From: Activation of the Rho/Rho Kinase Signaling Pathway Is Involved in Cell Death of Corneal Endothelium Invest. Ophthalmol. Vis. Sci.. 2016;57(15):6843-6851. doi:10.1167/iovs.16-20123 Figure Legend: Effect of inhibition of ROCK/MLC pathway on apoptosis of MCECs. (A) Cultured MCECs were washed gently with PBS and exposed to UV (100 J/m2), followed by further incubation for 12 hours in culture medium supplemented with the pan-caspase inhibitor Z-VAD-FMK. Representative phase contrast images show that UV-induced cell detachment was suppressed by caspase inhibition. Scale bar: 100 μm. (B) Activation of caspase-3 and PARP was evaluated by Western blot after 12 hours of UV stimulation. Ultraviolet exposure induced partially cleaved 19-kDa and fully cleaved 17-kDa products of caspase-3. The UV treatment also induced PARP cleavage in MCECs. By contrast, Z-VAD-FMK suppressed the appearance of the 17-kDa cleavage product of caspase-3 and the 89-kDa cleavage product of PARP, showing that damage to the MCECs caused by UV is mainly due to apoptosis. (C) Cleavage of caspase-3 was evaluated by Western blot after UV irradiation of MCECs. Y-27632 and blebbistatin (an inhibitor of MLC) treatment suppressed cleavage of caspase-3 in the UV-treated cells. (D, E) Monkey corneal endothelial cells were exposed to UV (100 J/m2) and then cultured in Dulbecco's modified Eagle's medium supplemented with Y-27632/blebbistatin for 24 hours. The presence of apoptotic cells was evaluated by staining the MCECs with Annexin V. Evaluation of the numbers of Annexin V–positive apoptotic cells produced in response to UV irradiation revealed a significant suppression by Y-27632 or blebbistatin treatment. Values are the averages of four independent images. Experiments were performed in triplicate. Nuclei were stained with DAPI. Scale bar: 50 μm. *P < 0.01. (F, G) Apoptotic cells were evaluated by TUNEL staining. The numbers of TUNEL-positive apoptotic cells were evaluated. Values are the averages of four independent images. Experiments were performed in triplicate. Nuclei were stained with DAPI. Scale bar: 50 μm. *P < 0.01. (H) Monkey corneal endothelial cells were stimulated with hydrogen peroxide by culturing with Dulbecco's modified Eagle's medium supplemented with hydrogen peroxide (1000 μM) for 24 hours. Phosphorylation of MLC was evaluated by immunostaining. Actin was stained by phalloidin and nuclei were stained with DAPI. Scale bar: 50 μm. (I, J) After stimulation by hydrogen peroxide (1000 μM) for 24 hours, MCECs were evaluated by phase contrast microscopy. Annexin V–positive apoptotic cells were evaluated by flow cytometry. Scale bar: 50 μm. *P < 0.01. Date of download: 10/20/2017 The Association for Research in Vision and Ophthalmology Copyright © 2017. All rights reserved.