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Characterizing primary and secondary senescence in vivo

Nature Aging volume 5, pages 1568–1588 (2025)Cite this article

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Abstract

There is robust evidence that senescence can be propagated in vitro through mechanisms including the senescence-associated secretory phenotype, resulting in the non-cell-autonomous induction of secondary senescence. However, the induction, regulation and physiological role of secondary senescence in vivo remain largely unclear. Here we generated senescence-inducible mouse models expressing either the constitutively active form of MEK1 or MKK6 and mCherry, to map primary and secondary senescent cells. Our models recapitulate characteristic features of senescence and demonstrate that primary and secondary phenotypes are highly tissue- and inducer-dependent. Spatially resolved RNA expression analyses at the single-cell level reveal that each senescence induction results in a unique transcriptional profile—even within cells of the same cell type—explaining the heterogeneity of senescent cells in vivo. Furthermore, we show that interleukin-1β, primarily derived from macrophages, induces secondary phenotypes. Our findings provide insight into secondary senescence in vivo and useful tools for understanding and manipulating senescence during aging.

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Fig. 1: Establishment of mouse models of inducible senescence.
Fig. 2: Bulk and single-cell RNA-seq analysis of senescence-induced cells and surrounding cells.
Fig. 3: Liver zonation changes in primary senescence.
Fig. 4: scRNA-seq analysis reveals the cell-to-cell heterogeneity of primary senescence in the liver.
Fig. 5: scRNA-seq analysis of secondary senescence.
Fig. 6: IL-1 mediates secondary senescence.
Fig. 7: caMEK1 and caMKK6 transcriptome are associated with aging.

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Data availability

Bulk RNA-seq and scRNA-seq data were deposited in the Gene Expression Omnibus under accession number GSE242709. The publicly available datasets used in this study are mm10 mouse reference genome (https://www.10xgenomics.com/support/jp/software/cell-ranger/latest/release-notes/cr-reference-release-notes#2020-a/), GSE145642 (Amor et al.30), GSE132040 (Tabula Muris Senis bulk RNA-seq data, 2020), GSE148080 (Esmaili et al.58), GSE106330 (Tong et al.47), GSE74843 (Fujii et al.48) and GSE149590 (Tabula Muris Senis single-cell data19). Source data are provided with this paper. All other data are available from the corresponding authors upon request.

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Acknowledgements

We thank J. Asahira and K. Okita for technical support with generating RNA-seq and scRNA-seq libraries; F. Nakasuka and Y. Shimada-Takayama for experimental support; S. Sakurai for support with analysis of RNA-seq data; S. Goulas for critical reading and constructive suggestions of the paper; and Single-Cell Genome Information Analysis Core (SignAC) in ASHBi for RNA-seq. This work was supported by Advanced Research & Development Programs for Medical Innovation (AMED-CREST) (grant number JP17gm1110004) and Japan Agency for Medical Research and Development (AMED) (grant number JP22bm1223002) to Y.Y. and T.Y.; AMED-CREST (grant numbers JP22gm1310002 and JP21gm1310011) to T.Y.; AMED (grant number JP24bm1123053) to T.Y.; Japan Science and Technology Agency (JST), Core Research for Evolutionary Science and Technology (CREST) (grant number JPMJCR2023) to T.Y.; JST Fusion Oriented REsearch for disruptive Science and Technology (FOREST) Program (grant number JPMJFR206C) to T.Y.; Core Center for iPS Cell Research (JP22bm0104001) to T.Y.; Core Center for Regenerative Medicine and Cell and Gene Therapy (grant number JP23bm1323001) to T.Y.; Japan Society for the Promotion of Science (JSPS) A3 Foresight Program Number (grant number JPJSA3F20230001) to T.Y.; AMED (grant numbers JP22ama221201, JP22gm1110004, JP22zf0127008 and JP223fa627001) to Y.Y.; and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI) (grant numbers 23H05485 and 23H00407) to Y.Y. ASHBi is supported by the World Premier International Research Center Initiative (WPI), Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Author information

Author notes

  1. Hirofumi Shibata

    Present address: Department of Otolaryngology Head and Neck Surgery, Gifu University Graduate School of Medicine, Gifu, Japan

Authors and Affiliations

  1. Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

    Yuko Sogabe, Hirofumi Shibata, Mio Kabata, Akito Tanaka, Kanae Mitsunaga, Kazunori Sunadome, May Nakajima-Koyama, Knut Woltjen & Takuya Yamamoto

