF-1-induced protrusions were scored as in A; n=3, *P<0.05 compared to non permeabilized cells on the same cover slip. (E) WAVE2 and ß-actin expression in WAVE2 shRNA-treated RAW/LR5 cells (heterogenous cell populations) was analyzed by western blotting with the appropriate antibodies and compared to mock shRNA treated cells (see inset) and WAVE2/ß-actin signal intensity ratios of the indicated blot were quantified. (F) Mock, WAVE2 shRNA-treated cells (white bars) or WAVE2 shRNA-treated cells transiently transfected with a human GFP-WAVE2 construct (black bars) were stimulated with CSF-1 and their ability to form F-actin-rich protrusions in response to CSF-1 was analyzed as in A and expressed as a percentage of the CSF-1 stimulation observed in mock shRNA-treated cells; n=3, *P<0.05 compared to mock shRNA-treated cells.





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