Fig 2: Nuclear accumulation of WRAP53 promoted DSB repair.(A) WRAP53 knockdown was performed by siRNA (siWrap53) transfection for 48 hours and neurons were subjected to OGD, as indicated in Fig. 1. Neurons were immunostained for ?H2AX and 53BP1 at 8 hours after OGD. Representative images are shown. DAPI, nuclear staining. Scale bar, 5 µm. Quantification of 53BP1 foci–positive neurons (percentage of neurons costaining ?H2AX and 53BP1 foci) and percentage of ?H2AX foci–positive and 53BP1 foci–negative neurons. The error bars are standard error deviation of 300 measurements from three different neuronal cultures (90 to 110 neurons per culture. *P < 0.05 versus normoxia; #P < 0.05 versus siControl OGD; two-way ANOVA followed by the Bonferroni post hoc test). (B) Neurons were transfected with plasmids encoding GFP, GFP-WRAP53, and MT-GFP-WRAP53 (MT sequence) for 24 hours and were subjected to OGD, as indicated in Fig. 1. Neurons were immunostained for GFP, ?H2AX, and 53BP1 at 8 hours after OGD. Representative images are shown. Scale bar, 5 µm. Quantification of 53BP1 foci–positive neurons (percentage of neurons costaining ?H2AX and 53BP1 foci) and percentage of ?H2AX foci–positive and 53BP1 foci–negative neurons. The error bars are standard error deviation of 30 to 35 measurements from three different neuronal cultures (10 to 15 GFP+ neurons per culture. *P < 0.05 versus GFP; #P < 0.05 versus GFP-WRAP53; one-way ANOVA followed by the Bonferroni post hoc test).

Fig 3: Oxidative stress is necessary to up-regulate WRAP53 expression and promote its traffic to the nucleus.Neurons were treated with the topoisomerase inhibitor, camptothecin, and the mitochondrial inhibitor, rotenone, for 8 and 4 hours, respectively. (A and B) WRAP53 and ?H2AX expression levels detected by Western blotting. GAPDH was probed as loading control. Representative blots are shown. Protein abundance quantification from three different neuronal cultures is shown in fig. S5B. (C and D) Neurons were transfected with WRAP53-GFP for 24 hours and were subjected to either OGD, as indicated in Fig. 1, or to camptothecin and rotenone treatments as described above. (C) Representative images of cortical neurons stained with GFP and MAP2 (neuronal marker). Scale bar, 25 µm. (D) Representative cross-sectional intensity profiles for GFP (green) and DAPI (blue) staining of GFP-WRAP53–transfected neurons.

Fig 4: WRAP53 knockdown enhanced DNA DSBs and neuronal susceptibility to OGD-induced apoptosis.WRAP53 knockdown was performed by siRNA (siWrap53) transfection for 48 hours, and then neurons were subjected to OGD, as indicated in Fig. 1. (A) Time course of ?H2AX and active caspase-3 expression levels detected by Western blotting. GAPDH was probed as loading control. Representative blots are shown. Protein abundance quantification from three different neuronal cultures is shown in fig. S2 (C and D). (B) The percentage of TUNEL-positive siControl and siWrap53-transfected cells was detected by flow cytometry at different time points after OGD. Data are means ± SEM from three different neuronal cultures (#P < 0.05 versus siControl OGD; two-way ANOVA followed by the Bonferroni post hoc test). (C and D) Analysis of caspase-3 activity and neuronal apoptosis in siControl and siWrap53-transfected neurons performed at different time periods after OGD. Data are means ± SEM from three different neuronal cultures (#P < 0.05 versus siControl OGD; two-way ANOVA followed by the Bonferroni post hoc test).

Fig 5: Mitochondrial generation of ROS triggers WRAP53 nuclear accumulation after OGD.Neurons were subjected to OGD in the presence of the mitochondrial ROS production inhibitor, MitoSNO (mitochondria-selective S-nitrosating agent). As control, neurons were incubated with SNAP, which do not affect mitochondrial S-nitrosation and respiration. (A) Mitochondrial ROS generation after OGD. The error bars are standard error deviation from three different neuronal cultures (#P < 0.05 versus SNAP OGD; two-way ANOVA followed by the Bonferroni post hoc test). (B) Time course of WRAP53 and ?H2AX expression levels as detected by Western blotting. GAPDH was probed as loading control. Representative blots are shown. Protein abundance quantification from three different neuronal cultures is shown in fig. S5D. (C and D) Neurons were transfected with GFP-WRAP53 for 24 hours and were treated with equal doses of SNAP or MitoSNO during OGD (3 hours), as indicated above. (C) Representative images of cortical neurons stained with GFP and MAP2 (neuronal marker). Scale bar, 25 µm. (D) Representative cross-sectional intensity profiles for GFP (green) and DAPI (blue) staining of representative GFP-WRAP53-transfected neurons. a.u., arbitrary units.