Enalapril mitigates senescence and aging-related phenotypes in human cells and mice via pSmad1/5/9-driven antioxidative genes
- The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, China
- Kunming Institute of Zoology, Chinese Academy of Sciences, China
- Zhanjiang Institute of Clinical Medicine, Central People's Hospital of Zhanjiang, Guangdong Medical University, China
Figures
Figure 1 with 1 supplement
Enalapril alleviates cellular senescence.
(A, D) Senescence-associated β-galactosidase (SA-β-Gal) staining (A) and statistical analysis of SA-β-Gal ratios (D) in IMR90 cells after enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (B, E) Ki67 immunofluorescence experiment (B) and statistical analysis of Ki67 intensity (E) after enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. (C) Western blot analysis showing the protein levels of p16 and p21 in IMR90 cells after enalapril treatment. (F) Heatmap showing cell cycle-related gene (green) upregulation and senescence-associated secretory phenotype (SASP) gene (purple) downregulation after enalapril treatment. (G) RNA expression of SASP factors in IMR90 cells after enalapril treatment, with blue bars representing the control group and red bars representing the enalapril-treated group. (H) Growth curves of IMR90 cells after enalapril treatment. The x-axis represents the passage time, while the y-axis represents population doubling (PD). A two-tailed t-test was employed; ns indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 1—source data 1
PDF file containing original western blots for Figure 1C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1C.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig1-data2-v1.zip
Figure 1—figure supplement 1
The expression of various senescence markers during IMR90 cell senescence.
(A) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and statistical analysis of SA-β-Gal ratios (right) during IMR90 cell senescence. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (B) Western blot analysis showing the protein levels of p16 and p21 in IMR90 cells at different passages. (C) Ki67 immunofluorescence experiment (left) and statistical analysis of Ki67 intensity (right) during IMR90 cell senescence. Scale bars, 80 μm. Enlarged scale bars, 40 μm. (D) RNA expression of senescence-associated secretory phenotype (SASP) factors during IMR90 cell senescence, with blue bars representing the young group and red bars representing the old group. A two-tailed t-test was employed, ns indicates no significant difference, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 1—figure supplement 1—source data 1
PDF file containing original western blots for Figure 1—figure supplement 1B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig1-figsupp1-data2-v1.zip
Figure 2 with 3 supplements
Phosphorylated Smad1/5/9 mediates the anti-senescence effect of enalapril.
(A) Western blot results for the detection of pSmad5, pSmad1/5/9, and other proteins after enalapril treatment. (B) GSEA showing the enrichment of the BMP signaling pathway after enalapril treatment. NES, normalized enrichment score. ES, enrichment score. (C) Profile of pSmad1/5/9 enrichment at the TSS regions in the control group (Ctrl) and the enalapril-treated group (Enalapril). (D) Heatmap of cell cycle (green) and SASP-related genes (purple) in the control group (Ctrl), BMP receptor inhibitor-treated group (LDN193189, LDN), and enalapril and BMP receptor inhibitor-cotreated group (EP+LDN). (E) Western blot analysis showing the protein levels of pSmad1/5/9, p16, and p21 in response to different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. (F) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and corresponding ratio statistical chart (right) of IMR90 cells treated with different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (G) Ki67 immunofluorescence experiment (left) and intensity statistical chart (right) of IMR90 cells treated with different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed; ns indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2A and E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2A and E.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-data2-v1.zip
Figure 2—figure supplement 1
Alterations in critical regulators of cellular senescence.
(A) Western blot analysis showing the changes in protein levels of key targets in classical senescence signaling pathways following enalapril treatment. (B) Western blot analysis showing the changes in Smad and BMP-related protein levels during IMR90 cell senescence.
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Figure 2—figure supplement 1—source data 1
PDF file containing original western blots for Figure 2—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
pSmad1/5/9 alleviates cellular senescence phenotypes.
