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Figure 1. Dysregulated glycine and oxalate metabolism in patients and mice with atherosclerosis (A) Schematic representation of glycine metabolic pathways. (B–E) Targeted metabolomics assessing the ratios of glycine to (B) serine, (C) threonine, (D) alanine, and (E) oxalate in serum from age- and sex- matched patients with or without sCAD (n = 24). (F) En face analysis of atherosclerotic lesions in male Apoe/ mice fed a standard diet (SD) or Western diet (WD) for 12 weeks (n = 5). (G–J) Targeted metabolomics assessing the ratios of glycine to (G) serine, (H) threonine, (I) alanine, and (J) oxalate in plasma from male Apoe/ mice fed a SD or WD for 12 weeks (n = 5). (K) Western blot analysis of <t>AGXT</t> protein abun- dance in livers from Apoe/ mice fed a SD or WD for 12 weeks (n = 5). Mann-Whitney U test for (B)-(E), (G), (H), and (K). Unpaired t test for (F), (I), and (J). Data are pre- sented as mean ± SEM. All points and p values are shown.

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Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 1. Dysregulated glycine and oxalate metabolism in patients and mice with atherosclerosis (A) Schematic representation of glycine metabolic pathways. (B–E) Targeted metabolomics assessing the ratios of glycine to (B) serine, (C) threonine, (D) alanine, and (E) oxalate in serum from age- and sex- matched patients with or without sCAD (n = 24). (F) En face analysis of atherosclerotic lesions in male Apoe/ mice fed a standard diet (SD) or Western diet (WD) for 12 weeks (n = 5). (G–J) Targeted metabolomics assessing the ratios of glycine to (G) serine, (H) threonine, (I) alanine, and (J) oxalate in plasma from male Apoe/ mice fed a SD or WD for 12 weeks (n = 5). (K) Western blot analysis of AGXT protein abun- dance in livers from Apoe/ mice fed a SD or WD for 12 weeks (n = 5). Mann-Whitney U test for (B)-(E), (G), (H), and (K). Unpaired t test for (F), (I), and (J). Data are pre- sented as mean ± SEM. All points and p values are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Primer sequences for Agxt genotyping:, forward: 50-ACACCTCCACTGTCCTG TCC-30 Integrated DNA Technologies N/A Primer sequences for Agxt genotyping:, reverse: 50-GGTCAGATCTGCCTGCTA CC-30 Integrated DNA Technologies N/A Primer for Sanger sequencing:, 50-GCAGAGCTAGCTGGGAAATG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, forward: 50-GCCTAGCCGAGGGAGAG CCG-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 1: 50-TGTGACTTGGGAGCTC TGCAGC-30 Integrated DNA Technologies N/A Primer sequences for Apoe genotyping:, reverse 2: 50-GCCGCCCCGACTGC ATCT-30 Integrated DNA Technologies N/A Primer sequences for qPCR, see Table S2 Integrated DNA Technologies N/A Recombinant DNA pAdDeltaF6 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV2/8 Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-GFP Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A pAAV-TBG-MCS Jiandie Lin’s lab, University of Michigan; Guo et al., 2017 N/A AGXT Human Tagged ORF Clone Origene CAT# RG212899 Software and algorithms ImageJ National Institutes of Health RRID:SCR_003070 https://imagej.nih.gov/ ij/download.html GraphPad Prism GraphPad Software Version 8.0 RRID:SCR_002798 https:// www.graphpad.com/scientific-software/ prism/ R statistics Hosted by Vienna, University of Economics and Business http://www.R-project.org/ RStudio RStudio Team (2020). https://www.rstudio.com/ RRID:SCR_000432 IBM SPSS software IBM Corporation Version 23.0; RRID:SCR_019096 https:// www.ibm.com/analytics/ spss-statistics-software FastQC Babraham Bioinformatics Version 0.11.8 RRID:SCR_014583 https:// www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Trimmomatic Bolger et al., 2014 Version 0.35 RRID:SCR_011848 http:// www.usadellab.org/cms/? page=trimmomatic HISAT2 Kim et al., 2015 Version 2.1.0.13; RRID:SCR_015530 http:// daehwankimlab.github.io/hisat2/ download/ HTSeq Anders et al., 2015 Version 0.6.0; RRID:SCR_005514 https:// htseq.readthedocs.io/en/master/install. html (Continued on next page) Cell Reports 36, 109420, July 27, 2021 e3 Article ll OPEN ACCESS

