GPNMB regulates EGFR mitochondrial translocation via HK2, influencing microglial respiratory chain and metabolic defects to promote polarization and stroke progression

Fig. 1. GPNMB is upregulated in microglia after MCAO in mice. A. Transcriptomic analysis of microglia isolated from a rat model of focal cerebral ischemia using the GEO dataset (GSE148350) shows significant upregulation of GPNMB. B. TTC staining of brain sections from sham-operated and MCAO mice. C. HE staining of brain tissues from sham and MCAO mice. D. Nissl staining of the hippocampus from sham and MCAO mice. E. Immunofluorescence staining for GPNMB in brain sections from sham and MCAO mice. F. Immunofluorescence staining for CD16/32 and CD206 microglial polarization markers CD16/32, IBA1, CD206 in brain sections from sham and MCAO mice, P = 0.0001, P = 0.0001. N = 5 animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 2. GPNMB knockout reduces ischemic injury and restores mitochondrial function in microglia. A. TTC staining of brain sections, P = 0.0001, P = 0.0001. B. HE staining of brain tissues from the same groups to assess neuronal damage. C. Nissl staining of hippocampal sections from mice. D. Immunofluorescence staining for GPNMB in microglia. E. Western blot analysis confirming GPNMB knockout and qPCR detection of GPNMB expression changes in microglia, P = 0.0001, P = 0.0001. F. Western blot analysis of GPNMB expression in hippocampal tissues from the same groups, P = 0.0001, P = 0.0005. G. ATP levels measured in hippocampal tissues, P = 0.0001, P = 0.0001. H. JC-1 staining and flow cytometry analysis to assess mitochondrial membrane potential in microglia, P = 0.0001, P = 0.0001. I. Western blot analysis of mitochondrial respiratory chain complex proteins (CYTB, MTCO2, ATP6) in hippocampal tissues, P = 0.0001, P = 0.0002; P = 0.0001, P = 0.0237; P = 0.0001, P = 0.0007. J. Immunofluorescence staining for CD16/32 and CD206 microglial polarization markers CD16/32, IBA1, CD206 in brain sections, P = 0.0001, P = 0.0001, P = 0.0001, P = 0.0003. N = 5 animals per group. *, P < 0.05; **, P <…

Fig. 3. GPNMB knockdown restores mitochondrial function and modulates microglial polarization. A. qPCR (P = 0.0001, P = 0.0009) and Western blot analysis (P = 0.0002, P = 0.0038) of GPNMB expression in microglial cells. B. CCK-8 assay to assess cell viability, P = 0.0003, P = 0.0056. C. Immunofluorescence staining for GPNMB. Nuclei are stained with DAPI (blue). D. ATP levels measured in microglial cells, P = 0.0001, P = 0.0037. E. JC-1 staining and flow cytometry analysis to assess mitochondrial membrane potential in microglial cells, P = 0.0001, P = 0.0017. F. Western blot analysis of mitochondrial respiratory chain complex proteins (CYTB, MTCO2, ATP6) in microglial cells, P = 0.0076, P = 0.0009; P = 0.0001, P = 0.0002; P = 0.0002, P = 0.0002. G. Immunofluorescence staining for CD16/32 and CD206 microglial polarization markers CD16/32, IBA1, CD206 in microglial cells, P = 0.0001, P = 0.0001; P = 0.0006, P = 0.0003. N = 3 repeated experiments. **, P < 0.01; ***, P < 0.001

Fig. 4. GPNMB interacts with EGFR and regulates its mitochondrial translocation. A. In STRING prediction of the interaction between GPNMB and EGFR using protein interaction databases. B. Co-IP analysis showing the direct binding of GPNMB and EGFR in microglial cells. C. Immunofluorescence staining of GPNMB and EGFR in microglial cells, showing their colocalization and EGFR phosphorylation after I/R. D. Western blot analysis of GPNMB, EGFR, and p-EGFR in si-NC and si-GPNMB-transfected microglial cells after I/R injury, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0002. E. Immunofluorescence staining showing the mitochondrial translocation of p-EGFR and colocalization of TOMM20/COX IV in si-NC and si-GPNMB-transfected microglial cells after I/R injury. F. Fractionation Western blot and Pearson’s coefficient quantifying mitochondrial localization of p-EGFR, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0001. N = 3 repeated experiments. ***, P < 0.001