  2. Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto, Japan

    Yuko Sogabe & Hiroshi Seno

  3. Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan

    Kazunori Sunadome & Takuya Yamamoto

  4. Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Michitada Hirano & Yasuhiro Yamada

  5. RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan

    Eisuke Nishida

  6. Medical-risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan

    Takuya Yamamoto

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Contributions

Y.S., Y.Y. and T.Y. designed and conceived of the study and wrote the paper. H. Seno supervised the experiments. Y.S., H. Shibata and M.H. generated vectors, cell lines and mice, and performed experiments. M.N.-K. and E.N. provided technical instruction for experiments. K.S. performed HybISS. K.W. and E.N. provided vectors. A.T. performed blastocyst injections. K.M. analyzed fluorescence-activated cell sorting data. Y.S. and K.S. analyzed RNA-ISH and HybISS data. Y.S., M.K. and T.Y. analyzed RNA-seq data.

Corresponding authors

Correspondence to Yasuhiro Yamada or Takuya Yamamoto.

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Nature Aging thanks Thomas von Zglinicki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Senescence induction in caMEK1 and caMKK6 MEFs.

a, Representative images of caMEK1 and caMKK6 MEFs derived from germline transmission in mice without Dox treatment and after 7 days of 2.0 µg/ml Dox treatment. Scale bars, 100 µm. Experiments were performed in biological triplicates. b, Western blot analysis for p-ERK, ERK, p-p38 and p38 in caMEK1 and caMKK6 MEFs at 3, 7 and 10 days after 2.0 µg/ml Dox treatment. ERK was specifically phosphorylated in caMEK1 MEFs, and p38 was specifically phosphorylated in caMKK6 MEFs. Experiments were performed in biological replicates. c, caMEK1 and caMKK6 MEFs cease proliferation after 2.0 µg/ml Dox treatment. Data are presented as the means ± s.d. of biological triplicates. Paired t test, two sided. d, Representative images of SA-β-gal staining of caMEK1 and caMKK6 MEFs with/without Dox treatment (2 µg/ml, 7 days). Insets are magnified pictures of the dashed areas. Scale bars, 50 µm. e, The SA-β-gal-positive ratios of caMEK1 and caMKK6 MEFs without and with Dox treatment are shown. Data are presented as the means ± s.d. of biological triplicates. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, two sided. f, qRT‒PCR analyses of the expression of senescence markers in caMEK1 and caMKK6 MEFs at 2, 4 and 7 days after 2 µg/ml Dox treatment. Data are presented as the means ± s.d. of biological triplicates. Expression levels relative to those of MEFs without Dox treatment are shown. One-way repeated-measures ANOVA with Dunnett’s multiple comparisons test, two sided.

Source data

Extended Data Fig. 2 caMEK1 and caMKK6 induced senescence in vivo.

