Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells

Chalcomoracin Prevents Vitreous-Induced Activation of AKT and Migration of Retinal Pigment Epithelial Cells

“…This experiment was performed as described in previous reports [29]. Briefly, when cells were grown to 90% confluence in 24-well plates, they were serum starved for 24 h, and then treated with vitreous (0-24 h) from rabbits with PVR (RV, diluted 1:3 in DMEM/F12) or with CMR (5 μM) for 8 h. Proteins from treated cells were extracted in an extraction buffer and separated by 10% SDS-polyacrylamide gel electrophoresis.…”

“…This assay was performed as described in previous reports [29]. After reached approximately 90% confluence, they were digested with 0.5% tripsin-EDTA, counted and plated into wells of 24-well plates at a density of 3 × 10 4 cells/well in a full culture medium.…”

“…This assay was performed as described in previous reports [29]. Briefly, when ARPE-19 was grown to confluence in 24-well plates, autoclaved 200 μl pipet tips were used to scratch the cells across the wells.…”

“…The scratched areas were photographed to capture the initial width and photographed again 24 h later. The data were analyzed using Image J and Adobe Photoshop CS4 software as described previously [29]. At least three independent experiments were performed.…”

“…This assay was performed as described in previous reports [29]. In brief, when cells reached 90% confluence, they were re-suspended at a density of 1 × 10 6 in 1.5 mg/ml of neutralized PureCol type I bovine collagen solution (Advanced BioMatrix, San Diego, CA) (pH 7.2) on ice.…”