Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging
- Laboratory of Cardiovascular and Aging Research, University of Fribourg, Switzerland
- Cardiology, Department of Endocrinology, Metabolism, and Cardiovascular System, Faculty of Science and Medicine, University of Fribourg, Switzerland
- Institute for Pathology, Cantonal Hospital, Switzerland
Figures
Figure 1 with 2 supplements
Age-associated increase in Arg2 levels in mouse heart.
(A) Arg2 mRNA levels of male and female young (3–4 months) and old (20–22 months) wild type (wt) heart tissues analyzed by qRT-PCR. Rps12 served as the reference (n=8–9 animals per group); (B) Representative histological images of heart interstitial and perivascular fibrosis in young and old wt and Arg2-/- female mice. Fibrosis is shown by the blue-colored Trichrome Masson’s staining. Scale bar = 50 µm. (n=5–7 mice per group); (C) Quantification of total fibrotic area in cardiac tissue (% of total area); (D) Hydroxyproline content of mouse heart from young and old wt and Arg2-/- female mice. (n=4 mice in each group); (E) Representative confocal images showing immunofluorescence staining of PDGF-Rα (green, fibroblasts marker) in young and old wt and Arg2-/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (F) Relative PDGF-Rα signal quantification of confocal images (n=4 per each group). (G) Representative confocal images showing immunofluorescence staining of p16 (green, senescent marker) in young and old wt and Arg2-/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (H) Percentage of p16+ nuclei in the four groups (n=4). The values shown are mean ± SD. Data are presented as the fold change to the young-wt group, except for panel D. *p≤0.05, **p≤0.01, ***p≤0.005, ****p≤0.001 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 1—figure supplement 1
Arg2 and Arg1 expression in male and female mice.
Arg1 mRNA levels of female (A) and male (B) young (3–4 months) and old (20–22 months) wild type (wt) heart tissues analyzed by qRT-PCR. Rps12 served as the reference (n=6–7 animals per group); Arg2 (C) and Arg1 (D) expression extrapolated from high throughput sequencing database of male mice of 6, 18, and 27 months old (GSE201207, Wolff et al., 2023). Data are presented as the fold change to the young-wt group. *p≤0.05 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 1—figure supplement 2
Collagen gene expression in female mice.
(A) Col1a1 and (B) Col3a1 mRNA levels of young (3–4 months) and old (20–22 months) wt and Arg2-/- female heart tissues analyzed by qRT-PCR. Rps12 served as the reference (n=6–9 animals per group). Data are presented as the fold change to the young-wt group. *p≤0.05, **p≤0.01 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 2 with 1 supplement
Age-associated elevation in inflammatory cytokines and apoptosis in heart is prevented in female Arg2-/- mice.
mRNA expression levels of (A) Adgre1, (B) Mcp-1, (C) Il1b, and (D) Tnfa in young and old wt and Arg2-/- female mouse hearts were analyzed by qRT-PCR. Rps12 served as the reference (n=6–12 mice per group); (E) Representative confocal images of young and old wt and Arg2-/- heart tissue showing co-localization of LYVE1 (green) and F4-80 (red, mouse macrophage marker), (F) Representative co-localization images of CCR2 (green) and F4-80 (red) in wt and Arg2-/- heart tissues. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm. Graph showing the quantification of (G) F4-80+ cells, (H) LYVE1+/F4-80+ cells, and (I) CCR2+/F4-80+ cells per mm2 in heart tissue of wt and Arg2-/- young and old mice (n=3 per each group). The values shown are mean ± SD. Data are expressed as fold change to the young wt group, except for panels G to I. *p≤0.05, **p≤0.01, ***p≤0.005, and ****p≤0.001 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2-/- gene knockout mice.
Figure 2—figure supplement 1
Validation of CCR2 and LYVE1 antibodies.
(A) Confocal microscopy illustration of immunofluorescence double staining of NOS2 (green) and F4-80 (red; macrophage marker). Scale bar = 50 µm; The negative control was generated by omitting the two primary antibodies. The enlarged region of interests (ROIs) 1 and 2 show the existence of both LYVE1+/F4-80+ and LYVE−/F4-80+ signal excluding the cross-reactivity between secondary antibodies. (B) Confocal microscopy illustration of immunofluorescence double staining of CCR2 (green) and F4-80 (red; macrophage marker). Scale bar = 50 µm; The negative control was again generated by omitting the two primary antibodies. ROIs 1 and 2 show the existence of both CCR2+/F4-80+ and CCR2−/F4-80+ signal excluding the cross-reactivity between secondary antibodies.