a, Representative histological images and immunostaining for mCherry and p-ERK in caMEK1 liver and colon without Dox treatment. Scale bars, 100 μm. b, Representative histological images and immunostaining for mCherry and p-p38 in caMKK6 liver and colon without Dox treatment. Scale bars, 100 μm. c, RNA in situ analysis of mCherry and Cdkn1a gene expression in caMEK1 and caMKK6 liver without Dox treatment. Scale bars, 50 μm. d, Immunofluorescence for mCherry and p21 in caMEK1 and caMKK6 colon without Dox treatment. p21-positive cells were rarely detected in the absence of Dox treatment. Scale bars, 50 μm. e, Immunostaining for p21 in caMEK1 and caMKK6 liver without Dox treatment. p21-positive cells were rarely detected in the absence of Dox treatment. Scale bars, 100 μm. f, Immunostaining for mCherry and p21 in caMEK1 and caMKK6 liver serial sections after 7 days of Dox treatment. Black asterisks indicate mCherry-positive/p21-positive hepatocytes, and white asterisks indicate mCherry-negative/p21-positive hepatocytes. Arrows indicate p21-negative hepatocytes for comparison. Scale bars, 50 μm. g, Quantification of the p21-positive ratio in mCherry-positive and mCherry-negative hepatocytes in caMEK1 and caMKK6 livers. h, Schematic illustration of the genomic construct to activate mCherry as control. i, Immunostaining for mCherry, p-ERK and p-p38 in control liver and colon after 7 days of Dox treatment. Scale bars, 100 μm. j, Immunostaining for mCherry and p21 in control liver and colon serial sections after 7 days of Dox treatment. Scale bars, 100 μm. k, Left: Immunostaining for BrdU in the caMEK1 and caMKK6 colon after 3 and 7 days of Dox treatment. Scale bars, 100 μm. Right: Quantification of the BrdU-positive rate in mCherry-positive crypts of the caMEK1 and caMKK6 colon after 3 and 7 days of Dox treatment. l, Left: Immunofluorescence for mCherry and Ki67 in caMEK1 and caMKK6 liver after 7 days of Dox treatment. Arrowheads indicate Ki67-positive hepatocytes and arrow indicates Ki67-positive non-parenchymal cells (NPCs). Scale bars, 50 μm. Right: Quantification of the Ki67-positive rate in mCherry-positive and mCherry-negative hepatocytes (upper) and the number of Ki67-positive NPCs per field in caMEK1 and caMKK6 liver. m, Left: Immunofluorescence for mCherry and γH2AX in caMEK1 and caMKK6 liver after 7 days of Dox treatment. Arrows indicate γH2AX-positive NPCs. Scale bars, 100 μm. Right: Quantification of the number of γH2AX-positive hepatocytes (upper) and NPCs (lower) per field in caMEK1 and caMKK6 liver. Experiments were performed with n = 3 mice per group (a-g, i-m). Insets are magnified pictures of the dashed areas (f, l, m). Data are presented as the means ± s.d. of biologically independent samples. Ordinary one-way ANOVA with Tukey’s (g and right upper of (l, m)) or Dunnett’s (k) multiple comparisons test, two sided. Unpaired t test, two sided (right lower of (l, m)).

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Extended Data Fig. 3 caMEK1 and caMKK6 induced senescence in vivo.

a, Left: Immunostaining for NFκB in the caMEK1 and caMKK6 liver after 7 days of Dox treatment. Scale bars, 100 μm. Right: Quantification of NFκB-positive cells (n = 3 per group). b, Left: Immunostaining for NFκB in the caMEK1 and caMKK6 colon after 7 days of Dox treatment. Scale bars, 100 μm. Right: Quantification of NFκB-positive ratio in caMEK1 and caMKK6 after 3 and 7 days of Dox treatment (n = 3 per group, except caMKK6 day3 (n = 2)). c, qRT‒PCR analyses of the expression of senescence markers in the liver and colon of caMEK1 and caMKK6 chimeric mice at 3 and 7 days after Dox treatment (caMEK1 No Dox, n = 6; caMEK1 Dox 3 d, n = 7; caMEK1 Dox 7 d, n = 7; caMKK6 No Dox, n = 7; caMKK6 Dox 3 d, n = 6; caMKK6 Dox 7 d, n = 8). Expression levels relative to those in the liver or colon in the absence of Dox treatment are shown. Values that deviated more than twice the standard deviation from the mean were considered outliers and excluded. d, Schematic illustration of the genetic constructs for HDTVI; caMEK1 and HDTVI; caMKK6 for tissue-specific expression in the liver (upper panel). The lower panel shows the experimental protocol. e, RNA in situ analysis of Cdkn1a and mCherry gene expression in the HDTVI; caMEK1 and caMKK6 liver. Scale bars, 50 μm. n = 3 per group of mice examined. f, Quantification of the SA-β-gal index in HDTVI; caMEK1 and HDTVI; caMKK6 liver (HDTVI; caMEK1 No Dox, n = 5; Dox 7 d, n = 6; HDTVI; caMKK6 No Dox, n = 4; Dox, n = 6). g, Schematic illustration of the genetic constructs Villin-Cre; caMEK1 and Villin-Cre; caMKK6 for tissue-specific expression in the colon (upper panel). The lower panel shows the experimental protocol. h, Immunostaining for p21 and BrdU in Villin-Cre; caMEK1 and Villin-Cre; caMKK6 colon serial sections after 7 days of Dox treatment. Scale bars, 100 μm. i, Quantification of the p21- and BrdU-positive cells in Villin-Cre; caMEK1 and Villin-Cre; caMKK6 colons at 3 and 7 days after Dox treatment (Villin-Cre; caMEK1 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4; Villin-Cre; caMKK6 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4). j, qRT‒PCR analyses of the expression of Cdkn2a and Cdkn1a in Villin-Cre; caMEK1 and Villin-Cre; caMKK6 colons at 3 and 7 days after Dox treatment (Villin-Cre; caMEK1 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4; Villin-Cre; caMKK6 No Dox, n = 3; Dox 3 d, n = 3; Dox 7 d, n = 4). Expression levels relative to those in the colon in the 3 days Dox treatment for Cdkn2a or the absence of Dox treatment for Cdkn1a are shown. Data are presented as the means ± s.d. of biologically independent samples. Unpaired t test, two sided (a, f). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, two sided (b, c, i, j).

