Supplementary Figure 2 from The Acetylenic Tricyclic Bis(cyano enone), TBE-31 Inhibits Non–Small Cell Lung Cancer Cell Migration through Direct Binding with Actin

PDF - 324K, TBE-31 inhibits stress fiber formation in Rat2 fibroblasts A subconfluent layer of Rat2 fibroblasts were grown on coverslips and incubated in media supplemented with 5 microM cytochalasin D at 37 degrees C for 30 min to depolymerize the actin cytoskeleton. Afterwards, the cells were rinsed with PBS to remove the previous treatments. Media supplemented with DMSO, 1 microM TBE-31, or 1 microM CDDO-Im was then reintroduced to the cells while the actin stress fibers re-polymerized in the absence of cytochalasin D. The cells were then fixed, permeabilized and immunostained with AlexaFluor 555 phalloidin to visualize actin filaments (red), and DAPI (blue) for the nuclei. Shown here are representative immunofluorescence images taken with an Olympus IX81 microscope at 40x magnification (n=3). Bar=20 microm. Inset: Representative areas were magnified to demonstrate actin stress fibers (white arrows) or areas of branched actin polymerization (blue arrowheads). Note that TBE-31 treatment reduced the length of stress fibers but did not inhibit branched actin polymerization. However, CDDO-Im treated Rat2 fibroblasts contained actin stress fibers comparable to vehicle-treated cells but contained reduced branched actin polymerization.