Figure 3
Age-associated elevation in apoptotic cardiomyocytes is prevented in female Arg2-/- mice.
(A) Representative confocal images and relative quantification of apoptotic cardiac cells in young and old wt and Arg2-/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (B) graph showing the quantification of the TUNEL-positive cells in old wt and Arg2-/- hearts; (C) Wheat germ agglutinin (WGA)-Alexa Fluor 488-conjugate was used to stain cell membrane, and separate cardiomyocytes and non-cardiomyocytes apoptotic cells. Cell distinction was based on cell size and shape. Scale bar = 10 µm; (D) Graphs showing the quantification of the TUNEL-positive cardiomyocytes (CM) and non-myocytes (NCM) in old wt and Arg2-/- hearts. The values shown are mean ± SD. Data are expressed as fold change to the young wt group, except for panel D. **p≤0.01, ***p≤0.005 and ****p≤0.001 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 4 with 1 supplement
Age-related endothelial-to-mesenchymal transition (EndMT) in heart tissue.
(A) Immunoblotting analysis of CD31 (endothelial marker), vimentin (mesenchymal marker), and SNAIL (master regulator of EndMT) in the heart of wt and Arg2-/- female young and old mice; tubulin and vinculin served as protein loading controls. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of the SNAIL (B), vimentin (C), and CD31 (D) signals on immunoblots (n=6–10 mice in each group); (E) Representative confocal images showing co-localization of CD31 (green) and vimentin (red) in young and old wt and Arg2-/- heart tissues. DAPI (blue) is used to stain nuclei. Scale bar = 20 µm. *p≤0.05 and **p≤0.01, between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 4—figure supplement 1
Age-related heart tissue hypertrophy.
Comparison between the heart weight to body weight ratio (HW/BW) of wt and Arg2-/- young and old mice (n=5–9 per group). ****p≤0.001, between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 5 with 1 supplement
Cellular localization of ARG2 in aging heart of female mice.
(A) Confocal microscopy illustration of immunofluorescence double staining of ARG2 (red) and TNNT (green; cardiomyocytes marker). Scale bar = 25 µm; (B) Bright-field microscopy images of isolated wt cardiomyocytes and primary cardiac fibroblasts. The immunoblot shows the level of ARG2 in both cardiomyocytes and fibroblasts upon exposure to hypoxia (1% O2) for 24 hr. C indicates freshly isolated cardiomyocytes used as control, N and H indicate normoxia and hypoxia conditions, and K indicates kidney tissue extract used as positive control. Tubulin served as protein loading control; (C) mRNA expression levels of Arg2 in cardiomyocytes (card) and non-cardiomyocytes (non-c) cells isolated from old wt and Arg2 -/- female mouse hearts. Gapdh served as the reference. (n=3–5 mice per group); (D to G) Representative confocal images of old wt mouse heart showing co-localization of (D) ARG2 (red) and MAC-2 (green, mouse macrophage marker), (E) ARG2 (red) and CD31 (green, endothelial marker), (F) ARG2 (red) and PDGF-Rα (green, fibroblasts marker), and (G) ARG2 and α-smooth muscle actin (α-SMA; green, smooth muscle cell/myofibroblasts marker); (H to M) Representative confocal images of human heart tissue showing co-localization of (H) ARG2 (green) and TNNT (red, cardiomyocytes marker), (I) ARG2 (red) and CD31 (green, endothelial marker), (J) ARG2 (red) and CD-68 (green, macrophage marker), (K) ARG2 (red) and vimentin (green, fibroblast marker), and (L–M) ARG2 (red) and α-smooth muscle actin (α-SMA; green), (L) myofibroblasts and (M) smooth muscle cell marker. DAPI (blue) stains cell nuclei. Scale bar = 20 µm. Each experiment was repeated with 3–5 animals.
Figure 5—figure supplement 1
ARG2 localization in mouse and rat tissues.
(A) Representative confocal images showing immunofluorescence staining of ARG2 (green) in old wt and Arg2-/- kidney tissue. DAPI (blue) is used to stain nuclei. Brightfield light microscopy was combined with fluorescent signals to assess cell morphology. Scale bar = 20 µm; (B) Representative confocal images showing immunofluorescence staining of ARG2 (green) in heart tissue of control rat (Sham), rat under myocardial infarction followed by artery ligation (MI), and relative negative control (omission of primary Ab). DAPI (blue) is used to stain nuclei. The images show ARG2 localization in non-myocytes cells. (C) Representative confocal images showing ARG2 (green) in old wt mouse tissue with relative negative control (omission of primary Ab). DAPI (blue) is used to stain nuclei.
