Mid-zone hepatocytes trade proliferation for survival via Atf4-Chop axis in early acute liver injury
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, Yunnan University, China
Figures
Figure 1
Mid-zone hepatocytes exhibit the most pronounced proliferative decline in early acute liver injury.
Wild-type C57BL/6J mice were intraperitoneally injected with 300 mg/kg acetaminophen (APAP) for 0, 3, 6, 12, and 24 hr. (A) Immunohistochemical staining of Ki67 to detect proliferating hepatocytes in liver sections at 0, 3, 6, 12, and 24 hr post-APAP. Representative images of pericentral (PC), middle (Mid), and periportal (PP) are shown. Red arrows indicate Ki67+ hepatocytes. (B) Quantitative analysis of Ki67+ hepatocytes across liver zones (PC, Mid, PP) at 0, 3, 6, 12, and 24 hr post-APAP. (C) Quantitative analysis of Ki67+ hepatocytes across 0, 3, 6, 12, and 24 hr post-APAP in each liver zone (PC, Mid, PP). (D) Changes of Ki67+ hepatocytes across liver zones (PC, Mid, PP) at 0, 3, 6, 12, and 24 hr post-APAP. Value change = number of Ki67+ hepatocytes at x hr post-APAP—the number of Ki67+ hepatocytes at 0 hr post-APAP. (a) denotes significance between PC and Mid regions, (b) denotes significance between PC and PP regions, and (c) denotes significance between Mid and PP regions. Data points represent mean hepatocyte counts from six high-power fields per zonal layer (n=3 mice/group) in B and C. Data are represented as means ± SD; one-way ANOVA (B, C, D). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 1—source data 1
Numerical data of Figure 1B–D.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
Mid-zone hepatocytes show distinct gene expression profiles.
(A) Heatmap of the top 10 differentially expressed genes in PP, Mid, and PC hepatocyte zones at 0, 3, 6, and 24 hr post-acetaminophen (APAP). To highlight relative expression shifts across zones, this heatmap is visualized using row-wise Z-score standardization, which scales each gene to a uniform range and emphasizes spatial switching patterns rather than absolute transcript abundance. Functional categories at the bottom were assigned using gene ontology (GO) terms (biological process, cellular component, molecular function); for genes annotated with multiple GO terms, the most representative or significantly enriched term was selected. (B) Immunofluorescence staining for glutamine synthetase (GS, a PC marker) with DAPI (blue) in liver sections at 0, 3, 6, and 24 hr after APAP administration. Quantification of the zonal distribution of GS-positive cells at the indicated time points. (C) Immunohistochemical detection of p62 (Sqstm1 protein, a midzone marker during early injury) in liver sections at 0, 3, 6, and 24 hr after APAP administration. Red arrows indicate representative p62-positive hepatocytes. Quantification of the zonal distribution of p62-positive cells at the indicated time points. (D) Immunohistochemical staining of Cyp2f2 (a PP marker) in liver sections at 0, 3, 6, and 24 hr after APAP administration. Quantification of the zonal distribution of Cyp2f2-positive cells at the indicated time points. Data are presented as mean ± SD. One-way ANOVA (B–D). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.
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Figure 2—source data 1
Numerical data of Figure 2B–D.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
The liver displays transcriptome-wide zonation in acetaminophen-induced liver injury.
(A) Schematic figure illustrating the experimental strategy for spatial transcriptome analysis. n=1 mouse/time point. (B) Representative H&E staining images illustrating liver morphology in mice following intraperitoneal injection of 300 mg/kg acetaminophen (APAP) at various time points (0, 3, 6, and 24 hr). These liver sections were subsequently used for spatial transcriptome analysis. Injured area is outlined by red line. Necrotic areas outlined by loss of cellular architecture on H&E. (C) Spatial map visualizing the spatiotemporal dynamics of hepatocyte zones (zones periportal [PP], mid-zonal [Mid], and pericentral [PC]) at 0, 3, 6, 24 hr post-APAP, respectively. (D and E) Uniform Manifold Approximation and Projection (UMAP) visualizing the spatiotemporal dynamics of hepatocyte zones (zones PP, Mid, and PC) at 0, 3, 6, 24 hr post-APAP, based on zonal distribution and time points, respectively. (F) Spatiotemporally resolved heatmaps of representative PC marker Glul, PP marker Cyp2f2 in zoom-in area. (G) Volcano plot illustrating the differentially expressed genes (DEGs) in mid-zone at 6 hr compared to 0 hr post-APAP. Gray dots denote genes that are not statistically significant. Red and blue dots represent genes that are upregulated and downregulated, respectively, in the sample tissue, at least a 0.5-fold difference from the matched control, with a false discovery rate (FDR) threshold of 0.05. (H) Scatter plot shows the dynamic changes of Sqstm1 mean expression log2(TPM) in hepatocyte zones (zones PP, Mid, and PC) at 0 and 6 hr post-APAP. TPM: transcripts per million. (I) Percentage of Ki67-positive spots analyzed at 0, 3, 6, and 24 hr post-APAP. (J) Average module score for S-phase (up) and G2/M-phase (down) genes across hepatocyte zones (zones PP, Mid, and PC) at 0, 3, 6, 24 hr post-APAP. (K) Expression levels of the S-phase gene Nasp (up) and the G2/M phase gene Cks1b (down) in each hepatocyte zone (zones PP, Mid, and PC) at 0, 3, 6, 24 hr post-APAP.