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Extended Data Fig. 4 RNA-seq analysis of caMEK1 and caMKK6 mosaic mice.

a, FACS sorting strategy to isolate hepatocytes (upper) and colonic epithelial cells (lower). b, PCA for bulk RNA sequencing of the liver (upper) and colon (lower). c, Gene Ontology (GO) term enrichment of genes upregulated and downregulated in the liver and colon of caMEK1 and caMKK6 mice compared with those of control mice (fold change > 2 or < -2, FDR < 0.05 and FPKM > 50). Representative GO terms and P values are shown (Fisher’s exact test, one-sided.). DAVID Bioinformatics Resources (v6.8) were used for the analysis. d, Heatmap showing the relative expression of 17 commonly upregulated genes in all of caMEK1 mCherry and caMKK6 mCherry of the liver and the colon. The color range shows the log2 scale. e, Heatmap showing the relative expression of genes shared with previously reported senescence models30 (listed in (d)). Bolded genes indicate 6 genes increased in both our study and the previous study. TIS; therapy-induced senescence in mouse lung adenocarcinoma KrasG12D; p53−/− cells that are triggered to become senescent by a combination of MEK inhibition and CDK4 and CDK6 inhibition, OIS; oncogene-induced senescence in mouse hepatocytes, mediated by the in vivo delivery of NrasG12V through hydrodynamic teil vein injection, RIS; replication-induced senescence in mouse hepatic stellate cells. RNA-seq data were obtained from GSE145642. The color range shows the log2 scale. f, Immunostaining for uPAR in the liver (left) and colon (right) of caMEK1 and caMKK6 chimeric mice after 7 days of Dox treatment. Insets are magnified images of the dashed areas. Scale bars, 100 μm. n = 3 per group of mice examined. g, Expression levels of Plaur and Serpine1 in bulk RNA-seq analysis in mCherry (m) and GFP (G) cells in the liver and colon of control, caMEK1 and caMKK6 mice. h, Left: RNA in situ analysis of mCherry, Cdkn1a and Plaur gene expression in caMEK1 and caMKK6 liver after 7 days of Dox treatment. Insets are magnified pictures of the dashed areas. Arrows indicate mCherry-positive Plaur-high hepatocytes. Scale bars, 50 μm. Upper right: Quantification of Plaur expression in No Dox, Dox on mCherry-positive and mCherry-negative cells. Cdkn1a-high, log2 (Cdkn1a dots/cell+1) > 3; Plaur-high, log2 (Plaur dots/cell+1) > 2. The center, box and whiskers represent the median, upper and lower quartiles and 1.5× interquartile range (IQR), respectively. Kruskal‒Wallis with Dunn’s multiple comparisons test, two sided. Lower right: Quantification of the Plar-high ratio in Cdkn1a-high cells (n = 3 per group). Ordinary one-way ANOVA with Tukey’s multiple comparisons test, two sided. i, Expression levels of Cd9 in bulk RNA-seq analysis in mCherry (m) and GFP (G) cells in the liver of control, caMEK1 and caMKK6 mice. Liver; Control, n = 2; caMEK1, n = 3; caMKK6, n = 2. Colon; Control, n = 2; caMEK1, n = 2; caMKK6, n = 3(g, i). Data are presented as the means ± s.d. of biologically independent samples(g-i).