Techniques: Western Blot, Clinical Proteomics, MANN-WHITNEY

Figure 2. AGXT deﬁciency exacerbates atherosclerosis in male Apoe/ mice (A) Western blot analysis conﬁrming the loss of AGXT in livers from male Agxt//Apoe/ mice (n = 6). (B) Glycine/oxalate ratio in plasma from male Agxt//Apoe/ mice and their Agxt+/+/Apoe/

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 2. AGXT deﬁciency exacerbates atherosclerosis in male Apoe/ mice (A) Western blot analysis conﬁrming the loss of AGXT in livers from male Agxt//Apoe/ mice (n = 6). (B) Glycine/oxalate ratio in plasma from male Agxt//Apoe/ mice and their Agxt+/+/Apoe/

Techniques: Western Blot, Clinical Proteomics

Figure 3. Oxalate homeostasis is main- tained and atherosclerosis is unaltered in fe- male Agxt//Apoe/ mice (A) Western blot analysis conﬁrming the loss of AGXT in livers from female Agxt//Apoe/ mice (n = 6). (B) GlGlycine/oxalate ratio in plasma from female Agxt//Apoe/ and Agxt+/+/Apoe/ mice (n = 10). (C–G) Female Agxt//Apoe/ and Agxt+/+/ Apoe/ mice were fed a WD for 12 weeks (n = 10): (C) plasma TC, (D) atherosclerosis in the aortic tree, (E) H&E staining, (F) ORO staining, and (G) Mac-2 immunohistochemistry of aortic sinus. Scale bars, 200 mm. Unpaired t test for (B), (D), (E), and (G). Mann- Whitney U test for (C) and (F). Data are presented as mean ± SEM. All points and p values are shown.

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 3. Oxalate homeostasis is main- tained and atherosclerosis is unaltered in fe- male Agxt//Apoe/ mice (A) Western blot analysis conﬁrming the loss of AGXT in livers from female Agxt//Apoe/ mice (n = 6). (B) GlGlycine/oxalate ratio in plasma from female Agxt//Apoe/ and Agxt+/+/Apoe/ mice (n = 10). (C–G) Female Agxt//Apoe/ and Agxt+/+/ Apoe/ mice were fed a WD for 12 weeks (n = 10): (C) plasma TC, (D) atherosclerosis in the aortic tree, (E) H&E staining, (F) ORO staining, and (G) Mac-2 immunohistochemistry of aortic sinus. Scale bars, 200 mm. Unpaired t test for (B), (D), (E), and (G). Mann- Whitney U test for (C) and (F). Data are presented as mean ± SEM. All points and p values are shown.

Techniques: Western Blot, Clinical Proteomics, Staining, Immunohistochemistry, MANN-WHITNEY

Figure 4. Dysregulated oxalate metabolism induces a pro-inﬂammatory response and CCL5 release (A–C) RNA sequencing of livers collected from male Agxt//Apoe/ and Agxt+/+/Apoe/ mice fed a WD for 12 weeks (n = 5): (A) volcano plot of DEGs (padj < 0.05, log2 fold change > 1) in male Agxt//Apoe/ versus Agxt+/+/Apoe/ mice (blue indicates downregulated; red indicates upregulated). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG)-based pathway analysis. The signiﬁcance of the enrichment was determined by a right-tailed Fisher’s exact test followed by a Benjamini-Hochberg multiple testing adjustment. (C) Heatmap-based representation of 50 DEGs linking inﬂammation, ﬁbrogenesis, and the metabolism of lipids and sterols/steroids with atherosclerosis.

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 4. Dysregulated oxalate metabolism induces a pro-inﬂammatory response and CCL5 release (A–C) RNA sequencing of livers collected from male Agxt//Apoe/ and Agxt+/+/Apoe/ mice fed a WD for 12 weeks (n = 5): (A) volcano plot of DEGs (padj < 0.05, log2 fold change > 1) in male Agxt//Apoe/ versus Agxt+/+/Apoe/ mice (blue indicates downregulated; red indicates upregulated). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG)-based pathway analysis. The signiﬁcance of the enrichment was determined by a right-tailed Fisher’s exact test followed by a Benjamini-Hochberg multiple testing adjustment. (C) Heatmap-based representation of 50 DEGs linking inﬂammation, ﬁbrogenesis, and the metabolism of lipids and sterols/steroids with atherosclerosis.

