Fig. 4. Colchicine stimulates PAI-1 synthesis in quiescent R22 cells. Growth-arrested cells were treated with DMSO (solvent control) or colchicine (10 µM, final concentration) for 4 hours. The cellular microtubule network and PAI-1 protein were visualized in fixed and permeabilized cells by indirect immunocytochemistry with antibodies to tubulin and PAI-1, respectively (A). Cells exposed to DMSO were well spread with a highly branched microtubule network. Colchicine treatment disrupted the microtubule skeleton and significantly reduced the cell-spread area (Fig. 3, legend). PAI-1 protein was virtually undetectable by immunocytochemistry (A) and resolved at only extremely low levels by western blotting of extracts of control cells (B). Abundant PAI-1 protein was easily identified, by contrast, by both techniques in 4 hour colchicine-stimulated cells (A,B). Nuclei were stained with DAPI.