Figure 5.

Inhibitory effect of Compounds NP‐G2‐011 and NP‐G2‐044 on stress fiber formation and focal adhesion turnover. (A) Cells with wild‐type fascin were treated with LPA to induce actin stress fiber formation. Addition of Compound NP‐G2‐011 or NP‐G2‐044 blocked LPA‐induced stress fiber formation. (B) 4T1 cells expressing fascin(E391A) mutant formed stress fibers upon LPA treatment. Stress fiber formation in these cells was resistant to Compound NP‐G2‐044 inhibition. (C) Quantification of data in A and B. Error bars, mean ± SEM. *p < 0.05; **p < 0.001. Data are representative of three similar experiments. Scale bar, 10 μm. (D) Confocal live cell images of MDA‐MB‐231 cells expressing GFP‐FAK were taken before photobleaching the ROIs (red box) containing focal adhesions. (E) Time lapse sequence of the selected ROIs showing a representative recovery after photobleaching (Pre, pre bleach). (F) Kinetics of recovery of GFP‐FAK after photobleaching of focal adhesions from cells pre‐treated with vehicle (Ctrl) or NP‐G2‐044 are illustrated. The measured fluorescence intensity was normalized and the mean values (18 independent ROIs from different cells for each condition) are shown along with the exponential fit (red line). (G) Corresponding recovery half‐time obtained from FRAP analyses of GFP‐FAK pre‐treated with vehicle (Ctrl) or NP‐G2‐044. (H) Confocal live cell image of a representative MDA‐MB‐231 cell expressing GFP‐FAK and a focal adhesion containing ROI (yellow box). (I) Kymograph of the corresponding focal adhesion over a period of 40 min. Acquisitions were taken every 30 s. (J) Quantitative assessment of focal adhesion disassembly in GFP‐FAK expressing cells pre‐treated with vehicle (Ctrl, blue square) or NP‐G2‐044 (red circle). (K) and (L) Assembly (K) and disassembly (L) rates were obtained by numerical differentiation of the corresponding time‐dependent signal. Error bars, mean ± s.e.m. Scale bars, 10 μm *p < 0.05; **p < 0.01.