Figure 6
ARG2 ablation reduces Il-1β protein levels in aging heart.
(A) Immunoblotting analysis of Il-1β (active form) in the heart of old wt and Arg2 -/- female mice; tubulin served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots; (B) The plot graph shows quantification of the Il-1β signals on immunoblots (n=6–7 mice in each group); (C) Representative confocal images showing Il-1β localization in old wt and Arg2 -/- heart tissues. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (D) Relative Il-1β signal quantification of confocal images (n=9 per each group); (E) Representative confocal images showing co-localization of MAC-2 (green, mouse macrophage marker), and Il-1β (red) in wt heart tissues. This experiment was repeated with 3 animals. Scale bar = 10 µm. *p≤0.05 and ***p≤0.005, between the indicated groups. wt, wild-type mice; Arg2 -/-, Arg2 gene knockout mice.
Figure 7 with 2 supplements
In vitro study of crosstalk between aging splenic macrophages and cardiomyocytes.
(A) Immunoblotting analysis of ARG2 and Il-1β (active form) in mouse splenic macrophages isolated from young and old wt and Arg2 -/- female mice; tubulin served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of ARG2 (B) and IL-1β (C) protein signals on immunoblots (n=3 mice in each group); (D) IL-1β levels in the conditioned medium from young and old wt and Arg2 -/- splenic macrophages were measured by ELISA; (E) Schematic representation of in vitro crosstalk study; (F) Representative confocal images of adult isolated mouse cardiomyocytes stimulated with conditioned media (CM) from young and old, wt and Arg2 -/- splenic cells (24 hr incubation). Wheat Germ Agglutinin (WGA)-Alexa Fluor 488-conjugate was used to stain cell membrane, and TUNEL was performed to identify apoptotic cells. DAPI is used to stain nuclei. Interleukin receptor antagonist (ILRA; 50 ng/ml) is used to prevent IL-1β binding to its receptor; (G) Quantification of TUNEL-positive cardiomyocytes (% in respect to total number of cells). Scale bar = 20 µm. *p≤0.05, **p≤0.01, ***p≤0.005 and ****p≤0.001 between the indicated groups. MØ, splenic macrophage; Y, young; O, old; wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 7—figure supplement 1
In vitro crosstalk between LPS-activated RAW 264.7 macrophages and cardiomyocytes.
(A) Immunoblotting analysis of ARG2 and Il-1β (precursor) in RAW 264.7 mouse macrophages upon transduction with the rAd/U6-LacZshRNA as the control or rAd/U6-Arg2shRNA to silence Arg2. RAW cells were polarized toward pro-inflammatory phenotype by incubation with lipopolysaccharide (LPS; 100 ng/ml) for 24 hr; tubulin served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of ARG2 (B) and Il-1β (C) protein signals on immunoblots (n=3); (D) Representative confocal images of adult isolated mouse cardiomyocytes stimulated with conditioned media (CM) from control RAW cells and RAW cells with Arg-ii silencing, with and without LPS (24 hr incubation). Wheat Germ Agglutinin (WGA)-Alexa Fluor 488-conjugate was used to stain cell membrane, and TUNEL was performed to identify apoptotic cells. DAPI is used to stain nuclei. Interleukin receptor antagonist (ILRA; 50 ng/ml) is used to prevent IL-1β binding to its receptor; (E) Quantification of TUNEL-positive cardiomyocytes (% in respect to total number of cells). Scale bar = 40 µm. **p≤0.01, ***p≤0.005 and ****p≤0.001 between the indicated groups.
Figure 7—figure supplement 2
Effects of ARG2 and NOS2 in regulation of IL-1β production in macrophages.
(A) Immunoblotting analysis of ARG2 and NOS2 in human wt THP1 (THP1wt) and Arg2 -/- THP1 (THP1Arg2-/-) cells stimulated with LPS (100 ng/ml, 24 hr). Tubulin served as a protein loading control. Mouse bone-marrow-derived macrophages (MØ) treated with LPS were used as positive control for NOS2 and ARG2 detection; Molecular weight (kDa) is indicated at the side of the blots; (B) qRT-PCR analyzing mRNA levels of il-1β in the cells. β-actin served as internal reference (n=6); (C), IL-1β levels in the conditioned medium from the THP1wt and THP1Arg2 -/- cells stimulated with LPS (100 ng/ml, 24 hr) measured by ELISA; (D) Immunoblotting analysis showing ARG2, NOS2, and IL-1β precursor protein levels in the wt and Nos2 knockout (BMDMNos2-/-) bone-marrow-derived macrophages (MØ) stimulated with LPS (100 ng/ml, 24 hr). GAPDH served as protein loading controls. Molecular weight (kDa) is indicated at the side of the blots (n=5 independent experiments). The plot graphs show the quantification of IL-1β (E), and ARG2 (F) protein levels on the immunoblots. *p≤0.05, **p≤0.01, ***p≤0.005, ****p≤0.001 between the indicated groups. (G) Schematic illustration of IL-1β production regulated by NOS2 and ARG2 in macrophages. The dotted line indicates inhibition. wt, wild-type; Arg2-/-, Arg2 gene knockout; Nos2−/−, NOS2 gene knockout; h ARG2, human ARG2; m ARG2, mouse ARG2.
