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

Figure 6

rHMGB1 induced downregulation of Drp1 and mitochondrial fusion was NRF2 independent. (a) Immunoblotting result showed that rHMGB1 upregulated the expression of NRF2, and CXCR4 antagonist AMD3100 abolished this effect. (b) Immunoblotting result demonstrated that NRF2 was successfully silenced with specific siRNAs (1# siRNA, 2# siRNA, and 3# siRNA). (c) Immunoblotting result showed that silencing the expression of NRF2 had no significant effect on the expression of Drp1. (d, e) The densitometric analysis of relative NRF2 expression of cells treated with rHMGB1, AMD3100, or siRNAs referenced to matching GAPDH. (f) The densitometric analysis of relative Drp1 expression of cells treated with siRNAs referenced to matching GAPDH. (g) Immunoblotting result showed that silencing the expression of NRF2 had no significant effect on reversing rHMGB1-induced downregulation of Drp1. (h) The densitometric analysis of relative Drp1 expression of cells treated with siRNAs and/or rHMGB1 referenced to matching GAPDH. (i) Confocal result showed that silencing the expression of NRF2 had no significant effect on mitochondrial dynamics or on reversing rHMGB1-induced mitochondrial fusion in EA.hy926 cells. Cells were subjected to fluorescent staining with MitoTracker Green FM and observed by a Leica SP8 confocal laser scanning microscope. Scale bar: 10 μm. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: negative control. Data were expressed as the ; , .

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