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Fig. 6. Wild-type p53 activity is required to mediate TNF-induced filopodia inhibition. (A) Effects of TNF on filopodia formation in actinomycin D-treated MEFs. Serum-arrested cells were pretreated or not with the transcription inhibitor actinomycin D, followed by a treatment with 100 ng ml^–1 TNF for various times as indicated, fixed and stained for F-actin, then quantified for the presence of filopodia. For each experiment, 100 cells were scored and results are the mean±s.d. of three independent experiments. (B) Quantification of MEFs having filopodia. MEFs were transfected or not with plasmids encoding p53 wt, p53-H175, p53-H273 or pEGFP, then serum arrested before treatment or not with TNF for 1 hour as indicated. After fixation and staining for F-actin, cells were analysed for the presence of filopodia as indicated in Fig. 3. (C) Representative actin staining of MEFs transfected MEFs with p53-H273. Serum arrested cells were treated (a and b) or not (c and d) with TNF for 1 hour. Cells were fixed, visualised for p53 expression as revealed by GFP staining (a) and stained for F-actin (b). Arrows indicate filopodia. Bar, 10 µm. (D) Actin staining of p53^–/–MEFs. Cells were either untreated (a) or treated with TNF for 1 hour (b), then fixed and stained for F-actin. In (c) and (d), p53^–/–MEFs were transfected with HA-tagged MKK3 before staining for MKK3 (c) or F-actin (d). Arrows indicate filopodia. Bar, 10 µm. (E) Quantification of p53^–/–MEFs having filopodia. The percentages of filopodia-positive cells relative to control cells are shown. p53^–/–MEFs were transfected or not with plasmids encoding pEGFP (Control), p53 wt, MKK3, or treated with TNF for 1 hour, as indicated. 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. (F) Abundance of Cdc42 and p38 in MEFs expressing p53 mutants and in p53^–/–MEFs. MEFs were transfected with pEGFP, p53H273, p53 H175, p53 wt and p53^–/–MEFs before preparation of total protein lysates, and analysis by gel electrophores was followed by immunoblotting analysis using antibodies to Cdc42 and p38. Each lane contains equal amounts of loaded proteins (40 µg).

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