Figure 2

RTN1-C promotes apoptosis during cerebral ischemia/reperfusion. (a) Representative immunofluorescence photomicrography showing the expression of RTN1-C protein and cleaved-caspase-3 protein in sham-operated rats (the upper panels), or MCAO rats (the bottom panels). The expressions of RTN1-C (green) are shown by white arrows and magnification in merged panels. cleaved-caspase-3 (red) and the nuclei (DAPI, blue). Scale bar=20 μm. (b) N2a cells were transfected with RTN1-C-GFP or GFP vector, then exposed to OGD for 3 h and reoxygenation for 4 h, or maintained under normal conditions. Cell viability was detected with CCK-8. n=5, ***P<0.001. (c) N2a cells were transfected with shRTN1-C or shNC (non-targeting shRNA), then exposed to OGD for 3 h and reoxygenation for 4 h, or normal conditions. Cell viability was detected with CCK-8. n=5, ***P<0.001. (d) The effectiveness of shRNA of RTN1-C was monitored by western blot. N2a cells were transfected with four different shRNA against RTN1-C (shRTN1-C- 637; shRTN1-C -833; shRTN1-C -785; shRTN1-C -587), shRNA against GAPDH and shNC (non-targeting shRNA) for 24 h, following by western blot analysis. (e) N2a cells were transfected with RTN1-C-GFP or GFP vector, then exposed to OGD for 3 h and reoxygenation for 4 h, or normal conditions. After OGD/R treatment, apoptosis in the cells was detected by flow cytometry with Annexin V-PE/7-AAD staining. n=3, *P<0.05; **P<0.01. (f) N2a cells were transfected with shRTN1-C or shNC (non-targeting shRNA), then exposed to OGD for 3 h and reoxygenation for 4 h, or normal conditions. After OGD/R treatment, apoptosis in the cells was detected by flow cytometry with Annexin V-PE/7-AAD staining. n=3, *P<0.05