(A) Western blot analysis showing the changes in protein levels following BMP4 treatment. (B) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and corresponding ratio analysis chart (right) following BMP4 treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (C) Ki67 immunofluorescence (right) and corresponding ratio analysis chart (left) following BMP4 treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, **p<0.01, ****p<0.0001.
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Figure 2—figure supplement 2—source data 1
PDF file containing original western blots for Figure 2—figure supplement 2A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 2A.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-figsupp2-data2-v1.zip
Figure 2—figure supplement 3
Reduction of pSmad1/5/9 correlates with cellular senescence.
(A) Western blot showing the protein levels after the addition of LDN193189. (B) Senescence-associated β-galactosidase (SA-β-gal) staining (left) after adding LDN193189 and its corresponding staining proportion statistical chart (right). Scale bars, 200 μm. Enlarged scale bars, 100 μm. (C) Immunofluorescence staining for Ki67 (left) after adding LDN193189, along with the corresponding intensity statistical chart (right). Scale bars, 80 μm. Enlarged scale bars, 40 μm. (D) Western blot showing the protein levels following BMPR1A knockdown. (E) Relative RNA levels of BMPR1A following BMPR1A knockdown. (F) SA-β-gal staining (left) and its corresponding staining proportion statistical chart (right) in the control group (Ctrl) and BMPR1A-knockdown groups (BMPR1A_KD) with enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (G) Ki67 immunofluorescence (left) and its corresponding staining proportion statistical chart (right) in the control group (Ctrl) and BMPR1A-knockdown groups (BMPR1A_KD) with enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, ns indicates no significant difference, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 2—figure supplement 3—source data 1
PDF file containing original western blots for Figure 2—figure supplement 3A, D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-figsupp3-data1-v1.zip
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Figure 2—figure supplement 3—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 3A,D .
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig2-figsupp3-data2-v1.zip
Figure 3 with 1 supplement
pSmad1/5/9 regulate inhibitor of DNA-binding proteins (ID)-induced inhibition of p16, p21, and senescence-associated secretory phenotype (SASP).
(A) Changes in the peaks of all transcription factors identified by CUT&Tag following enalapril treatment, with red dots indicating transcription factors with increased peak intensity (upregulated TFs). (B) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the ID1 region between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. (C) Western blot analysis showing the changes in the protein levels of ID1 and ID2 following enalapril treatment. (D) Changes in the RNA levels of ID1 and ID2 following enalapril treatment. (E) Western blot analysis showing the protein levels of ID1 and ID2 following treatment with a BMP receptor inhibitor (LDN193189, LDN). (F, I) Western blot showing the changes in the protein levels of p16 and p21 following the knockdown of ID1 or ID2 (F) or the inhibition of ID1 (I). (G) RNA expression of SASP factors after ID knockdown, with pink representing ID1 knockdown and orange representing ID2 knockdown. (H) Normalized average RNA expression levels of selected SASP factors and cell cycle arrest factors after enalapril treatment and ID knockdown. Positive values indicate upregulation, while negative values indicate downregulation. The values represent the expression levels relative to those of the Ctrl. (J) Western blot analysis showing pSmad1/5/9 levels following ID1 and ID2 knockdown. (K) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and SA-β-Gal ratio quantification (right) in the control group (Ctrl) and ID-knockdown groups (ID1_KD, ID2_KD) with or without enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (L) Ki67 immunofluorescence intensity in the control group (Ctrl) and ID knockdown groups (ID1_KD, ID2_KD), with or without enalapril treatment. A two-tailed t-test was employed, ns indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3C, E, F, I and J, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3C, E, F, I and J.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig3-data2-v1.zip
Figure 3—figure supplement 1
Knockdown of inhibitor of DNA-binding protein (ID) accelerates cellular senescence.
(A) Western blot showing changes in ID protein levels during cellular senescence. (B) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) following knockdown of ID1 and ID2, along with corresponding statistical chart (right). Scale bars, 200 μm. Enlarged scale bars, 100 μm. (C) Ki67 immunofluorescence (left) following knockdown of ID1 and ID2, along with corresponding statistical chart (right). Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, *p<0.05, ***p<0.001. (D) Ki67 immunofluorescence in control group (Ctrl) and ID knockdown groups (ID1_KD, ID2_KD), with or without enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. (E) Heatmap of SASP factor expression in mouse bone marrow cells following ID knockout from previously reported data (Fei et al., 2023).