Figure 3 with 1 supplement
Zonal metabolism of acetaminophen (APAP) leads to stress response in mid-zone hepatocytes.
(A) Schematic figure illustrating the metabolic process of liver injury caused by APAP. (B) Dot plot displays the log-normalized absolute transcript abundance of the Cyp family in hepatocyte zones (zones periportal [PP], mid-zonal [Mid], and pericentral [PC]) at 0, 3, 6, 24 hr post-APAP. (C) Immunohistochemical staining of Cyp1a2 to detect its dynamic changes in hepatocyte zones (from central vein [CV] to portal vein [PV]) at 0, 3, 6, 24 hr post-APAP. Injured area is outlined by black dashed lines. (D) Quantification of zonal distribution of Cyp1a2-positive cells in liver sections at 0, 3, 6, and 24 hr post-APAP, respectively. (E) Schematic figure illustrating expression changes of Cyp1a2 before and after APAP. (F) Immunofluorescence staining of Chop protein (green) to observe the dynamic changes in its expression across hepatocyte zones (from CV to PV) at 6 hr post-APAP with doses of 100, 300, or 500 mg/kg. Cyp1a2 protein (red) staining highlights the area around the CV. Cell nuclei are stained with DAPI (blue). (G) Quantification of zonal distribution of Chop-positive cells in liver sections from mice treated with varying doses of APAP—100, 300, or 500 mg/kg. (H) Schematic explaining why mid-lobular hepatocytes display a distinct gene expression program early after APAP injury. Cyp enzymes are expressed in a decreasing gradient from the CV to the PV. APAP is therefore metabolized primarily by pericentral Cyp-expressing hepatocytes; their necrosis shifts the main site of residual Cyp activity to the mid-lobular zone. High local APAP metabolism then drives a specific stress-response program, accounting for the unique early transcriptional signature of the mid-zone. Data are represented as means ± SD; one-way ANOVA (D, G). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 3—source data 1
Numerical data of Figure 3D and E.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
The zonal expression of the Cyp family dictates the zonal hepatocyte response to acetaminophen (APAP).
(A) Scatter plot showing the dynamic changes of Cyp2e1 and Cyp1a2 mean expression log2 (TPM), in hepatocyte zones (periportal [PP], mid-zonal [Mid], pericentral [PC]) at 0, 3, 6, and 24 hr post-APAP. TPM: transcripts per million. (B) Representative H&E staining images illustrating liver morphology in mice following intraperitoneal injection of various doses of APAP (100, 300, 500 mg/kg) at 6 hr post-APAP. Injured area is outlined by black dashed lines. Necrotic areas outlined by loss of cellular architecture on H&E. (C) The percentage of injured area is quantified. n=3 mice/group. Data are represented as means ± SD; one-way ANOVA (C). *p<0.05; ***p<0.001.
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Figure 3—figure supplement 1—source data 1
Numerical data of Figure 3—figure supplement 1C.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 3 supplements
The Atf4-Ddit3 axis emerges as pivotal in mid-zone during early acute injury.
