Recombinant High-Mobility Group Box 1 (rHMGB1) Promotes NRF2-Independent Mitochondrial Fusion through CXCR4/PSMB5-Mediated Drp1 Degradation in Endothelial Cells

Figure 5

Inhibition of PSMB5 abolished rHMGB1-induced decrease in Drp1 protein and mitochondrial fusion. (a) Representative immunoblotting bands of PSMB5 of cells treated with different concentrations of rHMGB1 and the matching internal standard β-Tubulin. (b) The densitometric analysis of relative PSMB5 expression of cells treated with different concentrations of rHMGB1 referenced to matching β-Tubulin. (c) Representative immunoblotting bands of Drp1 of cells preexposed to epoxomicin (10 μM) followed by rHMGB1 (1 μg/ml) and the matching internal standard GAPDH. (d) The densitometric analysis of relative Drp1 expression of cells preexposed to epoxomicin followed by rHMGB1 referenced to matching GAPDH. (e) Mitochondrial morphology of cells exposed to epoxomicin (10 μM) and subsequent rHMGB1 (1 μg/ml). Cells were subjected to fluorescent staining with MitoTracker Green FM and observed by a Leica SP8 confocal laser scanning microscope. The magnification is 630. Scale bar: 10 μm. (f) TEM images of mitochondria of EA.hy926 endothelial cells exposed to epoxomicin (10 μM) and subsequent rHMGB1 (1 μg/ml). The magnification is 5900. Scale bar: 1 μm. (g) The average mitochondrial area (μm²) under TEM changes of EA.hy926 endothelial cells treated with rHMGB1, epoxomicin, or both, compared with the control group. (h) The mitochondrial number changes under TEM in EA.hy926 endothelial cells treated with rHMGB1, epoxomicin, or both, compared with the control group. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TEM: transmission electron microscopy. Data were expressed as the . For comparison of the average mitochondrial area, at least 30 mitochondria of 10 cells per group were calculated; for comparison of mitochondrial number, at least 10 cells per group were counted. , , , and .

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