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Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig3-figsupp1-data2-v1.zip
Figure 4
pSmad1/5/9 increase the expression of antioxidative genes.
(A) Bar plot showing the enriched Gene Ontology (GO) terms and pathways associated with the CUT&Tag-upregulated peaks (left) and the upregulated RNA-seq genes (right) following enalapril treatment. (B) Heatmap showing the changes in the RNA expression of antioxidative genes. (C) Profile plot showing the increase in the pSmad1/5/9 binding signal of antioxidative genes in the transcription start site (TSS) region following enalapril treatment. (D) Western blot analysis showing the protein levels of representative antioxidative genes after enalapril treatment. (E) Relative RNA levels of TXN, GPX4, and PRDX5 during cellular senescence. The blue bars represent young cells, and the red bars represent senescent cells. (F, H) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the PRDX5 (F) and TXN (H) regions between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. (G) ChIP-qPCR results showing pSmad1/5/9 levels at many peaks following enalapril or combination treatment with enalapril and LDN193189. The y-axis represents the normalized pSmad1/5/9 signals relative to 10% input. pSmad1/5/9 enrichment for HPRT1 and HBB served as a negative control, and pSmad1/5/9 enrichment for ID1 and ID2 served as positive controls, as previously described. A two-tailed t-test was employed, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (I) Detection of reactive oxygen species (ROS) fluorescence levels via the DCFH-DA probe in young cells, senescent cells (Ctrl), cells treated with enalapril, and cells treated with a combination of enalapril and LDN193189. Scale bars, 200 μm.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4D.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig4-data2-v1.zip
Figure 5 with 2 supplements
Enalapril exerts its anti-aging effects on mouse organs via pSmad1/5/9-antioxidative genes.
(A) A schematic workflow of the mouse feeding and experimental procedures. Created with BioRender.com. (B) Heatmap of RNA levels of senescence-associated secretory phenotype (SASP) factors in various organs following enalapril treatment (EP). (C) Cytokine array analysis of secreted proteins (below) and relative quantitation (above) of SASP factors in control serum (Ctrl) and enalapril-treated serum (Enalapril). (D) Western blot analysis showing the protein levels of pSmad1/5/9 and antioxidative genes in various organs following enalapril treatment. (E) Heatmap of the RNA levels of BMP signaling pathway-related genes and antioxidative genes in the brain, kidney, and liver following enalapril (EP) treatment.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5D.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig5-data2-v1.zip
Figure 5—figure supplement 1
Effects of enalapril on the expression changes of genes related to organ physiological functions in mice.
(A) In multiple organs, enalapril increases the enrichment of gene set enrichment analysis (GSEA) pathways associated with improved physiological function. (B) Western blot analysis showing changes in Id1 protein levels in various organs of mice.
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Figure 5—figure supplement 1—source data 1
PDF file containing original western blots for Figure 5—figure supplement 1B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Effects of reduced pSmad1/5/9 levels on the expression changes of genes in mice.
(A) Cytokine array analysis of secreted proteins (above) and relative quantitation (below) of senescence-associated secretory phenotype (SASP) factors in enalapril-treated serum (Enalapril) and co-treated serum (Enalapril +LDN). (B) Western blot analysis showing the protein levels of pSmad1/5/9 and antioxidative genes in various organs following enalapril and LDN co-treatment.
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Figure 5—figure supplement 2—source data 1
PDF file containing original western blots for Figure 5—figure supplement 2B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 5—figure supplement 2B.
- https://cdn.elifesciences.org/articles/104774/elife-104774-fig5-figsupp2-data2-v1.zip
Figure 6 with 2 supplements
Enalapril mitigates age-related degenerative changes in aged mice.