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Extended Data Fig. 5 scRNA-seq analysis, subclustering and zonation of the caMEK1 and caMKK6 liver.

a, The scRNA-seq experimental protocol. The Dox concentration was 0.2 mg/ml for caMEK1 and 2.0 mg/ml for caMKK6 and the control. b, Expression levels of representative marker genes for each cell type in UMAP visualization. c, The proportion of NPCs (Cluster 7) in each condition. m, mCherry; G, GFP. d, The cell number and proportion of subclusters in control mCherry and control GFP. e, Heatmap showing the relative expression of subcluster-specific genes in the control, caMEK1 mCherry, caMKK6 mCherry, caMEK1 GFP and caMKK6 GFP. The color range shows the z score. f, KEGG pathway analysis of subcluster-specific genes in the control. Representative KEGG pathway and P values are shown (Fisher’s exact test, one-sided). DAVID Bioinformatics Resources (v6.8) were used for the analysis. g, Violin plot of mCherry in caMEK1 mCherry and caMKK6 mCherry.

Extended Data Fig. 6 HybISS analysis of zonation and secreted transcripts.

a, Schematic illustration of the genetic constructs AAV8-Cre; caMEK1 and AAV8-Cre; caMKK6 in a hepatocyte-specific manner. When using a mosaic dosage of AAV8-CAG-Cre, we observed approximately 20% mCherry-positive hepatocytes. The lower panel shows the experimental protocol for HybISS experiment. AAV8-Cre; caMEK1 No Dox, n = 1; Dox, n = 1; AAV8-Cre; caMKK6 No Dox, n = 1; Dox, n = 1. The Dox concentration used was 0.2 mg/ml for AAV8-Cre; caMEK1 and 2.0 mg/ml for AAV8-Cre; caMKK6. b, These coordinates were used to reconstruct the spatial zonation profiles of liver genes. The fields were manually segmented based on the Glul, Cyp2e1 and Cyp2f2 gene spots. c, Zonation segmentation map in HybISS. mCherry-positive PC- and PP-hepatocytes were distinguished with reference to the concentric zonation gene expression of mCherry-negative hepatocytes. Scale bars, 200 μm. d, Quantified profiles for zonation marker transcripts from HybISS in No Dox, Dox mCherry-positive and mCherry-negative in AAV8-Cre; caMEK1 and AAV8-Cre; caMEK6. The lines show the number of amplicons/1000 μm2. e, Violin plot of Wnt target genes in control, caMEK1 mCherry and caMKK6 mCherry. PC, pericentral; PP, periportal.

Extended Data Fig. 7 caMEK1 and caMKK6 liver demonstrated heterogeneity at the single-cell level.

a, Heatmap showing the relative expression levels of secreted genes upregulated in caMEK1 mCherry compared with the control. The color range shows the z score. Dot plots showing relative expression in caMEK1 mCherry compared with the control. Dot size denotes the percentage of cells expressing the gene, and color denotes the logFC of average expression across all cells of that type. b, Expression levels of hepatic stem cell marker genes in caMEK1 mCherry visualized by UMAP. c, Immunostaining for AFP in caMEK1 chimeric mouse livers after 7 days of Dox treatment. Scale bars, 100 μm. n = 3 mice per group were examined. d, Violin plot of Serpine1 and Zfp36l2 in caMEK1 mCherry. e, RNA in situ analysis of Serpine1 and mCherry gene expression in caMEK1 and caMKK6 chimeric mice. Scale bars, 200 μm. n = 3 mice per group were examined. CV, central vein; PN, portal node. f, Violin plot of cell cycle-related genes (Cdkn2b and Ccnd1) and inflammatory response-associated genes (Cd14, Il1rn and Ifngr1) in caMEK1 mCherry. g, Expression levels of representative subcluster-specific genes (subcluster 0 (PP), Hsd17b13; subcluster 2 (PC), Serpina7 and Serpine1) in caMKK6 mCherry visualized by UMAP. h, The proportion of Serpine1 and/or Cd9 high expressing cells in Cdkn1a-high cells in in scRNA-seq. Cdkn1a-high, Expression Level > 1; Serpine1-high, Expression Level > 0; Cd9-high, Expression Level >0.5.

