Fig 2: The effects of the selective activator/inhibitor of PSMD12 and NF-κB, Betulinic acid/benzoxathiole, on the silenced RPS27A in I/R mice A, The mRNA expression of PSMD12 in the cerebral cortical tissues of I/R-injured mice in response to overexpression or silencing of RPS27A as determined by RT-qPCR. B, The protein expression of PSMD12, IκBα and NC-p65 and the phosphorylation level of IκBα in the cerebral cortical tissues of I/R-injured mice in response to overexpression or silencing of RPS27A as determined by Western blot. C, RT-qPCR to measure the mRNA expression of PSMD12 in the cerebral cortical tissues of the I/R-injured mice in response to silenced PSMD12 or betulinic acid. D, Behavioral evaluation of cerebral injury in the I/R-injured mice in response to silenced PSMD12 or betulinic acid. E, TTC staining to detect the cerebral infarct size of I/R-injured mice in response to silenced PSMD12 or betulinic acid. F, Nissl and TUNEL staining to detect the neuron cell injury and apoptosis in I/R-injured mice in response to silenced PSMD12 or betulinic acid, respectively. G, Detection of the levels of GSH, MDA and ROS in the cerebral cortical tissues of the I/R-injured mice in response to silenced PSMD12 or betulinic acid. H, The protein expression of PSMD12, IκBα and NC-p65 and the phosphorylation level of IκBα in the cerebral cortical tissues of mice in response to silenced PSMD12 or betulinic acid as determined by Western blot. I, ELISA to measure the release of inflammatory factors in the cerebral cortical tissues of I/R-injured mice in response to silenced PSMD12 or betulinic acid. J, The content of neutrophils in the ischemic hemispheres of I/R-injured mice in response to silenced PSMD12 or betulinic acid as determined by flow cytometry. K, Immunofluorescence staining to detect the number and distribution of neutrophils in the whole brain of I/R-injured mice in response to silenced PSMD12 or betulinic acid. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the sham group or control group. ## p < 0.01 vs. the RPS27A-oe-NC group. && p < 0.01 vs. the RPS27A-sh-NC group

Fig 3: Improvement of I/R-induced Brain Damage by RPS27A Silencing in Mice. A, RT-qPCR to measure the mRNA expression of RPS27A in the cerebral cortical tissues of sham-operated mice and I/R-injured mice with overexpression or silencing of RPS27A. B: Immunofluorescence detection of the subcellular localization of RPS27A in transfected cells, microglial cells, and neurons. C, Behavioral evaluation of neurological injury of sham-operated mice and I/R-injured mice with overexpression or silencing of RPS27A. D, TTC staining to detect area of cerebral infarction in sham-operated mice and I/R-injured mice with overexpression or silencing of RPS27A. E, Nissl staining to detect neuron injury in cerebral cortical tissues of sham-operated mice and I/R-injured mice with overexpression or silencing of RPS27A. F, TUNEL staining to detect neuron apoptosis in the cerebral cortical tissues of sham-operated mice and I/R-injured mice with overexpression or silencing of RPS27A. G, Measurement of GSH, MDA and ROS production in the cerebral cortical tissues of sham-operated mice and I/R-injured mice with overexpression or silencing of RPS27A. n = 6. *** p < 0.001 vs. the sham-operated mice. ## p < 0.01, ### p < 0.001 vs. the RPS27A-oe-NC group. & p < 0.05, && p < 0.01, &&& p < 0.001 vs. the RPS27A-sh-NC group

Fig 4: Schematic illustration of the molecular mechanism of RPS27A for inflammatory factor secretion by microglia and immune infiltration in cerebral I/R injury. RPS27A activates the NF-κB pathway through the PSMD12/NF-κB axis, thereby promoting the release of inflammatory factors in microglia, which induces immune infiltration in I/R-injured brain tissues and finally exacerbates cerebral I/R injury in mice

Fig 5: Effects of RPS27A on inflammatory factors in the brain of I/R-injured mice. A, ELISA to detect release of inflammatory factors in cerebral cortical tissues of I/R-injured mice in response to overexpression or silencing of RPS27A. B, Flow cytometry to determine immune cells in ischemic hemisphere of I/R-injured mice in response to overexpression or silencing of RPS27A. C, Immunofluorescence staining to measure number and distribution of neutrophils (indicated by red) in the whole brain of I/R-injured mice in response to overexpression or silencing of RPS27A. D, Flow cytometry to determine immune cells in peripheral blood collected from eyeballs of I/R-injured mice in response to overexpression or silencing of RPS27A. n = 6. ** p < 0.01 vs. the sham group. ## p < 0.01 vs. the RPS27A-oe-NC group. & p < 0.05, &&& p < 0.001 vs. the RPS27A-sh-NC group