Figure 8 with 1 supplement
Crosstalk between splenic macrophages and cardiac fibroblasts and cell-autonomous effect of ARG2 in cardiac fibroblasts.
(A) Collagen production measured as hydroxyproline content in mouse wt fibroblasts treated with conditioned media (CM) from old, wt, and Arg2 -/- splenic cells (96 hr incubation) (n=3 mice in each group). The values shown are mean ± SD. mRNA expression levels of (B) Fib, (C) Col3a1, and (D) Tgfb1 in wt fibroblasts treated with conditioned media (CM) from old wt and Arg2 -/- splenic cells (96 hr incubation) were analyzed by qRT-PCR. Gapdh served as the reference. (n=4 mice per group); (E) Hydroxyproline content in fibroblasts treated with CM from old wt splenic cells (96 hr incubation). ILRa (50 ng/ml) is used to prevent IL-1β binding to its receptor (n=4 independent experiments). (F) Immunoblotting analysis of ARG2 and vimentin in human cardiac fibroblasts (HCFs) upon Arg2 gene overexpression. GAPDH served as protein loading control; (G) qRT-PCR analysis of mRNA expression levels Arg2 in HCF cells; (H) The plot graph shows the quantification of the vimentin signals on immunoblots shown in panel F. (n=3 independent experiments); (I) qRT-PCR analysis of mRNA expression levels of Col3a1 in HCF cells. Gapdh served as the reference. (n=6 independent experiments); (J) Representative confocal images of human cardiac fibroblasts (HCF) upon transfection with rAd-CMV-Con/ Arg2 for 48 hr. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/l) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. (K) Mitosox signal quantification (n=3 independent experiments); (L) qRT-PCR analysis of mRNA expression levels of Col3a1 in HCF cells treated as indicated. Gapdh served as the reference. (n=5 independent experiments). (M) Immunoblotting analysis of ARG2 and HIF-1α in human cardiac fibroblasts (HCFs) upon 1% hypoxia incubation for 48 h. Tubulin served as a protein loading control; (N) Representative confocal images of HCFs under normoxia (21% O2) or hypoxia (1% O2) for 48 hr. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/l) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. (O) Representative confocal images of HCFs upon transfection with shRNA for Arg2 gene silencing under normoxia or hypoxia. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). DAPI (blue) stains cell nuclei. Scale bar = 25 µm. (P) MitoSOX signal quantification of images shown in panel O (n=3 independent experiments). Data are expressed as fold change to respective control group. *p≤0.05, **p≤0.01, ***p≤0.005 and ****p≤0.001 between the indicated groups. MØ, splenic macrophage; Con, control.
Figure 8—figure supplement 1
Crosstalk between splenic macrophages and cardiac fibroblasts.
(A) Collagen production measured as hydroxyproline content in mouse wt fibroblasts treated with conditioned media (CM) from young and old wt splenic cells (96 hr of incubation) (n=3 mice in each group). mRNA expression levels of (B) Mmp2, (C) Mmp9, and (D) Col1a1 in wt fibroblasts treated with conditioned media (CM) from old wt and Arg2 -/- splenic cells (96 hr of incubation) were analyzed by qRT-PCR. Gapdh served as the reference. (n=4 mice per group). Data are expressed as fold change to respective control groups. *p≤0.05 between the indicated groups. MØ, splenic macrophage; CM, conditioned media; wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 9
Aging wt macrophages induces EndMT in HUVEC.
