Figure 3.

Influence of PAK4 transient or stable overexpression on CMAC size and number. (A) Images from control MCF-7 cells or MCF-7 cells transiently expressing HA-PAK4-WT 3 h after replating onto VN, stained for integrin αvβ5 and HA-tag as indicated. Bar, 20 μm. (B) Quantification of CMAC numbers at the cellular periphery. The results are displayed as means ± SEM of the number of peripheral CMACs within 5 μm of the cell border per cell; between three independent experiments. (C and D) Quantification of CMAC sizes at the cellular periphery. The sizes of the CMACs are categorized into small (≤1 μm²), medium (from >1 to ≤2 μm²), and large (>2 μm²) adhesions. Values represent the percentage distribution (C) or number per cell (D) for each group expressed as mean ± SEM between three independent experiments. (E and G) MCF-7 cells stably expressing Flag-BAP or Flag-PAK4-WT were plated onto VN and fixed after 6 h. Cells were stained with anti-αvβ5 (E) or anti-vinculin (G) antibodies and costained with rhodamine-phalloidin. Bar, 25 μm. (F and H) Quantification of the number of CMACs at the cellular periphery as described in Materials and Methods. Graphs show means of CMACs per cell ± 95% confidence intervals; n = 48 (F) and n = 40 (H). In addition to effects by PAK4 on CMACs, we observed fewer actin stress fibers and also an induction of filopodia (arrows) in some WT PAK4-overexpressing cells compared with controls. p values indicated according to unpaired two-tailed t test.