Fig. 4.

Acod1 deficiency reduces apoptosis and necrosis. A) Aortic root segments were stained for Sirius Red to B) quantify red collagen staining area as a percentage of the plaque area (blue outline), expressed as an average of the three valve cusps in Acod1^+/+ → Ldlr^−/− (left) and Acod1^−/− → Ldlr^−/− (right) mice (n = 19/22 for Acod1^+/+ → Ldlr^−/−/Acod1^−/− → Ldlr^−/−, unpaired t-test). C) Necrotic core outlined (red) on one of the three aortic root valve cusps stained with toluidine blue which was then D) quantified as a percentage of the total plaque and expressed as an average of the three cusps (n = 18/22, unpaired t-test). E) Cell viability in unstimulated (black) or LPS (100 ng/mL) stimulated (red) FCs after 24 h, measured via flow cytometry analysis of fixable viability dye (FVD) eFluor™ 780 (n = 5, two-way ANOVA). F) P53 signaling pathway enrichment from a gene set enrichment analysis of dataset GSE145950 by Swain et al., 2020 comparing LPS-stimulated Acod1^+/+ and Acod1^−/− BMDMs. G) Flow cytometry quantification of an Annexin V/7AAD apoptosis and necrosis assay on in vitro cultured Acod1^+/+ and Acod1^−/− BMDMs stimulated with LPS (100 ng/mL) over 72 h (n = 3, two-way ANOVA). H) Gene expression heatmap of reactive oxygen species-related markers and I) subsequent gene set enrichment analysis comparing LPS stimulated Acod1^+/+ and Acod1^−/− BMDMs from dataset GSE145950 by Swain et al., 2020. J) qPCR derived gene expression of redox markers from mouse aortic arches (n = 15/16, unpaired t-test). Gating strategies for flow cytometry is provided in Supplemental Fig. 4. Data points display individual mice and error bars show SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)