Figure 4.

Impaired ruffling and lamellipodia formation in DGKζ-null fibroblasts. Representative images of wt (+/+) and DGKζ-null (−/−) fibroblasts stained with AlexaFluor 488-conjugated phalloidin showing lamellipodia (A, arrows), peripheral membrane ruffles (B, arrows), and circular ruffles (C). The cells shown in A were grown in media containing 10% serum, whereas cells in B and C were serum-starved overnight and then stimulated with 10 ng/ml PDGF for 5 min. Bars, 20 μm. The graph to the right of each pair of images shows the quantification of each structure in wt and null cells. In each case, values are the mean ± SEM from at least three independent experiments. A minimum of 200 cells were counted per condition. The asterisks denote a significant difference between wt and null cells (p < 0.05) by Student's t test. (D) Reintroduction of wt DGKζ rescues circular ruffling. Representative images of DGKζ-null cells infected with adenoviruses bearing HA-tagged versions of wt DGKζ (HA-DGKζ wt) or a kinase-dead mutant (HA-DGKζ kd). Bars, 50 μm. The graph shows the quantification of circular ruffling in infected null cells fixed and stained to reveal actin. Values are the mean ± SEM from two independent experiments. The asterisk denotes a significant difference (p < 0.005) by Student's t test. More than 200 cells were counted per condition.