(A) The IOPs that were tracked daily after subconjunctival injection with Y-27632 or 0.9% saline. Cells Primary and immortal TM cells in medium were photographed using microscopy. Immunofluorescence staining revealed that both primary and immortal TM cells expressed TM biomarkers, including MMP3, TIMP3 and COL IV proteins (Physique 2A). The staining of unfavorable control group can be seen in Supplementary Material. We also Rabbit Polyclonal to OR6C3 compared the expression of myocilin, a glucocorticoid-inducible gene in the TM cells. Western blot showed the expressions of myocilin in primary and immortal TM cells were increased after DEX treatment (Physique 2B) and Biotin-HPDP the intensity of the visualized bands illustrated that DEX induced the expression of myocilin (? 0.05, Figure 2C). Cell morphology, immunofluorescence analysis, and western blot confirmed that these cell lines and isolated cells from human TM tissue had characteristics of TM cells. Open in a separate window Physique 2 Characterization of primary human trabecular meshwork (pTM) cells and immortal trabecular meshwork (TM) cells. (A) The morphology of pTM, immortal human trabecular meshwork cells (iHTM) and glaucomatous human trabecular meshwork cells (GTM3) in observed by phase contrast microscope. Positive staining of biomarkers, including TIMP3 (red), MMP3 (red) and COL IV (green) for TM cells. Cell nuclei were stained with DAPI (blue). Bar = 50 m. (B) Effect Biotin-HPDP of dexamethasone (DEX) for 5 days on induced the expression of myocilin in pTM, iHTM and GTM3 cells. (C) Intensity of visualized bands of myocilin proteins in control and DEX-treated cells from pTM and immortal TM cells. The results were quantified from three impartial experiments (= 3) by Image Lab software, and the expression of myocilin proteins was significantly higher in these TM cells after DEX treatment by unpaired 0.05. Y-27632 Modulated Cytoskeleton Characteristics and Promoted the Proliferation of iHTM Cells and GTM3 Cells 0.05, Figure 3B), whereas the CCK8 analysis of GTM3 cells revealed that treatment with all tested concentrations of Y-27632 caused significant increases in cell number compared with the control condition (??? 0.001, Figure 3C). Open in a separate window Physique 3 Effect of different concentrations of Y-27632 on cytoskeleton (F-actin) and cellularity in iHTM cells and GTM3 cells. (A) Immunofluorescence staining of F-actin (green). The nuclei were counterstained with DAPI (blue). The amplified part of the figures was in the upper right corner. Bar = 50 m. (B,C) Cell proliferation was analyzed using CCK-8 assay (= 6 impartial replicate experiments). Statistical analyses were performed using one-way ANOVA with Dunnetts test. * 0.05 and *** 0.001. Y-27632 Promoted the Proliferation of pTM Cells 0.05 and ?? 0.01, Physique 4A). The effect of Y-27632 was more obvious after 48 h than after 24 h. As shown in Physique 4B, positive Ki67 staining was present in a subset of pTM cells (pink), and the percentage of these positive cells was associated with the duration of treatment with Y-27632. The number of pTM cells that were positive for Ki67 was significantly greater than that in Biotin-HPDP the control condition (?? 0.01 and ??? 0.001, Figure 4C). Open in a separate window Physique 4 Y-27632 promoted the proliferation of pTM cells. (A) The cell numbers of pTM cells incubated with 100 M of Y-27632 for 24 and 48 h. Three impartial experiments were carried out (= 3). Bar = 50 m. (B) Immunofluorescent staining was performed with anti-Ki67 antibody (red). The nuclei were stained with DAPI (blue). (C) The percentage of Ki67-positive pTM cells (pink fluorescence in nuclei, arrows highlight several, but not all). Three random fields in each three coverslips were counted. Data were analyzed by unpaired -test. * 0.05, ** 0.01, and *** 0.001. Y-27632 Promoted the Phagocytosis of GTM3 Cells As shown in Physique 5A, fluorescent beads (green) were present in the cytoplasm, and the fluorescence intensity significantly differed between Y-27632-treated and control GTM3 cells (?? 0.01 and ??? 0.001, Figure 5B); the control iHTM cells also showed higher fluorescence than the Y-27632-treated GTM3 cells. FACS analyses confirmed that the intensity of the Y-27632 group was higher than that of the control GTM3 group (Physique 5C). These results indicate that Y-27632 promotes the phagocytosis of GTM3 TM cells. Open in a separate window Physique 5 Y-27632 promoted phagocytosis of GTM3 cells. (A) Photograph of TM cells 2 h after exposure to fluorescent beads. Phagocytosed particles fluoresced green. Bar = 50.