To investigate whether Nrdp1 is implicated in OGD-induced apoptosis in neurons, we transfected cerebral cortical neurons with Ad-control, Ad-Nrdp1 or Ad-si-Nrdp1. As shown in Figure 3, the transfection efficiency reached more than 90% at 24 h after transfection (Figure 3A), and western blot analysis showed that incubating the neurons with Ad-si-Nrdp1 and Ad-Nrdp1 for 48 h significantly reduced (~90% reduction) and increased (~2-fold increase) Nrdp1 protein levels, respectively (Figures 3B,C). We assessed the effect of Ad-si-Nrdp1 and Ad-Nrdp1 on OGD-induced neuronal death by measuring LDH release (indicating late apoptosis and necrosis) and TUNEL staining (indicating apoptosis). The data showed that cell death and apoptosis did not differ across the groups under basal conditions (Figures 3D,E and Supplementary Figure S2). However, after OGD treatment, inhibition of Nrdp1 significantly attenuated neuronal death and apoptosis as compared with the Ad-control, while transfection with the Ad-Nrdp1 greatly enhanced OGD-induced neurons death and apoptosis (Figures 3D,E and Supplementary Figure S2).