Figure 4.

Effects of ROCK-is on the mRNA expression of ECMs in 3D spheroids of human corneal stroma fibroblasts (HCSFs) and representative immune-fluorescein images showing their expressions. In the absence or presence of 1 μM or 10 μM ROCK-is, ripasudil (Rip), or KD025, 3D HCSFs spheroids at Day 6 (n = 10–15, each) were subjected to qPCR analysis, and the mRNA expression of ECM molecules including COL1, COL4, COL6, and FN was estimated (A). Alternatively, those 3D spheroids (n = 4–8 each) were immune-stained with specific antibodies to ECMs including collagen 1 (COL 1), collagen 4 (COL 4), collagen 6 (COL6), or fibronectin (FN) designated by green, DAPI by blue, and phalloidin by red under above conditions. Representative confocal images are shown (B) (Scale bar: 100 μm). The staining intensities of the ECMs of the spheroids that were stained as above are plotted in (C). All experiments were performed in duplicate using fresh preparations. Data are presented as arithmetic means ± standard error of the mean (SEM). *P < 0.05, **P < 0.01, ***P < 0.005, ***P < 0.001 (ANOVA followed by a Tukey’s multiple comparison test).