Figure 2. ZO-1, ZONAB, and F-actin expression in TrypLE-prepared BCECs with or without Y-27632 on days 1, 3 (subconfluence), and 7 (confluence). In the TrypLE-prepared bovine corneal endothelial cells (BCECs) without Y-27632, TJ protein ZO-1 developed gradually from the nucleus to the cell–cell contact and formed a continuous hexagonal pattern. ZONAB expression exhibited increasing staining in the nucleus, as in ZO-1 expression at the cellular borders (A, B: day 1; D, E: day 3; G, H: day 7). The F-actin cytoskeleton was gradually arranged into a dense peripheral band at the cell margin and cortical actin mat with prominent perinuclear staining from days 1 to 7 (C, F, I). The effect of Y-27632 on the expression of the ZO-1 (A’, D’, G’), ZONAB (B’, E’, H’), and F-actin cytoskeleton (C’, F’, I’) was similar to that without Y-27632. The cellular morphology became irregular (G, G’), and there was a marked decrease in the amount of F-actin in the presence of Y-27632 compared with the BCECs without Y-27632 on day 7 (I, I’). However, the ZONAB expression patterns were similar in the BCECs with and without Y-27632 (B, E, H, B’, E’, H’).