(A) Immunoblotting analysis of VE-Cadherin (endothelial marker), N-Cadherin and vimentin (both mesenchymal markers), and ARG2 in HUVEC cells upon incubation with conditioned media (CM) from young and old wt and Arg2-/- splenic cells (4 days incubation); GAPDH served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of Arg-II (B), N-Cadherin (C), Vimentin (D), and VE-Cadherin (E) protein levels (n=3 independent experiments); (F) Immunoblotting analysis of VE-Cadherin, N-Cadherin, Vimentin, and ARG2 in HUVEC cells upon incubation with CM from old wt splenic cells (4 days incubation); Interleukin receptor antagonist (IL-Ra; 50 ng/ml) is used to prevent IL-1β effect. GAPDH served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of Arg2 (G), N-Cadherin (H), Vimentin (I), and VE-Cadherin (J) protein signals on immunoblots (n=4 independent experiments); Data are expressed as fold change to respective control group. *p≤0.05, **p≤0.01 and ***p≤0.005 between the indicated groups. MØ, splenic macrophage; wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 10 with 2 supplements
ARG2 ablation improves heart function recovery from global ischemia/reperfusion injury (I/R-I).
(A) Protocol applied for ex vivo Langendorff-heart functional assessment of old wt and age-matched Arg2-/- female mice. After baseline recordings, 20 min global ischemia is followed by 30 min of reperfusion; (B) Ex vivo Langendorff-heart assessment of old female wt (black line) and Arg2-/- (red line) hearts. The graphs show the functional recovery of the left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVED), and maximal rate of contraction (dP/dtmax) and relaxation (dP/dtmin). The data are expressed as % of recovery in respect to baseline values and represent the mean ± SD of data from 5 mice per group; (C) Representative sections of wt and Arg2-/- female hearts stained with 2,3,5-TTC. The healthy/normal tissue appears deep red. The arrows indicate the white infarct tissue showing the absence of living cells. (D) Relative quantification of infarcted areas. *p≤0.05 and **p≤0.01 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 10—figure supplement 1
Short ischemia/reperfusion induces negligible myocardial infarct in female mice.
(A) Representative sections of wt and Arg2-/- hearts stained with 2,3,5-TTC and (B) relative quantification of infarcted areas. 20 minutes global ischemia is followed by 30 minutes reperfusion. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 10—figure supplement 2
Arg2 ablation improves heart function recovery from global ischemia/reperfusion injury (I/R-I) in male mice.
(A) Protocol applied for ex vivo Langendorff-heart functional assessment of old wt and age-matched Arg2-/- male mice. After baseline recordings, 15 min global ischemia is followed by 30 min of reperfusion; (B) Ex vivo Langendorff-heart assessment of old male wt (black line) and Arg2-/- (red line) hearts. The graphs show the functional recovery of the left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVED), and maximal rate of contraction (dP/dtmax) and relaxation (dP/dtmin). The data are expressed as % of recovery in respect to baseline values and represent the mean ± SD of data from 5 mice per group; (C) Representative sections of wt and Arg2-/- male hearts stained with 2,3,5-TTC and (D) quantification of infarcted areas. *p≤0.05 between the indicated groups. wt, wild-type mice; Arg2-/-, Arg2 gene knockout mice.
Figure 11
Schematic illustration of the role of Arg-II and underlying mechanisms in cardiac aging.
Additional files
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Supplementary file 1
Baseline comparison between wt and Arg2-/- mice under Langendorff recordings.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp1-v1.docx
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Supplementary file 2
List of RT-PCR primer sequences.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp2-v1.docx
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Supplementary file 3
List of antibodies used in the study with specific dilutions for immunoblotting and immunofluorescence staining.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp3-v1.docx
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Supplementary file 4
Short tandem repeat (STR) profiling of RAW 264.7, which shows 98.6% concordance with reference cell lines in the Cellosaurus database, confirming the cell line identity.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp4-v1.pdf
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Supplementary file 5
Short tandem repeat (STR) profiling of THP1 wt, which shows 100% concordance with reference cell lines in the Cellosaurus database, confirming the cell line identity.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp5-v1.pdf
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Supplementary file 6
Short tandem repeat (STR) profiling of THP1 Arg2 knockout, which shows 100% concordance with reference cell lines in the Cellosaurus database, confirming the cell line identity.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp6-v1.pdf
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Supplementary file 7
Short tandem repeat (STR) profiling of wt bone-marrow-derived macrophage cell line.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp7-v1.pdf
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Supplementary file 8
Short tandem repeat (STR) profiling of Nos2 knockout bone-marrow-derived macrophage cell line.
- https://cdn.elifesciences.org/articles/94794/elife-94794-supp8-v1.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/94794/elife-94794-mdarchecklist1-v1.pdf
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Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging
eLife 13:RP94794.
https://doi.org/10.7554/eLife.94794.3
