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Fig. 1. Cdc42 is required for the rapid induction of TNF{alpha}-mediated filopodia formation, but not for their subsequent decrease. (A) Representative actin staining of MEFs following TNF{alpha}-treatment. Serum-starved MEFs were treated with 100 ng ml–1 TNF{alpha} for the indicated time, then fixed and stained for F-actin. Arrows depict filopodia. Bar, 10 µm. (B) Quantification of MEFs having filopodia shown in (A). The percentages of filopodia-positive cells following TNF{alpha}-treatment, relative to control cells, are shown. Values are means±s.d. of three independent experiments. Significance in induction of filopodia formation caused by TNF{alpha} compared with untreated: P=0.016 (Student's t-test). (C) Effect of inhibition of Cdc42 activity on TNF{alpha}-induced filopodia formation. MEFs were transfected with either GFP-tagged Cdc42-N17 (dominant negative form of Cdc42) (a and b) or with the control plasmid pEGFP (c and d). Serum-arrested transfected MEFs were treated with 100 ng ml–1 TNF{alpha} for 10 minutes, then fixed and analysed for F-actin organisation (b and d) and GFP staining (a and c). Arrows depict filopodia. Bar, 10 µm. (D) Quantification of Cdc42-N17 transfected MEFs having filopodia. MEFs transfected with GFP-tagged Cdc42-N17 were treated as in (A) with TNF{alpha} for the indicated times. The percentages of filopodia-positive cells following TNF{alpha}-treatment are shown. Values are means±s.d. of three independent experiments. (E) Cdc42 activity in TNF{alpha}-treated MEFs. Serum-arrested MEFs were treated with 100 ng ml–1 TNF{alpha} for various times as indicated, then lysed and the GTP-bound form of Cdc42 was assayed as described in Materials and Methods.





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