Sertad1 is upregulated and mainly localized in neurons after MCAO/R. (A) The expression levels of Sertad1 mRNA were analyzed by RT-qPCR (n = 6). (B) The expression levels of Sertad1 protein were analyzed by Western blot (n = 6). (C) The immunofluorescence signal of Sertad1 (green) was colocalized with NeuN (neurons marker, red) in the peri-infarct region of the cortex and hippocampus after MCAO/R. Scale bars = 30 μm and 100 μm. **P < 0.01 compared with the sham group.|@|~(^,^)~|@|Downregulation of MCAO/R-induced Sertad1 by AAV-shSertad1. (A) Immunofluorescence analysis was used to determine the silencing efficiency of AAV-shSertad1 in the peri-infarct region of the cortex and hippocampus 2 days after MCAO/R. Sections were stained for Sertad1 (red), NeuN (blue), and EGFP (green). Scale bars = 30 μm and 100 μm. (B and C) The silencing efficiency of AAV-shSertad1 2 days after MCAO/R was confirmed by RT-qPCR and Western blot (n = 6). **P < 0.01.|@|~(^,^)~|@|Sertad1 inhibition ameliorated stroke outcomes. (A) TTC staining was used to determine the infarct volume in the mice that underwent the indicated treatment at 48 h after MCAO/R (n = 6). (B) Neurological function in the mice that underwent the indicated treatment was measured using the neurological deficit score at 48 h after MCAO/R (n = 18). (C and D) TUNEL assay was used to determine the dead neurons in the peri-infarct region of the cortex and hippocampus of mice at 48 h after MCAO/R. Sections were stained for TUNEL (red), NeuN (blue), and EGFP (green) (n = 6). Scale bars = 30 μm and 100 μm. (E) The expression levels of Cleaved-caspase3 protein in the peri-infarct region of the cortex and hippocampus of mice at 48 h after MCAO/R were analyzed using Western blot (n = 6). **P < 0.01.|@|~(^,^)~|@|Effect of OGD/R on cell viability, Sertad1 expression and apoptosis in HT22 cells. (A) HT22 cells were cultured in a glucose-free DMEM solution and maintained in a hypoxic chamber with 94% N₂, 1% O₂, and 5% CO₂ at 37°C for 2, 4, 8, or 12 h. Then, they were reoxygenated for 24 or 48 h in a complete medium. The HT22 cells in the normal group were subjected to OGD for 0 h and reoxygenated for 24 h or 48 h in a complete medium. Cell viability was determined using CCK-8. (B and C) The expression levels of Sertad1 mRNA and protein were analyzed using RT-qPCR and Western blot. (D) After OGD for 8 h and reperfusion for 24 h, the cell death of HT22 cells was measured by TUNEL. Scale bar = 50 μm. (E) The expression levels of Cleaved-caspase3 protein in HT22 cells after OGD/R were analyzed using Western blot. Data were presented as the mean ± SD obtained from three independent experiments for three well replicates. **P < 0.01 compared with the normal group. ^##P < 0.01 compared with the normal group.|@|~(^,^)~|@|OGD/R-induced Sertad1 upregulation was involved in neurological injury in HT22 cells. (A) HT22 cells were transfected with the scramble control siRNA (siCon), Sertad1 siRNA (siSertad1), pcDNA3.1 negative control plasmid (pcDNA3.1), or Sertad1-3flag expression plasmid (Sertad1 ov) for 48 h and then subjected to OGD for 8 h and reperfusion for 24 h. The expression levels of Sertad1 protein were analyzed by Western blot. (B) Cell viability was analyzed using CCK-8 assay. (C and D) Cell death was analyzed using TUNEL assay. (E) The expression levels of Cleaved-caspase3 protein were analyzed using Western blot. Data were presented as the mean ± SD obtained from three independent experiments. Scale bar = 50 μm. **P < 0.01.|@|~(^,^)~|@|Sertad1 promoted the CDK4/p-Rb pathway by directly binding CDK4 in HT22 cells during OGD/R. (A) HT22 cells were transfected with the scramble control siRNA (siCon), Sertad1 siRNA (siSertad1), pcDNA3.1 negative control plasmid (pcDNA3.1), or Sertad1-3flag expression plasmid (Sertad1 ov) for 48 h and then subjected to OGD for 8 h and reperfusion for 24 h. Western blot was used to determine the expression levels of CDK4, p-Rb, Rb, B-Myb, and Bim proteins. (B) HT22 cells were transfected with pcDNA3.1 negative control plasmid (pcDNA3.1) or Sertad1-3flag expression plasmid (Sertad1 ov). Whole cell lysates were precipitated with the indicated antibody. Whole cell lysates and precipitated proteins were analyzed by Western blot (WB) with the indicated antibodies. Data were presented as the mean ± SD obtained from three independent experiments. **P < 0.01.|@|~(^,^)~|@|Sertad1 contributed to OGD/R-induced neurological injury in the HT22 cells by regulating the CDK4/p-Rb pathway. (A) HT22 cells were transfected with the pcDNA3.1 negative control plasmid (pcDNA3.1) or Sertad1-3flag expression plasmid (Sertad1 ov). After 8 h, the medium was changed to fresh medium with DMSO, 20 nM ON123300, or 50 nM ON123300, and HT22 cells were further incubated for 48 h. HT22 cells were then subjected to OGD for 8 h and reperfusion for 24 h. Western blot was used to determine the expression levels of CDK4, p-Rb, Rb, B-Myb, and Bim proteins. (B and C) Cell viability and cell death was analyzed by CCK-8 and TUNEL assays, respectively. Scale bar = 50 μm. (D) The expression levels of Cleaved-caspase3 protein were analyzed using Western blot. Data were presented as the mean ± SD obtained from three independent experiments. **P < 0.01.|@|~(^,^)~|@|Sertad1 regulated the CDK4/p-Rb pathway during stroke. (A) Western blot was used to determine the effects of Sertad1 on the expression levels of p-Rb, Rb, B-Myb, and Bim proteins in brain tissues from the peri-infarct region of the cortex and hippocampus 48 h after MCAO/R. (B) Schematic of the mechanism of Sertad1 in neuronal injury during stroke. Data were presented as the mean ± SD obtained from three independent experiments. **P < 0.01.

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