Fig 2: FBXO3 facilitated inflammation probably through binding and degrading HIPK2 in HT22 cells after OGD/R stimulation. (A,C) WB analysis of HIPK2 in HT22 cells at OGD4 h/R 24 h with or without siRNA treatment. (B,D) WB analysis of HIPK2 in HT22 cells at OGD4 h/R 24 h with DMSO or BC-1215 treatment. (E) Co-Immunoprecipitation (Co-IP) analysis of FBXO3 and HIPK2 in HT22 cells. (F) Representative immunofluorescence images (200×, scale bar = 100 µm) of FBXO3 (red) and HIPK2 (green) in HT22 cells at OGD4 h/R 24 h with or without siRNA treatment. Statistics for each group are expressed as mean ± SD (n = 6). * p < 0.05 (values in Control group versus MCAO group), # p < 0.05 (values in NC group versus si-FBXO3 group), && p < 0.01 (values in DMSO group versus BC-1215 group).

Fig 3: Inhibition of FBXO3 alleviated inflammatory response induced by I/R injury in vitro. (A,B) WB analysis of inflammatory cytokines including IL-1ß, IL-18, and TNFa in HT22 cells at OGD 4 h/R 24 h with or without siRNA treatment. (C,D) ELISA analysis of inflammatory cytokines including IL-1ß and IL-18 in HT22 cells at OGD 4 h/R 24 h with or without siRNA treatment. (E,F) WB analysis of inflammatory cytokines including IL-1ß, IL-18, and TNFa in HT22 cells at OGD 4 h/R 24 h with DMSO or BC-1215 treatment. (G,H) ELISA analysis of inflammatory cytokines including IL-1ß and IL-18 in HT22 cells at OGD 4 h/R 24 h with DMSO or BC-1215 treatment. Statistics for each group are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, **** p < 0.0001 (values in Control group versus MCAO group), # p < 0.05, ## p < 0.01, ### p < 0.001 (values in NC group versus si-FBXO3 group), & p < 0.05, && p < 0.01, &&& p < 0.001 (values in DMSO group versus BC-1215 group).

Fig 4: Schematic illustration of FBXO3 driving neuroinflammation to aggravate neuronal damage in cerebral I/R injury by degrading HIPK2.

Fig 5: Interference of FBXO3 rescued the neurological outcomes after MCAO/R in vivo. (A–C) WB and qPCR analysis of FBXO3 in the peri-infarcted cortex of MCAO/R rats with or without siRNA treatment and in sham rats. (D) Representative TTC (2,3,5-triphenyltetrazolium chloride) staining images of coronal sections of MCAO/R rats with or without siRNA treatment and sham rats. (E) Quantification of infarct volumes of coronal sections of MCAO/R rats with or without siRNA treatment and sham rats by ImageJ (total lesion volume/total brain volume × 100%). (F) Neurological deficit scores of MCAO/R rats with or without siRNA treatment and sham rats. (G) HE and Nissl staining (400×, scale bar = 50 µm) in the peri-infarcted cortex of MCAO/R rats with or without siRNA treatment and in sham rats. The arrows indicate representative Nissl bodies. Statistics for each group are expressed as mean ± SD (n = 6). ** p < 0.01, **** p < 0.0001 (values in Sham group versus MCAO group), and # p < 0.05, #### p < 0.0001 (values in NC group versus si-FBXO3 group).