(A) Changes in the body weights of the mice after enalapril feeding. (B, C) Movement trajectory (B) and spontaneous alternation rate (C) in the Y-maze of the mice after enalapril treatment. (D, E) Changes in the grip strength (D) and rotarod fall time (E) of the mice after enalapril treatment. (F, G) Heatmap of movement trajectories (F) and the number of entries into the central area (G) in the open-field test after enalapril treatment. (H-M) Alterations in the serum levels of aspartate transaminase (AST) (H), creatinine (CREA) (I), lactate dehydrogenase (LDH) (J), triglycerides (TGs) (K), low-density lipoprotein cholesterol (LDL) (L), and high-density lipoprotein cholesterol (HDL) (M) after enalapril treatment in mice. (N) Senescence-associated β-galactosidase (SA-β-Gal) staining of the brain, liver, and spleen after enalapril treatment. Scale bars, 20 μm. (O, P) Periodic Acid-Schiff (PAS) staining (O) and Congo red staining (P) of the brain after enalapril treatment. Scale bars, 50 μm. (Q) Sirius red staining of the kidney after enalapril treatment. Scale bars, 50 μm. (R) Oil Red O staining of the spleen, kidney, and liver after enalapril treatment. Scale bars, 20 μm. (S) Hematoxylin and eosin (H&E) staining of the liver after enalapril treatment. Scale bars, 50 μm.
Figure 6—figure supplement 1
Effects of reduced pSmad1/5/9 levels on aging-related behaviors in mice.
(A, D) Changes in the rotarod fall time (A) and grip strength (D) of the mice after enalapril and LDN treatment. (B, C) The number of entries into the central area (B) and heatmap of movement trajectories (C) in the open-field test after enalapril and LDN treatment. (E, F) Spontaneous alternation rate (E) and movement trajectory (C) in the Y-maze of the mice after enalapril and LDN treatment.
Figure 6—figure supplement 2
Enalapril ameliorates pathological phenotypes in aged mice.
(A–C) Senescence-associated β-galactosidase (SA-β-Gal) staining quantification chart of the brain (A), liver (B), and spleen (C) after enalapril treatment. (D, E) Periodic Acid-Schiff (PAS) staining (D) and Congo red staining (E) quantification chart of the brain after enalapril treatment. (F) Sirius red staining quantification chart of the kidney after enalapril treatment. (G–I) Oil Red O staining quantification chart of the spleen (G), kidney (H), and liver (I) after enalapril treatment. (J) Hematoxylin and eosin (H&E) staining quantification chart of the liver after enalapril treatment.
Figure 7
Summary schematic.
It shows pSmad1/5/9-mediated combating cellular senescence of enalapril, and improvement of organ function in mice. Specifically, enalapril increases the level of pSmad1/5/9 and Smad4, promoting the expression of downstream genes, such as antioxidative and cell cycle-related genes, thereby promoting cell proliferation and reducing reactive oxygen species (ROS). Moreover, enalapril mitigates age-related degenerative changes, including enhanced memory, improved kidney function, increased liver metabolism, and other organ functions. Created with BioRender.com.
Additional files
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Supplementary file 1
List of primers used in RT-qPCR.
- https://cdn.elifesciences.org/articles/104774/elife-104774-supp1-v1.docx
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Supplementary file 2
List of primers used in pSmad1/5/9 ChIP-qPCR.
- https://cdn.elifesciences.org/articles/104774/elife-104774-supp2-v1.docx
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Supplementary file 3
List of shRNA target sequences used in knockdown experiment.
- https://cdn.elifesciences.org/articles/104774/elife-104774-supp3-v1.docx
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Supplementary file 4
List of antibodies.
- https://cdn.elifesciences.org/articles/104774/elife-104774-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104774/elife-104774-mdarchecklist1-v1.docx
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Enalapril mitigates senescence and aging-related phenotypes in human cells and mice via pSmad1/5/9-driven antioxidative genes
eLife 14:RP104774.
https://doi.org/10.7554/eLife.104774.3
