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Fig. 4. Rottlerin inhibits cell spreading of REFs on FN but does not interfere with focal adhesions of REFs pre-spread on FN. (A) REF cells were serum starved overnight, trypsinized and either treated with rottlerin at 1 µM (a,b) or 3 µM (c,d) or DMSO (e) in suspension for 20 minutes, then plated on FN-coated coverslips for 2.5 hours. For some cultures, rottlerin-containing medium was replaced with fresh serum-free medium 30 minutes after seeding on the FN substrate to ascertain whether the rottlerin effects were reversible (b,d). Cells were subsequently fixed and stained for F-actin. Bar, 50 µm. The graph (f) shows the cell area (mean ± s.d.) obtained from 40 cells selected at random from a representative experiment. (B) Serum-starved REF cells were spread on FN for 2 hours, then treated with rottlerin at 1 µM (a) or 3 µM (d) for 30 minutes. Cells were fixed, permeabilized and double stained for F-actin (red) and paxillin (green). Arrows indicate cells with a protrusive phenotype, arrowhead indicates a cell with no obvious filamentous actin organization. Bar, 50 µm. The graph (c) depicts the percentage of cells (± s.d.) with stress fibres, protrusive and no F-actin structures under the various rottlerin treatments. Black bars, DMSO; white bars, 1 µM rottlerin; hatched bars, 3 µM rottlerin. At least 30 cells were scored from each of two independent experiments.
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