Figure 2
Astrocyte elevated gene-1 (AEG-1) mediates astrocyte migration during wound healing. Human astrocytes were transfected with AEG-1 specific siRNA (siAEG-1) or non-targeting, scrambled siRNA (siCon) by nucleofection and plated for 48 h. Messenger RNA was isolated at 24 h and 120 h post recovery and AEG-1 levels were measured by real-time polymerase chain reaction (PCR) (***P <0.001, (A)). In parallel experiments, immunoblotting was performed for AEG-1 at 48 h; β-actin was used as normalizing loading control (**P <0.001, (B)). Transfected astrocytes were plated to confluence for 48 h into OrisTM migration assay plates and then injured by removal of the cell-seeding stopper. Migrating astrocytes were visualized with Hoechst nuclear stain at various time points. Micrographs are shown for siCon-transfected (C1,C3,C5,C7) and siAEG-1-transfected astrocytes (C2,C4,C6,C8). The number in the lower right corner of the white box (C1-C8) show number of cells in the area of the injury. Separate images from four replicate wells were analyzed to quantify the number of cells present (***P <0.001, (D)). Representative data from three individual donors assayed in triplicate is shown.