(A) Heatmap displays the area under the curve (AUC) scores of transcription factor (TF) motifs, estimated per dot by single-cell regulatory network inference and clustering (SCENIC), highlighting gene regulatory networks and differentially activated TF motifs in hepatocyte zones (periportal [PP], mid-zonal [Mid], pericentral [PC]) at 6 hr post-acetaminophen (APAP). Columns represent TF motifs, rows represent dots, and color intensity indicates AUC scores. (B) Heatmap shows inferred TFs' activity across different zonal regions at 6 hr post-APAP. Activity was quantified as regulon enrichment scores using AUCell. The color scale represents regulon activity scores. (C) Violin plots show regulon activity of Atf4 and Ddit3 in each zonation at 6 hr post-APAP. Activity was quantified as regulon enrichment scores using AUCell. (D) Gene ontology (GO) pathway analysis reveals the top 15 enriched pathways for genes co-regulated by Atf4 and Ddit3 in Mid hepatocyte zones at 6 hr post-APAP. (E) Heatmap showing the target genes’ expression of Atf4 (left) and Ddit3 (right) in hepatocyte zones (zones PP, Mid, and PC) at 6 hr post-APAP. (F) Immunohistochemistry staining of p-eIF2A in liver sections from mice at 0 hr and 3 hr post-APAP. p-eIF2A-positive hepatocytes are indicated by red arrows. The number of p-eIF2A-positive cells per field of view (FOV) is quantified. n=3 mice/group. (G) Heatmap displays stress response and ER stress-related genes from differentially expressed genes (DEGs) identified in Mid hepatocyte zones at 0 and 6 hr post-APAP. Data are represented as means ± SD; unpaired Student’s t-test (F). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 4—source data 1
Numerical data of Figure 4F.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
The Atf4-Ddit3 axis emerges as pivotal in mid-zone hepatocytes during the initial stages of acute injury.
(A) Heatmap displays the area under the curve (AUC) scores of transcription factor (TF) motifs, estimated per dot by single-cell regulatory network inference and clustering (SCENIC), highlighting gene regulatory networks and differentially activated TF motifs in hepatocyte zones (periportal [PP], mid-zonal [Mid], pericentral [PC]) at 3 hr post-acetaminophen (APAP). Columns represent TF motifs, rows represent dots, and color intensity indicates AUC scores. (B) The heatmap shows inferred TFs’ activity across different zonal regions at 3 hr post-APAP. Activity was quantified as regulon enrichment scores using AUCell. The color scale represents regulon activity scores. (C) Violin plots show regulon activity of Atf4 (left) and Ddit3 (right) in each zonation at 3 hr post-APAP. Activity was quantified as regulon enrichment scores using AUCell. (D) Gene regulatory network displays Atf4 and its target genes, as well as Ddit3 and its target genes at 3 hr post-APAP, respectively. Edge color indicates the regulatory strength of TFs on target genes, as measured by AUCell scores. (E) Gene ontology (GO) pathway analysis reveals the top 15 enriched pathways for genes co-regulated by Atf4 and Ddit3 in Mid hepatocyte zones at 3 hr post-APAP. (F) GO pathway analysis reveals the top 15 enriched pathways for upregulated genes in Mid hepatocyte zones at 3 hr compared to the rest of the hepatocyte zones post-APAP. (G) Heatmap displays stress response and ER stress-related genes from differentially expressed genes (DEGs) identified in Mid hepatocyte zones at 0 and 3 hr post-APAP.
Figure 4—figure supplement 2
Validation of zonal hepatocyte responses and transcriptional changes post-acetaminophen (APAP) using the GSE223561 scRNA-seq dataset.
(A) Uniform Manifold Approximation and Projection (UMAP) visualization of hepatocytes colored by cell-type annotations. (B) UMAP visualization of hepatocytes colored by time post-APAP treatment. (C) UMAP visualization of hepatocytes colored based on the expression of centrally zonated genes (Glul, Cyp2e1, Cyp2a5), periportal genes (Alb, Cyp2f2, Sds), and mid-zonal genes (Hmox1, Atf3, Ubc). (D) Gene ontology (GO) pathway analysis showing the top 10 enriched pathways for upregulated genes in mid-zonal hepatocytes at 3 hr post-APAP. (E) GO pathway analysis showing the top 10 enriched pathways for upregulated genes in mid-zonal hepatocytes at 6 hr post-APAP. (F) Heatmap illustrating the area under the curve (AUC) scores of transcription factor (TF) motifs (top 50), as estimated per cell using single-cell regulatory network inference and clustering (SCENIC). Differentially activated motifs in each zonal region are shown for 3 hr post-APAP. (G) Heatmap illustrating the AUC scores of TF motifs (top 50) estimated per cell using SCENIC, highlighting differentially activated motifs in each zonal region at 6 hr post-APAP.
