Fig. 6: Phosphorylation of RIPK3 in serine 204 regulates mitochondrial fission via DRP1.

(a) IF staining of phosphorylated RIPK3 (pRIPK3) in murine aortic sections of animals treated as indicated (scale bar = 20 µm). (b) Hematoxylin/Eosin (H&E) and IF staining of pRIPK3 (red) and CD68 (green) in healthy or aneurysmal human tissue (scale bar H&E = 500 µm, IF = 20 µm). Merge is shown in yellow and nucleus is stained with DAPI in blue. Dotted lines define the magnified region. L lumen. (c) Immunoblot of pRIPK3 and quantification of healthy or aneurysmal human tissue. n = 3 per group. **P = 0.0037. (d) IF staining of pRIPK3 in BMDM stimulated with HMGB1 (10 ng ml⁻¹). Nucleus is stained with DAPI in blue (scale bar = 10 µm). (e) Schematic representation of RIPK3 phosphorylation sites. Targeted mutations designed in synthetic modified RNA (modRNA) are illustrated. WT, wild type; S, serine; A, Alanine; N-T, N-terminal; C-T, C-terminal; S204>A, modification of serine 204; S232>A modification of serine 232; DM, double serine mutations. (f) Representative histograms of flow cytometry analysis of Mitotracker and quantification of MFI in BMDM transfected with Ripk3 modRNA as indicated. n = 4 (DM) and 5 (Control, WT, 204>A and 232>A). *P < 0.05, **P < 0.01. Error bars represent s.e.m. (g) Immunoblot analysis and quantification of pDRP1 in BMDM transfected with Ripk3 modRNA as indicated. n = 3. *P < 0.05, **P < 0.01. Data is presented as mean, error bars represent s.e.m. P values were calculated using two-tailed unpaired t-tests (c) or one-way ANOVA (f, g).