Extended Data Fig. 8 IL1 and Notch signaling pathway in caMEK1 and caMKK6.

a, Expression levels of Hes1, Jag1 and Dll1 in bulk RNA-seq analysis in mCherry (m) and GFP (G) cells in the liver of control (n = 2), caMEK1 (n = 3) and caMKK6 (n = 2) mice. Data are presented as the means ± s.d. of biologically independent samples. b, Violin plot of Hes1 expression in scRNA-seq in control, caMEK1 and caMKK6 mCherry, GFP Cdkn1a-high and GFP Cdkn1a-low cells. Cdkn1a-high, Expression Level > 1; Cdkn1a-low, Expression Level < 0.5. c, Schematic illustration of RNA in situ quantification analysis. mCherry-negative Proximate cells having a nuclear center of gravity less than 75 pixels from the nuclear center of gravity of mCherry-positive cells. d, Left: RNA in situ analysis of mCherry, Cdkn1a and Hes1 gene expression in caMEK1 and caMKK6 chimeric mice after 7 days of Dox treatment. Insets are magnified pictures of the dashed areas. Arrows indicate Cdkn1a-high proximate hepatocytes with elevated Hes1 expression and arrowheads indicate Cdkn1a-low remote hepatocytes without elevated Hes1 expression. Scale bars, 50 μm. Right: Quantification of Hes1 expression in No Dox, Dox on mCherry-positive and mCherry-negative Proximate (Prox) and Remote, Cdkn1a-high and low cells (n = 3 per group). Cdkn1a-high, log2 (Cdkn1a dots/cell+1) > 3; Cdkn1a-low, log2 (Cdkn1a dots/cell+1) ≦ 1. The center, box and whiskers represent the median, upper and lower quartiles and 1.5× interquartile range (IQR), respectively. Kruskal‒Wallis with Dunn’s multiple comparisons test, two sided. *P < 0.0001. The tables show the percentages of Cdkn1a high- and low-expressing cells in proximal and remote cells. Fisher’s exact test, two sided. e, Upstream analysis of genes upregulated in caMEK1 GFP and caMKK6 GFP in bulk RNA-seq analysis. Representative upstream regulator and P values are shown (Fisher’s exact test, one-sided.). IPA software was used for analysis. f, Expression levels of Il1r1 in bulk RNA-seq analysis in mCherry (m) and GFP (G) of control (n = 2), caMEK1 (n = 3) and caMKK6 (n = 2) in the liver. Data are presented as the means ± s.d. of biologically independent samples. g, Expression levels and distribution of Il1b (left) and Clec4f (right). h, Left: Quantification of the Il1b-positive cell ratio in hepatocytes (Hep, n = 7,523 cells) and nonparenchymal cells (NPC, n = 155 cells). Middle: Expression level of Il1b in Clec4f-negative (n = 131 cells) and Clec4f-positive cells (n = 24 cells) in Cluster 7. Right: Expression level of Il1b in Clec4f-positive cells in mCherry-positive (n = 5 cells) and GFP-positive cells (n = 19 cells). Data are presented as the means ± s.d. of biologically independent samples. Unpaired t test, two sided.

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Extended Data Fig. 9 IL1 mediates secondary senescence.

a, Left: RNA in situ analysis of Ilr1 and mCherry gene expression in AAV8-Cre; caMEK1 and AAV8-Cre; caMKK6 after 7 days of Dox treatment. Insets are magnified pictures of the dashed areas. Arrows indicate mCherry-negative Il1r1-high hepatocytes. Scale bars, 50 μm. Right: Quantification of Ilr1 in No Dox, Dox mCherry-positive, mCherry-negative proximate (Prox) and mCherry-negative remote cells (AAV8-Cre; caMEK1 No Dox, n = 5; Dox, n = 4; AAV8-Cre; caMKK6 No Dox, n = 5; Dox, n = 5). The center, box and whiskers represent the median, upper and lower quartiles and 1.5× interquartile range (IQR), respectively. Kruskal‒Wallis with Dunn’s multiple comparisons test, two sided. *P < 0.0001. b, Left: RNA in situ analysis of Il1b and Clec4f gene expression in AAV8-Cre; caMEK1 and AAV8-Cre; caMKK6 after 7 days of Dox treatment without/with clodronate liposomes. Insets are magnified pictures of the dashed areas. Scale bars, 50 μm. Right: Quantification of Il1b- and Clec4f-positive areas. AAV8-Cre; caMEK1 No Dox, n = 5; Dox, n = 4; Dox + clodronate liposome, n = 4; AAV8-Cre; caMKK6 No Dox, n = 5; Dox, n = 5; Dox + clodronate liposome, n = 5. Data are presented as the means ± s.d. of biologically independent samples. Ordinary one-way ANOVA with Sidak’s multiple comparison test, two sided. c, Quantification of mCherry-positive cells. Approximately 30-40% mCherry-positive hepatocytes were observed after Dox treatment. AAV8-Cre; caMEK1 No Dox, n = 5; Dox, n = 4; Dox + IL1R1 inhibitor, n = 5; Dox + clodronate liposome, n = 4; AAV8-Cre; caMKK6 No Dox, n = 5; Dox, n = 5; Dox + IL1R1 inhibitor, n = 3; Dox + clodronate liposome, n = 5. Data are presented as the means ± s.d. of biologically independent samples. Ordinary one-way ANOVA with Sidak’s multiple comparison test, two sided. d, Quantification of the SA-β-gal index in AAV8-Cre; caMEK1 and AAV8-Cre; caMKK6 after 7 days of Dox treatment without/with IL1R1 inhibitor and clodronate liposomes. AAV8-Cre; caMEK1 No Dox, n = 4; Dox, n = 4; Dox + IL1R1 inhibitor, n = 5; Dox + clodronate liposome, n = 4; AAV8-Cre; caMKK6 No Dox, n = 4; Dox, n = 5; Dox + IL1R1 inhibitor, n = 3; Dox + clodronate liposome, n = 5. Data are presented as the means ± s.d. of biologically independent samples. Ordinary one-way ANOVA with Sidak’s multiple comparison test, two sided.

