Fig. 4

GCN2 regulated autophagy, cell cycle arrest and apoptosis in retinoblastoma upon arginine deprivation. A Immunoblot analyses revealed that GCN2 was induced upon arginine deprivation in both Y79 and WERI-Rb-1 cells. B Fluorescence microscopy image showing GFP signals in cells and immunoblot data showing the efficiency of GCN2 knockdown in Rb cells. Scale bar, 300 μm. C Y79 and WERI-Rb-1 cells were treated with rhArg for 72 h. The data are shown as the means ± SEMs, n = 4–5. Differences were assessed by the Mann‒Whitney test: ns = not significant. D Representative images and colony counts of GCN2-knockdown WERI-Rb-1 cells. E Immunoblot analyses showing the changes in autophagy activity (conversion of LC3-I to LC3-II) in Rb cells following arginine deprivation. F Flow cytometry analyses of cell cycle arrest. The bar chart shows the mean ± SEM of the percentage of cells in each cell cycle phase after 72 h of exposure of Rb cells to rhArg (n = 4). Immunoblot analyses showing the changes in cyclin A and cyclin B. G Flow cytometry analyses of apoptosis. The bar chart shows the mean ± SEM of the percentage of early apoptotic cells 72 h after arginine deprivation (n = 4)