AD-01 and FKBPL mediate cytoskeletal changes

<p><b>and AD-01 disrupts the RhoA-Rac1 dynamics.</b> (<b>A</b>) Confocal images (60x) representing the changes in phalloidin/F-actin dynamics in MDA-MB-231 upon wounding and treatment with AD-01 (10⁻⁹M) for 24 h; n = 3. Treated monolayers were fixed and stained with TRITC-phallodin (red) and DAPI (blue). Intense actin staining was accompanied by the loss in cell direction and communication. (<b>B</b>) Images (40x) representing disruption in tubulin distribution in HMEC-1 cell monolayers, wounded and treated with rFKBPL 750 ng/ml for 5 h. Fixed monolayers were stained for tubulin, followed by FITC conjugated secondary antibody (green) and nucleus with PI (red). (<b>C</b>) RhoA expression was increased after wounding and treatment with AD-01 for 24 h, resulting in a concomitant increase in the downstream actin binding proteins vinculin and profilin. Cells monolayers were treated with AD-01 (± wounding) for 24 h and total cell lysates were subjected to immuno blotting as indicated. (<b>D</b>) Treatment with AD-01 (10⁻⁹M) for 10/60 min inhibited fMLP induction (30 sec) of GTP-Rac-1 in HMEC-1 cells. Cell lysates of treated monolayers were subjected to Rac GTPase pull down assay; n = 3. (<b>E</b>) Representative western blots and quantitative densitometric analysis demonstrating an inhibition of cofilin phosphorylation after treatment with AD-01. HA treatment up-regulated cofilin phosphorylation maintaining its inactive state. HMEC-1 cell monolayers were treated with AD-01 for 3 h or HA for 10 min, and the extracted membrane fractions were subjected to immunoblotting with cofilin/p-cofilin; n = 3.