Regulation of cytoskeletal organization and junction assembly in low-density cultures. (A and B) HDMEC cells were transfected with siRNAs as indicated and then incubated with Blebbistatin, CK666, or SMIFH2 for the last 24 h before fixation. The cells were then stained for F-actin to visualize cytoskeletal organization (A) or JAM-A to monitor junction assembly (B). (C and D) Cells were plated at low density and then transfected with siRNAs. Before reaching full confluence, the cells were fixed and stained as indicated. Shown are images of areas where cells were already confluent or partially confluent, and images of cells without or with only little cell–cell contact. If indicated, the cells were incubated with the ROCK inhibitor Y27632 for 24 h before fixation. Bars, 20 µm.