Fig. 3. Neural progenitor cells (NPCs) overexpressing arginine decarboxylase (ADC) genes (ADC-NPCs) accelerate cell cycle exit via aberrant nucleocytoplasmic shuttling of phospho-retinoblastoma protein (p-pRB) and E2F1 during ischemic stress. (A) Immunocytochemistry, and correlation analysis (by Pearson's correlation coefficient (PCC)) of CDK4 (B), phospho-retinoblastoma protein (p-pRB) (C), pRB (D), and E2F1 (E). CDK4 accumulated in the nuclei of the ADC-NPCs following oxygen-glucose deprivation (OGD) injury (B). p-pRB increased significantly in the ADC-NPCs under normal and OGD conditions (C). However, there were no significant changes in the levels of pRB in the cytoplasmic fractions of the wild-type NPCs (wt-NPCs) and ADC-NPCs under normal conditions (D). The levels of E2F1 were significantly reduced in the nuclear fractions of the ADC-NPCs under normal and OGD conditions (E). Fluorescence intensity (Manders' overlap coefficient, MOC) graphs representing CDK4 (F), p-pRB (G), pRB (H), and E2F1 (I) proteins. (J) Representation of nucleocytoplasmic shuttling of cell cycle-related components in the ADC-NPCs. (K) Western blot of cytoplasmic and nuclear protein expression. Quantification graphs of CDK4 (L), p-pRB (M), pRB (N), and E2F1 (O). As with the immunocytochemical staining results, hyperphosphorylated pRB (p-pRB Ser 807/811) increased significantly in the cytoplasmic fraction of the ADC-NPCs under normal and OGD conditions. However, the levels of E2F1 were significantly reduced in the cytoplasmic fraction of the ADC-NPCs under normal and OGD conditions. The error bars represent the mean±SEM.
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