Fig 2: RTN1-C induces mitochondrial-associated apoptosis during OGD/R. (a) Representative western blot analysis of cytosolic cytochrome C (cyt-Cyto C), mitochondrial cytochrome C (mito-Cyto C) and cleaved-caspase-3 in N2a cells exposed to OGD/R treatment or normal condition, after transfection with RTN1-C-FLAG or shRTN1-C. GAPDH was used as a loading control. Fractionation experiment was performed before western blot. (b) Histograms showing quantification of images in a. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05; **P<0.01; ***P<0.001. (c) Representative western blot analysis of mitochondrial and cytosolic Bcl-xL in N2a cells transfected with RTN1-C-FLAG or empty vector for 24 h, then the cells exposed to OGD/R treatment or normal condition. COX IV and GAPDH were used as the markers of mitochondria and cytosol, respectively. (d) Histogram showing quantification of images in c. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05; **P<0.01; ***P<0.001. (e) Representative western blot analysis of mitochondrial and cytosolic Bcl-xL in N2a cells transfected with shRTN1-C or shNC for 24 h, then the cells exposed to OGD/R treatment or normal condition. COX IV and GAPDH were used as the markers of mitochondria and cytosol, respectively. (f) Histogram showing quantification of images in e. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05; **P<0.01. (g) Immunoprecipitation of endogenous Bcl-xL and RTN1-C. N2a cells were treated with OGD/R and the lysates were incubated with anti-RTN1-C antibody. Bcl-xL and RTN1-C were detected by immunoblotting with anti-Bcl-xL and anti-RTN1-C antibody, respectively. (h) Representative western blot analysis of mitochondrial and cytosolic Bcl-xL in N2a cells exposed to OGD/R treatment after transfection with RTN1-C-FLAG or shRTN1-C. COX IV and GAPDH were used as the markers of mitochondria and cytosol, respectively. (i) Histogram showing quantification of images in h. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05

Fig 3: RTN1-C downregulation using its specific shRNA reduced neural apoptosis in MCAO rats. (a) Rats were given intracerebroventricular injections with LV-shNC or LV-shRTN1-C for 7 days, then the rats were subjected to a middle cerebral artery occlusion (MCAO) for 2 h, followed by 12 or 24 h of reperfusion. The efficiency of LV-shRTN1-C in the brain tissues was detected by western blot. (b) Representative immunofluorescence staining of cleaved-caspase-3 in MCAO rats after given intracerebroventricular injections with LV-shNC or LV-shRTN1-C for 7 days. Cleaved-caspase-3 (red), RTN1-C (green) and DAPI (blue), Scale bar=20 µm. (c) Quantification of green fluorescence intensity of RTN1-C immunostaining in (b). Data are expressed as mean±S.D. n=5, ***P<0.001. (d) Representative TTC stains of 6 corresponding coronal brain sections. Ischemic infarctions appear white. (e) Brain infarct volumes were quantified. n=3, *P<0.05. (f) Neurological Bederson score assessed at day 1 after reperfusion in MCAO rats. n=5, **P<0.01

Fig 4: RTN1-C is upregulated during cerebral ischemia/reperfusion. Brain samples were collected from normal and ischemia/reperfusion (I/R) rat brain tissues. The male SD rats (n=3) were subjected to a middle cerebral artery occlusion (MCAO) for 2 h, followed by 12 or 24 h of reperfusion. (a) Representative western blot of three splice variants of RTN1 in the brain tissue samples from sham-operated and I/R rats. (b) Immunofluorescent staining of RTN1-C on brain sections from I/R rats. The upper panel is brain sections from sham-operated groups; the middle panel is the brain section from the group with MCAO for 2 h followed by 12 h of reperfusion; the lower panel is the brain section from the group with MCAO for 2 h followed by 24 h of reperfusion. Immunofluorescence was performed with anti-RTN1-C (green). DAPI was used to stain the nuclei (blue). The arrows highlight the RTN1-C expression. Scale bar=20 μm. (c) Representative immunofluorescent staining of RTN1-A on the brain sections of I/R rats. Immunofluorescence was performed with anti-RTN1-C (green). DAPI was used to stain the nuclei (blue). Scale bar=20 μm. (d) Representative western blot of RTN1-C in the brain tissue samples from I/R, ischemic postconditioning (IPC), Remifentanil postconditioning (RPC), and sham-operated rats

Fig 5: RTN1-C induces ER stress-associated apoptosis during OGD/R. (a) Representative western blot analysis of Grp78, cleaved-caspase-12, CHOP and cleaved-caspase-3 in N2a cells exposed to OGD/R treatment or normal conditions, after transfection with RTN1-C-FLAG or shRTN1-C. GAPDH was used as a loading control. (b) Histogram showing quantification of images in a. The results were normalized to GAPDH expression, and the quantitative data were expressed as the mean±S.D. from different assays (n=3). *P<0.05; **P<0.01; ***P<0.001. (c) Representative western blot analysis of Grp78, cleaved-caspase-12, CHOP and cleaved-caspase-3 in N2a cells transfected with RTN1-C-GFP or GFP vector in the absence or presence of 4-PBA for 6 h before OGD/R treatment. GAPDH was used as a loading control. (d) Histogram showing quantification of images in c. The results were normalized to GAPDH expression, and the quantitative data were expressed as the mean±S.D. from different assays (n=3). *P<0.05; **P<0.01; ***P<0.001