Activation of Rho GTPases, induction of IL-2, and formation of F-actin by IMiDs. (A) CD4⁺ T cells were treated with pomalidomide or lenalidomide for 6 hours at the indicated concentration in the presence of anti-CD3 antibody (A) or without anti-CD3 antibody (B). Cells were preincubated for 30 minutes with ROCK1 inhibitor or Rac1 inhibitor followed by pomalidomide or lenalidomide incubation. IL-2 level was measured by Mesoscale Discovery System. Columns and error bars represent mean (± SEM). (C) Pomalidomide selectively activates RhoA, Rac1. Small GTPase activities in CD4⁺ cells were measured by pull-down assay in the presence of pomalidomide at indicated concentrations for 30 minutes. Quantitation of pull-down protein is normalized to actin and expressed as percent control relative to DMSO treatment. (D) Representative experiment shows that the staining of F-actin by rhodamine-phalloidin, from nonstimulated (DMSO) or stimulated (pomalidomide) CD4⁺ T cells for indicated concentrations in the presence or absence of ROCK1 and Rac1 inhibitors. Cells were preincubated for 30 minutes in the presence of 10 μM ROCK1 inhibitor or Rac1 inhibitor followed by DMSO or pomalidomide stimulation. F-actin staining of 300 cells from 3 random fields was quantified in each experiment condition (right panel). Data represent 3 independent experiments. Columns and error bars represent mean ± SEM (Student t test, *P < .01).