Toxicity assessment on confluent primary cell culture.

<p>Toxicity assessment on confluent primary cell culture.</p

% EdU positive cells and relative ECD after wound healing in vitro.

<p>% EdU positive cells and relative ECD after wound healing in vitro.</p

<i>In vitro</i> cell adhesion assay.

<p>(<b>A</b>) No differences in cell adhesion are observed between the different groups after 2 hours of incubation. After an incubation of 6 hours a higher number of HCEC adheres onto fibronectin matrix compared with lower incubation time. Y-27632 enhances cells adhesion after 6 hours compared to control medium. Medium (Low or High) is not involved in cell adhesion process. Values are means +/− SEM (n = 9). *P<0.05 (Student’s <i>t</i>-test). (<b>B</b>) Micrographs of Phalloidin staining. An actin remodeling after ROCK inhibitor treatment can be observed. (Magnification 40x) (n = 9).</p

<i>In vitro</i> morphological assessment.

<p>(<b>A</b>) Zo-1 immunostaining reveals a continuous or partially segmented expression at intercellular junction in both conditions. This expression pattern is completely lost after Y-27632 addition in Low or High Medium culture with more diffuse intracellular staining. (<b>B</b>) Actin staining reveals radial and circumferential actin bundles in control groups. Y-27632 induces cytoskeleton reorganization, characterized by a redistribution of actin microfilament on the periphery of the cells and apparition of some membrane ruffles. (Magnification 40x) (n = 9).</p

<i>In vitro</i> proliferation assessment during wound healing.

<p>(<b>A</b>) EdU incorporation evaluation during wound healing process. EdU staining can be observed in all conditions. Magnification 10x. (<b>B</b>) Proliferation rate (%) corresponding to the number of positive EdU nuclei out of the total number of nuclei (EdU+Dapi nuclei). High Medium increases EdU incorporation compared with Low Medium in control cultures but also in Y-27632 treated cultures during wound healing process. Furthermore, Y-27632 treatment decreases the number of EdU positive cells compared to control groups with a decrease of 23.6 and 30.7% in Low and High Medium respectively. (<b>C</b>) Relative ECD corresponding to the ratio between the number of nuclei and the ROI measurement area. Cells incubated in High Medium have a superior Relative ECD in control and treated group compared with cells incubated in Low Medium of respectively 26.4% and 29.6%. Y-27632 treatment decreases the Relative ECD of 17.9% in Low Medium and of 14.2% in High medium compared to controls. Values are means +/− SEM (n = 9). **P<0.001 and ***P<0.0001 (Student’s <i>t</i>-test).</p

<i>Ex vivo</i> morphological assessment.

<p>(<b>A</b>) Zo-1 immunostaining. In control corneas, endothelial cells appear as a mosaic of cells with polygonal shape. Addition of Y-27632 induces a morphological change with a loss of the polygonal shape and an irregular cell border, suggesting a disruption of the tight junctions. (<b>B</b>) Actin staining. In control corneas, actin filaments are assembled into large radial and circumferential bundles, with a main localization along the membrane of the endothelial cells. After treatment the distribution of F-actin are altered, with only a residual staining associated with the cell periphery. The formation of circular membrane ruffles of variable size and actin content can also be observed.</p

<i>Ex vivo</i> toxicity-viability and apoptosis assessment.

<p>(<b>A</b>) Toxicity evaluation by Ethidium Homodimer and Hoechst staining. Some dead cells are present in all conditions. (Magnification 10x.) (<b>B</b>) Mortality rate (%) corresponding to the number of positive Ethidium nuclei out of the total number of nuclei (Hoechst+Ethidium nuclei). No differences are observed between groups. (<b>C</b>) Relative Endothelial Cell Density (ECD) corresponding to the ratio between the number of nuclei and the area of the Region Of Interest (ROI). No differences are observed between groups. (<b>D</b>) Cell viability evaluation by Calcein AM and Hoechst staining. Magnification 4x. (<b>E</b>) Viability rate (%) corresponding to the ratio between the surface of positive Calcein cells and the field measurement area. No differences are observed between groups. (<b>F</b>) Apoptosis evaluation by Caspase3 immunostaining. Caspase3 positive cells were rarely observed. No differences are observed between groups. (Magnification 40x.) Values are means +/− SEM (n = 3 pairs). No differences are observed between groups.</p

<i>In vitro</i> wound healing kinetic.

<p>(<b>A</b>) Representative micrographs of the wound healing kinetic. Wounds are totally closed whatever the conditions incubation after 24 hours. Compared to controls, Y-27632 enhances endothelial wound closure and cells adopt a fibroblastic-like appearance 6 hours after treatment. Magnification 10x. (<b>B</b>) Relative wound closure kinetic. Y-27632 significantly increases endothelial wound closure compared to controls (P = 0.0004). Globally no differences are observed between the Low and the High Medium in the wound healing process. a-d: P<0.05; a: Low Medium Control vs. Low Medium +Y-27632; b: High Medium Control vs. High Medium +Y-27632; c: Low Medium Control vs. High Medium Control; d: Low Medium +Y-27632 vs. High Medium +Y-27632 (2way Anova test; n = 9).</p

<i>In vitro</i> toxicity-viability and apoptosis assessment.

<p>(<b>A</b>) Toxicity and viability evaluation by triple staining Hoechst (nuclei) Ethidium (dead cells) and Calcein (viable cells). Methanol-fixed cultures act as positive controls for cell death. Few dead cells are present in all conditions and Calcein staining shows no differences in the cell viability after Y-27632 treatment. (<b>B</b>) Mortality rate (%) corresponding to the number of positive Ethidium nuclei out of the total number of nuclei (Hoechst+Ethidium nuclei). No differences are observed between all groups. (<b>C</b>) Relative Endothelial Cell Density (ECD) corresponding to the ratio between the number of nuclei and the Region Of Interest (ROI) measurement area. No differences are observed between all groups. (<b>D</b>) Apoptosis evaluation by Caspase3 immunostaining. No staining is observed in all groups. (Magnification 10x). Values are means +/− SEM (n = 9).</p

<i>Ex vivo</i> proliferation assessment.

<p>(<b>A</b>) Proliferation evaluation by Endothelial Cell Density (ECD) determination. ECD after corneas procurement is comparable in the control and the treated corneas. A cell loss of 12% and 9% for control and treated corneas respectively is observed after corneas storage during 14+/−2 days with no significant differences between both groups. A similar cell loss is observed in control and Y-27632 treated corneas after 13±3 days of preservation with respectively 8% and 9%. The threshold of 2000 cells/mm² conventionally used to deliver grafts for penetrating keratoplasty is shown as a green dotted line. Values are means +/− SEM (n = 6 pairs). (<b>B</b>) Proliferation evaluation by EdU incorporation and Ki67 immunostaining. Only few EdU and Ki67 positive cells were detected in both groups. (Magnification 40x).</p