Pomalidomide and lenalidomide induce stress fiber formation in Swiss 3T3 cells through activation of RhoA. (A) Swiss 3T3 fibroblasts were incubated in serum-free medium with 0.2% NaHCO₃ for 20 hours before stimulation with 0.01% DMSO, 20 ng/mL LPA, 3 ng/mL PDGF, 100 ng/mL bradykinin, 1 μM pomalidomide, or 1 μM lenalidomide for 30 minutes. Where indicated, 30 μM ROCK1 inhibitor or 30 μM Rac1 inhibitor were added to cells 12 hours before the above stimulations. The cells were then fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with Alexa 594 phalloidin to visualize F-actin (red). (B) Serum-starved cells were stimulated as indicated in a time course and were followed by Alex-594 phalloidin staining. Each bar represents the percentage of cells with stress fiber clearly observed under each condition, which were quantified in randomly chosen 100 cells per a 22 × 22-mm glass coverslip. Results shown here are the mean of 3 independent experiments. Error bars indicate ± SE. (C) Serum-starved cells were treated with 1 μM pomalidomide for the indicated time. Rho GTPases activity was analyzed by the GTPase pull-down assay as described in “Methods.” Quantification for each treatment condition is shown on the right panel.