Figure 7
Figure 7. WASP mediates the transition to filopodia in PSTPIP1-R405C cells. (A) The F-actin polymerizing ability of PSTPIP1-WT and -R405C rescue cells was determined by stimulating serum-starved cells with chemokine (C-C motif) ligand 2. F-actin was saturated with rhodamine phalloidin and extracted with methanol; fluorescence was determined with a plate reader. Fluorescence intensity was normalized to the level of PSTPIP1-WT cells for each experiment. (B) F-actin content assay performed as in (A). Prior to stimulation, cells were pretreated for 30 minutes with dimethyl sulfoxide (DMSO) or 10 μM wiskostatin. Stimulation was performed in the presence of DMSO or 20 μM wiskostatin (Wisko) for 5 minutes. DMSO-treated WT was the reference value for statistical comparison except where indicated by a bar. (C-F) Macrophages nucleofected with GFP-Cdc42 WT (WT) or GFP-Cdc42 Q61L (CA) were plated on fibrinogen-coated coverslips overnight and stained with rhodamine phalloidin, anti-vinculin antibody, and anti-GFP antibody to visualize podosomes and Cdc42 expression. (C) Representative images of podosomes and filopodia in Cdc42-nucleofected cells. GFP-positive cells were used for (D) quantification of the average number of podosomes per cell, (E) the percentage of cells that made podosome rosettes, and (F) the percentage of cells forming filopodia. (G-J) THP-1 shPST + R405C rescue cells were plated on Alexa Fluor 568–conjugated gelatin for 3 hours with the indicated concentration of (G) wiskostatin or (I) GM6001. Representative images are shown. (H) The amount of degradation in the presence of wiskostatin is shown as a percentage of cell area (0 μM, n = 112; 2.5 μM, n = 134; 5 μM, n = 138). (J) The amount of degradation in the presence of GM6001 is shown as a percentage of cell area (0 μM, n = 125; 10 μM, n = 131). All values are mean ± SEM from 3 (B,H,J) or 4 (A,D-F) independent experiments. *P < .05; ***P < .001 as determined by (A) unpaired Student t test, (B) one-way ANOVA with Sidak’s multiple comparison test, (D-F) paired Student t test, or (H,J) ANOVA with repeated measures using a compound symmetry correlation structure.

WASP mediates the transition to filopodia in PSTPIP1-R405C cells. (A) The F-actin polymerizing ability of PSTPIP1-WT and -R405C rescue cells was determined by stimulating serum-starved cells with chemokine (C-C motif) ligand 2. F-actin was saturated with rhodamine phalloidin and extracted with methanol; fluorescence was determined with a plate reader. Fluorescence intensity was normalized to the level of PSTPIP1-WT cells for each experiment. (B) F-actin content assay performed as in (A). Prior to stimulation, cells were pretreated for 30 minutes with dimethyl sulfoxide (DMSO) or 10 μM wiskostatin. Stimulation was performed in the presence of DMSO or 20 μM wiskostatin (Wisko) for 5 minutes. DMSO-treated WT was the reference value for statistical comparison except where indicated by a bar. (C-F) Macrophages nucleofected with GFP-Cdc42 WT (WT) or GFP-Cdc42 Q61L (CA) were plated on fibrinogen-coated coverslips overnight and stained with rhodamine phalloidin, anti-vinculin antibody, and anti-GFP antibody to visualize podosomes and Cdc42 expression. (C) Representative images of podosomes and filopodia in Cdc42-nucleofected cells. GFP-positive cells were used for (D) quantification of the average number of podosomes per cell, (E) the percentage of cells that made podosome rosettes, and (F) the percentage of cells forming filopodia. (G-J) THP-1 shPST + R405C rescue cells were plated on Alexa Fluor 568–conjugated gelatin for 3 hours with the indicated concentration of (G) wiskostatin or (I) GM6001. Representative images are shown. (H) The amount of degradation in the presence of wiskostatin is shown as a percentage of cell area (0 μM, n = 112; 2.5 μM, n = 134; 5 μM, n = 138). (J) The amount of degradation in the presence of GM6001 is shown as a percentage of cell area (0 μM, n = 125; 10 μM, n = 131). All values are mean ± SEM from 3 (B,H,J) or 4 (A,D-F) independent experiments. *P < .05; ***P < .001 as determined by (A) unpaired Student t test, (B) one-way ANOVA with Sidak’s multiple comparison test, (D-F) paired Student t test, or (H,J) ANOVA with repeated measures using a compound symmetry correlation structure.

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