Techniques: RNA Sequencing

Figure 5. Oxalate overload induces mito- chondrial dysfunction and overproduction of superoxide, leading to CCL5 release in macrophages (A) qPCR analyses of genes regulating redox ho- meostasis in livers from male Agxt//Apoe/

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 5. Oxalate overload induces mito- chondrial dysfunction and overproduction of superoxide, leading to CCL5 release in macrophages (A) qPCR analyses of genes regulating redox ho- meostasis in livers from male Agxt//Apoe/

Techniques:

Figure 6. AAV-mediated overexpression of AGXT reduces oxidative stress and inﬂammation AAV-AGXT or AAV-GFP was injected into male Apoe/ mice and the mice were fed a WD for 12 weeks (n = 8). (A) Western blot analysis conﬁrming the over- expression of AGXT in livers from mice treated with AAV-AGXT. (B) Plasma glycine/oxalate ratio. (C and D) qPCR analyses of genes regulating (C) redox homeostasis and (D) inﬂammatory re- sponses. Gene expression levels were normalized to 18S. (E and F) Plasma concentrations of (E) CCL2 and (F) CCL5. (G) DHE ﬂuorescence and Mac-2 immunoﬂuores- cence in the aortic sinuses. Scale bars, 50 mm. Unpaired t test for (B), (E), and (G), and Mann- Whitney U test for (F). Statistical differences in gene expression in (C) and (D) were tested using unpaired t test or Mann-Whitney U test, depending on normality tests. *P < 0.05, **P <0.01, ***P <0.001 versus mice injected with AAV-GFP. Data are presented as mean ± SEM. All points and p values are shown.

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 6. AAV-mediated overexpression of AGXT reduces oxidative stress and inﬂammation AAV-AGXT or AAV-GFP was injected into male Apoe/ mice and the mice were fed a WD for 12 weeks (n = 8). (A) Western blot analysis conﬁrming the over- expression of AGXT in livers from mice treated with AAV-AGXT. (B) Plasma glycine/oxalate ratio. (C and D) qPCR analyses of genes regulating (C) redox homeostasis and (D) inﬂammatory re- sponses. Gene expression levels were normalized to 18S. (E and F) Plasma concentrations of (E) CCL2 and (F) CCL5. (G) DHE ﬂuorescence and Mac-2 immunoﬂuores- cence in the aortic sinuses. Scale bars, 50 mm. Unpaired t test for (B), (E), and (G), and Mann- Whitney U test for (F). Statistical differences in gene expression in (C) and (D) were tested using unpaired t test or Mann-Whitney U test, depending on normality tests. *P < 0.05, **P <0.01, ***P <0.001 versus mice injected with AAV-GFP. Data are presented as mean ± SEM. All points and p values are shown.

Techniques: Over Expression, Injection, Western Blot, Clinical Proteomics, Gene Expression, MANN-WHITNEY

Figure 7. AAV-mediated overexpression of AGXT reduces atherosclerosis independent of circulating cholesterol AAV-AGXT or AAV-GFP was injected into male Apoe/ mice and the mice were fed a WD for 12 weeks (n = 8). (A) Plasma TC. (B) Atherosclerosis in the aortic tree. (C–E) H&E staining (D), ORO staining (D), and Mac- 2 immunohistochemistry (E) of aortic sinus. Scale bars, 200 mm. (F) Proposed model of dysregulated oxalate metabolism as a driver and therapeutic target in atherosclerosis. Unpaired t test for (A)–(E). Data are presented as mean ± SEM. All points and p values are shown.

Journal: Cell reports

Article Title: Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

doi: 10.1016/j.celrep.2021.109420

Figure Lengend Snippet: Figure 7. AAV-mediated overexpression of AGXT reduces atherosclerosis independent of circulating cholesterol AAV-AGXT or AAV-GFP was injected into male Apoe/ mice and the mice were fed a WD for 12 weeks (n = 8). (A) Plasma TC. (B) Atherosclerosis in the aortic tree. (C–E) H&E staining (D), ORO staining (D), and Mac- 2 immunohistochemistry (E) of aortic sinus. Scale bars, 200 mm. (F) Proposed model of dysregulated oxalate metabolism as a driver and therapeutic target in atherosclerosis. Unpaired t test for (A)–(E). Data are presented as mean ± SEM. All points and p values are shown.

Techniques: Over Expression, Injection, Clinical Proteomics, Staining, Immunohistochemistry

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