Figure 5.

ASAP1^BARPH induces the formation of cellular projections in NIH3T3 fibroblasts. Transient overexpression of ASAP1^BARPH induces the formation of long projections on the surface of NIH3T3 fibroblasts. NIH3T3 fibroblasts were transfected with different Flag-tagged ASAP1 constructs and costained for F-actin and Flag-ASAP1 fragments using rhodamine-phalloidin and anti-Flag antibody, respectively. A, F-actin is shown in red and Flag-ASAP1 proteins in green in the merged images. Scale bars throughout represent 10 μm and 5 μm in whole-cell and inset images, respectively. White arrows indicate examples of actin-containing projections. B, quantification (mean ± S.E.M.) of cells with long projections (at least 10 actin projections of >5 μm) per ASAP1 construct from 3–4 experiments. An average of 60∼100 cells positive for Flag immunostaining for each construct was scored blindly for exhibiting long projections in each experiment. C, quantification of the projection length by F-actin staining (n = 589 [pCI], n = 522 [ASAP1^FL], n = 967 [ASAP1^BARPH]). D, quantification of the total numbers of actin projection of >0.2 μm in cells transfected with different ASAP1 constructs from 3 experiments (n = 16 [pCI], n = 16 [ASAP1^FL], n = 16 [ASAP1^BARPH]). E, Quantification of the projection length by anti-Flag immunostaining (n = 324 [ASAP1^FL], n = 993 [ASAP1^BARPH]). F, quantification of the percentage of projection length filled by actin (n = 324 [ASAP1^FL], n = 993 [ASAP1^BARPH]). Statistics are based on the indicated number of projections (n) from 16 cells and three experiments. Of note, ASAP1^FL did not fill the entire length of the projections in ∼60% of the analyzed cases. This results in values of >100% for the projection length filled by actin. In contrast, ASAP1^BARPH did not fill the entire length of the projections in ∼10% of the analyzed cases. ****, p < 0.0001, ***, p < 0.001, and *, p < 0.05, using one-way ANOVA with Dunnett's multiple-comparison test (B, C, and D) and Student's t test (E and F).