Fig. 5: HMGB1-induced RIPK3 promotes mitochondrial fission in macrophages. | Nature Communications

Fig. 5: HMGB1-induced RIPK3 promotes mitochondrial fission in macrophages.

From: Lung-derived HMGB1 is detrimental for vascular remodeling of metabolically imbalanced arterial macrophages

Fig. 5

Oxygen consumption rate (OCR) and respiration quantifications of WT and Ripk3−/− BMDM treated with HMGB1 (a, b). n = 3 (WT + HMGB1, Ripk3−/− and Ripk3−/−+HMGB1) and 7 (WT). *P < 0.05, ***P < 0.001. OCR profiles and quantification of WT and Ripk3−/− BMDM treated with HMGB1 in the presence of GSK’872 (10 µM, 1 h) (c, d). n = 5 per group. **P < 0.01, ***P < 0.001. Representative histograms of Mitotracker and quantification of the mean fluorescence intensity (MFI) of WT or Ripk3−/− BMDM treated with HMGB1 (e). n = 3 (Ripk3−/− and Ripk3−/−+HMGB1), 5 (WT + HMGB1) and 6 (WT). *P < 0.05, ***P < 0.001. Representative histograms of Mitotracker and quantification of MFI of WT BMDM treated with lung extracts of PBS-treated mice (CTL) or ALI lung extracts (ALI) pre-incubated with anti-HMGB1 (α-HMGB1) or with control IgG (f). n = 3 per group. *P < 0.05. (g) Representative electron microscopy images of mitochondria in WT or Ripk3−/− BMDM treated with HMGB1 or PBS (CTL). Scale bar =  500 nm. Quantification of mitochondria number per cell (h) in WT or Ripk3−/− as indicated. n = 11 (WT), 10 (WT + HMGB1), 12 (Ripk3−/−) and (17 Ripk3−/−+HMGB1). **P < 0.01. (i) IF staining of Mitotracker and quantification of mean mitochondrial area in WT or Ripk3−/− BMDM treated with HMGB1 (10 ng ml−1). Scale bar = 10 µm. n = 4 per group. *P = 0.0358. (j) Immunoblot of pDRP1 and quantification. n = 3 per group. *P < 0.05. (k) Representative IF of CD68 (green) and pDRP1 (red) staining in aortas of mice as indicated (scale bar = 20 µm). Inset show magnified images (scale bar = 10 µm) (i) Schematic representation of Mdivi-1 treatment in mice. i.n., intranasal; i.p., intraperitoneal. Aneurysm incidence (m), quantification of maximal aortic diameter (n), representative color Doppler ultrasound images of aorta and M-mode (arrow) (o) and Verhoeff-Van Gieson staining (p) of aortic sections from vehicle or Mdivi-1 treatment. Arrows indicates elastin breaks. n = 4 (Mdivi-1) and 6 (Vehicle). ***P = 0.0002. Data is presented as mean, error bars represent s.e.m. P values were calculated using two-tailed unpaired t-tests (i, n) or one-way ANOVA (b–h, j).

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