Figure 4—figure supplement 3
Zonal hepatocyte responses and transcriptional reprogramming in human acetaminophen (APAP)-induced liver injury, analyzed using the GSE223561 dataset.
(A) Uniform Manifold Approximation and Projection (UMAP) of all single-nucleus RNA-sequencing (snRNA-seq) nuclei, colored by individuals (healthy controls versus APAP patients; n=9 and n=10, respectively). (B) UMAP of all snRNA-seq nuclei, annotated by major hepatic cell lineages. (C) UMAP of the hepatocyte subset, colored by disease status. (D) UMAP of hepatocytes showing expression levels of representative zonation markers: pericentral (PC) (Glul, Cyp2e1, Cyp2a5), periportal (PP) (Alb, Cyp2f2, Sds), and mid-zonal (Mid) (Igfbp2, Hamp, Hamp2). (E) Heatmap comparing zonal expression patterns of genes involved in stress response/cell death, cell cycle/proliferation, and DNA repair across PP, Mid, and PC hepatocyte clusters in healthy and APAP groups from the snRNA-seq dataset. (F) Heatmap showing the spatial distribution of the same gene categories across PP, Mid, and PC zones in healthy and APAP patient samples from the spatial transcriptomics (ST) dataset.
Figure 5
The Atf4-Ddit3 axis protects hepatocytes from liver injury.
(A) Immunohistochemical staining of Atf4 in liver sections at 0 and 6 hr post-acetaminophen (APAP). Red arrows indicate Atf4-positive hepatocytes. Zonal distribution of Atf4-positive cells in liver sections at 6 hr post-APAP is quantified. The statistic is the percentage of Atf4-positive hepatocytes in each layer over the total number of Atf4-positive hepatocytes. n=3 mice. (B) Schematic figure illustrating the overexpression of Atf4 via AAV in hepatocytes of wild-type C57BL/6J mice. Overexpression of EGFP is used as a control. (C) H&E staining showing liver morphology from mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 6 hr post-APAP. The injured area is outlined by black dashed lines. The percentage of injured area is quantified. n=3 mice/group. Necrotic areas outlined by loss of cellular architecture on H&E. (D) Serum levels of ALT are measured in mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 6, 24, 48, and 72 hr post-APAP. n=6 mice/group. (E) Immunohistochemistry staining of Atf4 in liver sections from mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 24 hr post-APAP. Atf4-positive hepatocytes are indicated by red arrows. The number of Atf4-positive cells per field of view (FOV) is quantified. n=9 mice/group. (F) Immunofluorescence staining of Ki67 (red) was performed to assess proliferating hepatocytes in mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 72 hr post-APAP. Cell nuclei were stained with DAPI (blue). The number of Ki67-positive cells per FOV is quantified. n=6 mice/group. (G) Immunohistochemistry staining of p-eIF2A in liver sections from mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 24 hr post-APAP. p-eIF2A-positive hepatocytes are indicated by red arrows. The number of p-eIF2A-positive cells per FOV is quantified. n=6 mice/group. Data are represented as means ± SD; unpaired Student’s t-test (C–G). One-way ANOVA (A). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 5—source data 1
Numerical data of Figure 5A and C–G.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig5-data1-v1.xlsx
Figure 6 with 2 supplements
The Atf4-Ddit3 axis protects the liver by pausing proliferation via Btg2.