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Extended Data Fig. 10 caMEK1 and caMKK6 are associated with aging.

a, Gene expression patterns of caMKK6 mCherry liver were compared with Nras-induced OIS data of mouse liver (GSE145642)30. NC, 1000; up, top 100; down, top 100. b, Gene expression patterns of caMEK1 mCherry and caMKK6 mCherry colonic epithelial cells were compared with mouse intestines with Braf-induced OIS (GSE106330)47. NC, 1000; up, top 100; down, top 100. c, The gene expression patterns of the caMEK1 mCherry and caMKK6 mCherry colon were compared with human SSAP data48. SSAP up and down signatures were generated based on the list of classifier genes of human organoids from SSAP (130 upregulated and 20 downregulated genes). A randomly extracted set of 1,000 unchanged genes were used as unchanged controls. d, Expression levels of Lgr5 in bulk RNA-seq analysis of mCherry (m) and GFP (G) cells from control (n = 2), caMEK1 (n = 2) and caMKK6 (n = 3) colon tissues. Data are presented as the means ± s.d. of biologically independent samples. e, Upper: RNA in situ analysis of Lgr5 gene expression in caMEK1 and caMKK6 chimeric mice colon. Scale bars, 50 μm. Lower: Quantification of Lgr5-positive cells per crypt in mCherry-positive crypts and mCherry-negative crypts. f, Gene expression patterns of caMEK1 mCherry and caMKK6 mCherry in the liver were compared with aged mouse liver data51. NC, 1000; up, 965; down, 1,570. g, Gene expression patterns of caMEK1 mCherry and caMKK6 mCherry in the liver were compared with aged mouse liver data52. NC,1,000; up, 813; down,332. h, Gene expression patterns of the caMEK1 mCherry and caMKK6 mCherry colons were compared with aged mouse colon data19. NC, 1,000 (randomly extracted); up, top 100; down, top 100. The mouse colon aging signature was generated from the reanalyzed data obtained from Zang et al.49. i, Venn diagram showing the overlap of the protein expression signature from aged human plasma61 (FDR < 0.05 from 2,925 proteins) with the caMEK1 mCherry colon (upper) or caMKK6 mCherry colon (lower). j, Left: Immunostaining for ChgA in caMEK1 and caMKK6 chimeric mice colon. Scale bars, 100 μm. Right: Quantification of the ChgA-positive cells per crypt in mCherry-positive crypts and mCherry-negative crypts. Box plots show the relative expression levels of NC, unchanged; up, upregulated; down, downregulated genes. The center, box and whiskers represent the median, upper and lower quartiles and 1.5× interquartile range (IQR), respectively. Kruskal‒Wallis with Dunn’s multiple comparisons test, two sided (a-c, f-h). Data are presented as the means ± s.d. of biologically independent samples (n = 3 per group). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, two sided (e, j).

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Sogabe, Y., Shibata, H., Kabata, M. et al. Characterizing primary and secondary senescence in vivo. Nat Aging 5, 1568–1588 (2025). https://doi.org/10.1038/s43587-025-00917-y

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