Fig. 3. Inhibition of TNF-induced filopodia formation depends on p38 kinase activity. (A) Quantification of TNF-treated MEFs having filopodia in the presence of SB 203580. The percentages of filopodia-positive cells treated with TNF in the presence of the p38 kinase inhibitor SB 203580 relative to control cells are shown. Cells were fixed, stained for F-actin, then quantified for the presence of filopodia, as described in Materials and Methods. For each experiment, 100 cells were scored and results are expressed as the means±s.d. of three independent experiments. (B) Representative actin staining of transfected MEFs. MEFs were transiently transfected with GFP-labelled Cdc42-V12 (visualised in a and d) or with HA-tagged MKK3, or both. 20 hours later cells were observed for F-actin staining (c, e and g) and for MKK3 expression (b and f). Bar, 10 µm. (C) Representative actin staining of transfected MEFs following TNF treatment. MEFs were transiently transfected with HA-tagged MKK3, then co-treated with TNF and SB 203580. After fixation, cells were visualised and stained for MKK3 expression (a) and for F-actin (b). (D) Quantification of MEFs having filopodia shown in MEFs from B and C, transfected or not with MKK3, were pretreated or not with SB 203580 and/or with 100 ng ml^–1 TNF for the indicated time. Cells were fixed, stained for F-actin then quantified for the presence of filopodia. MEFs were scored positively when presenting at least five filopodia. For each experiment, 100 cells were scored and values are the means±s.d. of three independent experiments. (E) Quantification of MEFs having filopodia shown in MEFs from B, transfected or not with Cdc42-V12, were pretreated or not with SB 203580 and/or with TNF for the indicated time. Scoring and analysis were performed as in D.