(A) Heatmap of Ddit3-binding signals in chromatin domains. (B) Overlap of Ddit3-binding genes by Cut&Run with differentially expressed genes (DEGs) in mid-zone was shown. These genes were referred to as Ddit3-binding DEGs. (C) Integrative Genomics Viewer (IGV) plot shows the Cut&Run peaks of Ddit3 and input on Btg2-site, Ifrd1-site, Fos-site, and Egr1-site. (D) Immunohistochemical staining of Btg2 in liver sections at 0 and 6 hr post-acetaminophen (APAP). Red arrows indicate Btg2-positive hepatocytes. (E) Schematic figure illustrating overexpression of Ddit3 via AAV in hepatocytes of wild-type C57BL/6J mice. Overexpression of EGFP is used as a control. (F) Immunohistochemical staining of Btg2 in liver sections from mice overexpressing either AAV-TBG-EGFP or AAV-TBG-Ddit3 at 12 hr post-APAP. Btg2-positive hepatocytes are indicated by red arrows. Btg2-positive hepatocytes in liver sections are quantified. n=4 mice/group. (G) Schematic figure illustrating overexpression of Btg2 via AAV in hepatocytes of wild-type C57BL/6J mice. Overexpression of EGFP is used as a control. (H) H&E staining shows morphology of livers from AAV-TBG-EGFP or AAV-TBG-Btg2 overexpressing mice at 6 hr post-APAP. Injured area is outlined by black dashed lines. The percentage of injury area is quantified. n=3 mice/group. Necrotic areas outlined by loss of cellular architecture on H&E. (I) Serum levels of ALT are measured at 6 hr post-APAP in AAV-TBG-EGFP or AAV-TBG-Btg2 overexpressing mice. n=3 mice/group. (J) Immunohistochemical staining of Ki67 in liver sections from mice overexpressing either AAV-TBG-EGFP or AAV-TBG-Btg2 at 72 hr post-APAP. Ki67-positive hepatocytes are indicated by red arrows. Ki67-positive cells in liver sections are quantified. n=4 mice/group. (K) Schematic figure illustrating knockdown of Btg2 via AAV in hepatocytes of wild-type C57BL/6J mice. IP479-EFS-sgEGFP is used as a control. (L) Serum levels of ALT are measured at 6 hr post-APAP in IP479-EFS-sgEGFP or IP479-EFS-sgBtg2 knockdown mice. n=3 mice/group. (M) H&E staining shows morphology of livers from IP479-EFS-sgEGFP or IP479-EFS-sgBtg2 knockdown mice at 6 hr post-APAP. Injured area is outlined by black dashed lines. The percentage of injury area is quantified. n=3 mice/group. Necrotic areas outlined by loss of cellular architecture on H&E. (N) Immunohistochemical staining of Ki67 in liver sections from IP479-EFS-sgEGFP or IP479-EFS-sgBtg2 knockdown mice at 72 hr post-APAP. Ki67-positive cells in liver sections are quantified. n=3 mice/group. Data are represented as means ± SD; unpaired Student’s t-test (F, H–N). **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 6—source data 1
Numerical data of Figure 6F, H–J, and L–N.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Transduction efficiency and confounding effects of AAV-TBG overexpression.
(A) Western blot analysis of Cyp2e1 protein in whole liver lysates from mice transduced with 1.2×1011 viral genome copies of AAV-TBG-EGFP, AAV-TBG-Atf4, or AAV-TBG-Btg2. Cyp2e1/GAPDH ratios are shown. n=4 mice/group. (B) Transduction efficiency of AAV-TBG-EGFP, AAV-TBG-Atf4, and AAV-TBG-Btg2 in mouse liver was measured by immunostaining for the transgene proteins. n=4–8 mice/group. (C) Correlation between transduction efficiency of AAV-TBG-Atf4 or AAV-TBG-Btg2 and serum ALT levels in the respective AAV-injected mice. Pearson correlation coefficient r and p-values are indicated. (D) Btg2 mRNA expression in liver tissues from IP479-EFS-sgEGFP and IP479-EFS-sgBtg2 mice. n=9 mice/group. Data are represented as means ± SD; Pearson correlation (C); unpaired two-tailed Student’s t-test (B, D). *p<0.05; **p<0.01; ****p<0.0001.
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Figure 6—figure supplement 1—source data 1
Numerical data of Figure 6—figure supplement 1B–D.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig6-figsupp1-data1-v1.xlsx
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Figure 6—figure supplement 1—source data 2
PDF file containing original western blots for Figure 6—figure supplement 1A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig6-figsupp1-data2-v1.pdf
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Figure 6—figure supplement 1—source data 3
Original files for western blot analysis displayed in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig6-figsupp1-data3-v1.zip
Figure 6—figure supplement 2
The ISR-Btg2 axis emerges in CCL4-induced acute liver injury.
