Fig. 6

Shp1 directly mediates the GroPIns4P-induced actin ruffle formation. a Representative confocal microscopy images of serum-starved NIH3T3 cells untreated (untreated) or treated with 50 μM of GroPIns4P for 5 min alone (GroPIns4P) or in presence of the Shp1 inhibitors TPI-1 (TPI-1 + GroPIns4P) or NSC-87877 (NSC-87877 + GroPIns4P). The cells were fixed and processed for immunofluorescence analysis with FITC-labelled phalloidin. Zoom 1, 2 and 3: higher magnification images of the membrane area. b Quantification of actin ruffle formation (as percentage of untreated cells) of cells treated as in a (see the Methods). c Representative confocal microscopy images of NIH3T3 cells untransfected or transfected with the Shp1-C455S mutant for 8 h, starved for 24 h and then untreated (untreated) or treated with 50 μM GroPIns4P (GroPIns4P) or 10 ng/ml PDGF (PDGF) for 5 min. The cells were fixed and stained with an anti-Shp1 antibody and FITC-labelled phalloidin. d Quantification of actin ruffle formation (as the percentage of untreated cells) of cells treated as above. e Quantification of actin ruffle formation (as the percentage of untreated cells) of cells transfected with Shp1-WT, Shp1-S118A/R138E/S140A and Shp1-S12A/R32E/S34A mutants for 8 h, starved for 24 h and then treated with 50 μM GroPIns4P for the indicated times. Data are expressed as the means (±SD) of at least three independent experiments. ***P < 0.001; **P < 0.02; *P < 0.05 (Student’s t-test) calculated for each treatment versus untreated samples (untrasfected). Scale bars, 10 μm