Fig. 5. GPNMB regulates EGFR mitochondrial translocation and HK2 activation, influencing microglial cell polarization. A. qPCR (P = 0.0001, P = 0.0003) and western blot (P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0012, P = 0.0106) analysis showing the levels of GPNMB, EGFR, and HK2 in groups as indicated. B. CCK-8 assay measuring cell viability in groups as indicated, P = 0.0001, P = 0.0049; P = 0.0088. C. ATP levels measured using a commercial kit in groups as indicated, P = 0.0001, P = 0.0001; P = 0.0013. D. JC-1 staining and flow cytometry assessment of mitochondrial membrane potential in microglial cells in groups as indicated, P = 0.0001, P = 0.0001; P = 0.0002. E. Western blot analysis detecting the levels of respiratory chain complex proteins (CYTB, MTCO2, and ATP6) in microglial cells, P = 0.0092, P = 0.0031; P = 0.0065; P = 0.0003, P = 0.0005; P = 0.0035; P = 0.0001, P = 0.0001; P = 0.0001. F. Immunofluorescence staining of CD16/32 and CD206 microglial polarization markers CD16/32, IBA1, CD206 in groups as indicated, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0167, P = 0.0001; P = 0.0001. N = 3 repeated experiments. *, P < 0.05; **, P < 0.01;…

Fig. 6. EGFR mitochondrial translocation affects HK2 expression, mediating microglial cell polarization. A. Western blot analysis of EGFR, p-EGFR and HK2 levels in groups as indicated, P = 0.0001, P = 0.0001; P = 0.0002, P = 0.0291, P = 0.0156. B. CCK-8 assay measuring cell viability in groups as indicated, P = 0.0021, P = 0.0356, P = 0.0155. C. ATP levels measured using a commercial kit in the groups as indicated, P = 0.0001, P = 0.0002, P = 0.0013. D. JC-1 staining and flow cytometry assessment of mitochondrial membrane potential in microglial cells in groups as indicated, P = 0.0001, P = 0.0137, P = 0.0003. E. Western blot analysis detecting the levels of respiratory chain complex proteins (CYTB, MTCO2, and ATP6) in microglial cells as indicated, P = 0.0001, P = 0.0002, P = 0.0001; P = 0.0005, P = 0.0003, P = 0.0004; P = 0.0001, P = 0.0001, P = 0.0003. F. Immunofluorescence staining of CD16/32 and CD206 microglial polarization markers CD16/32, IBA1, CD206 in groups as indicated, P = 0.0001, P = 0.0001, P = 0.0001; P = 0.0002, P = 0.0001, P = 0.0002. N = 3 repeated experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 7. GPNMB regulates EGFR mitochondrial translocation, influencing HK2 expression and mediating microglial cell polarization during stroke progression. A. HE staining assessing tissue damage in the MCAO model with and without GPNMB knockout and EGFR activation as indicated. B. Nissl staining evaluating hippocampal damage in groups as indicated. C. Western blot analysis detecting the expression levels of GPNMB, EGFR, and HK2 in the hippocampal region across groups as indicated, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0019, P = 0.0001; P = 0.0001, P = 0.0012, P = 0.0001. D. JC-1 staining and flow cytometry assessing mitochondrial membrane potential in microglial cells from various experimental conditions as indicated, P = 0.0001, P = 0.0001, P = 0.0001. E. Western blot analysis measuring the levels of respiratory chain complex proteins (CYTB, MTCO2, and ATP6) in the hippocampal region, P = 0.0084, P = 0.0011, P = 0.0011; P = 0.0001, P = 0.0013, P = 0.0010; P = 0.0001, P = 0.0003, P = 0.0001. F. Immunofluorescence staining of CD16/32 and CD206 microglial polarization markers CD16/32, IBA1, CD206 in groups as indicated, P = 0.0001, P = 0.0001, P = 0.0088; P = 0.0002, P = 0.0001, P =…

Fig. 8. Double-perturbation confirms the GPNMB–EGFR–HK2 axis in stroke progression. A. HE staining assessing tissue damage in the MCAO model across experimental groups (MCAO, GPNMB knockout, EGFR activation, and HK2 inhibition). B. Nissl staining detecting hippocampal region damage under the same conditions. C. Western blot analysis detecting expression levels of GPNMB, EGFR, and HK2 in hippocampal tissue across groups as indicated, P = 0.0001; P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0001, P = 0.0001. D. ATP levels measured in hippocampal tissue from different experimental groups, P = 0.0001, P = 0.0001, P = 0.0011. E. JC-1 staining and flow cytometry assessing mitochondrial membrane potential in hippocampal microglial cells under various treatments, P = 0.0001, P = 0.0001, P = 0.0001. F. Western blot analysis measuring respiratory chain complex proteins (CYTB, MTCO2, and ATP6) in hippocampal tissue, P = 0.0001, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0001, P = 0.0001; P = 0.0001, P = 0.0001, P = 0.0001. G. Immunofluorescence staining detecting microglial polarization markers CD16/32 and CD206 across experimental groups, P = 0.0018, P = 0.0024, P = 0.0015; P = 0.0001, P = 0.0001, P =…