Thrombin-induced increase in ROCK-II transcription and protein in HMEC-1 cells. (A) Time-dependent changes in the expression levels of genes linked to Rho pathways. cDNA microarrays were performed as described in “Materials and methods.” The graph represents individual profiling of the genes described in Table S1. (B) Validation of microarray data with real-time quantitative PCR analysis of ROCK mRNA: changes in ROCK-II mRNA (▴) and ROCK-I mRNA (•) were normalized to that observed with 18S ribosomal mRNA, as described in “Materials and methods.” Results were analyzed by the Δ^ΔCt method that reflects the difference in threshold for the target gene relative to that of 18S in each sample, and are expressed as fold induction of ROCK-II or ROCK-I mRNA expression in thrombin-stimulated HMEC-1 cells compared with time-matched untreated HMEC-1 cells in 3 independent experiments. (C) Representative experiments of time-dependent modulation in ROCK-II and ROCK-I proteins in thrombin-treated HMEC-1 cells. Each cell lysate (20 μg) was resolved on 4% to 12% SDS-PAGE gradient under reducing conditions. The blots were probed with anti–ROCK-II monoclonal antibody (top panel) or anti–ROCK-I polyclonal rabbit antibody (center panel) or with antitubulin as loading controls (bottom panel). (D) Cytoskeleton rearrangement in response to thrombin. Serum-starved HMEC-1 cells were cultured on glass chambers and stained for actin or vinculin, as described in “Materials and methods.” After 4 hours of stimulation, images were acquired on an inversed fluorescent microscope and were captured using the Lucias software for Nikon. Control (i, ii, v, vi) indicates unstimulated cells; thrombin (iii, iv, vii, viii), HMEC-1 cells stimulated with 1 IU/mL thrombin for 4 hours. When required, 1μM Y27632 was added 1 hour before stimulation and was maintained in the culture medium throughout the stimulation step. Objective magnification, 40×/0.60 NA.