(A) H&E staining shows morphology of livers from mice at 18 hr post oil and CCL4 injection. Injured area is outlined by black dashed lines. The percentage of injury area is quantified. n=4 mice/group. Necrotic areas outlined by loss of cellular architecture on H&E. (B) Serum levels of ALT are measured at 18 hr post-CCL4 mice. n=4 mice/group. (C) Immunohistochemistry staining of p-eIF2A in liver sections from mice at 18 hr post oil and CCL4 injection. p-eIF2A-positive hepatocytes are indicated by red arrows. n=4 mice/group. (D) Immunohistochemistry staining of Atf4 in liver sections from mice at 18 hr post oil and CCL4 injection. Atf4-positive hepatocytes are indicated by red arrows. n=4 mice/group. (E) Immunofluorescence staining of Chop protein (green) in liver sections from mice at 18 hr post oil and CCL4 injection. Cyp1a2 protein (red) staining highlights the area around the CV. Cell nuclei are stained with DAPI (blue). n=4 mice/group. (F) Immunohistochemistry staining of Btg2 in liver sections from mice at 18 hr post oil and CCL4 injection. Btg2-positive hepatocytes are indicated by red arrows. n=4 mice/group. (G) Immunofluorescence staining of Ki67 (red) was performed to assess proliferating hepatocytes in mice at 18 hr post oil and CCL4 injection. Cyp1a2 protein (red) staining highlights the area around the CV. Cell nuclei were stained with DAPI (blue). n=4 mice/group. Data are represented as means ± SD; unpaired Student’s t-test (A, B). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 6—figure supplement 2—source data 1
Numerical data of Figure 6—figure supplement 2A and B.
- https://cdn.elifesciences.org/articles/108646/elife-108646-fig6-figsupp2-data1-v1.xlsx
Figure 7
The Atf4-Ddit3 axis mediates integrated stress protection in mid-zone hepatocytes at the expense of proliferative capacity during early AILI.
Schematic model depicting the adaptive response of mid-zone hepatocytes to acute liver injury, wherein activation of the Atf4-Ddit3 pathway upregulates Btg2 to induce transient cell-cycle arrest, balancing survival and regeneration.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit polyclonal anti-Ki67 antibody | Abcam | Cat# ab15580; RRID:AB_443209 | IF (1:1000) IHC (1:400) |
| Antibody | Rabbit Glutamine Synthetase Polyclonal antibody | Proteintech | Cat# 11037–2-AP; RRID:AB_2110650 | IF (1:200) |
| Antibody | Mouse monoclonal Anti-Cyp1a2 antibody | SantaCruz | Cat# sc-53241; RRID:AB_629359 | IF (1:50) IHC (1:50) |
| Antibody | Rabbi CHOP; GADD153 Polyclonal antibody | Proteintech | Cat# 15204–1-AP; RRID:AB_2292610 | IF (1:1000) IHC (1:200) |
| Antibody | Monoclonal ATF-4 (D4B8) Rabbit mAb | Cell Signalling Technology | Cat# 11815 S; RRID:AB_2616025 | IHC (1:200) |
| Antibody | Rabbit BTG2 Polyclonal antibody | Proteintech | Cat# 22339–1-AP; RRID:AB_2879078 | IHC (1:100) |
| Antibody | Polyclonal Phospho-eIF2α (Ser51) (D9G8) XP Rabbit mAb | Cell Signalling Technology | Cat# 3398 s; RRID:AB_2096481 | IHC (1:50) |
| Antibody | Goat anti-Rabbit IgG (H+L) Secondary Antibody, Biotin | Invitrogen | Cat# 65–6140; RRID:AB_2533969 | IHC (1:1000) |
| Antibody | Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG(H+L) | Jackson ImmunoResearch | Cat# 115-035-003; RRID:AB_10015289 | IHC (1:1000) WB (1:2000) |
| Antibody | Peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) | Jackson ImmunoResearch | Cat# 111-035-003; RRID:AB_2313567 | WB (1:2000) |
| Antibody | Avidin, NeutrAvidin, Horseradish Peroxidase conjugate | Invitrogen | Cat# A2664 | IHC (1:2000) |
| Antibody | Alexa Fluor 488-conjugated Affinipure Goat Anti-Mouse IgG +IgM(H+L) | Jackson ImmunoResearch | Cat# 115-545-044; RRID:AB_2338844 | IHC (1:400) |
| Antibody | Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | Cat# 115-585-003; RRID:AB_2338871 | IHC (1:200) |
| Antibody | Alexa fluor 568-goat anti-rabbit IgG(H+L)cross-adsorbed secondary antibody | Invitrogen | Cat# A11011; RRID:AB_143157 | IHC (1:1000) |
| Antibody | Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | Cat# 111-545-003; RRID:AB_2338046 | IHC (1:400) |
| Antibody | CYP2E1-Specific Polyclonal antibody | Proteintech | Cat# 19937–1-AP; RRID:AB_10646444 | WB (1:5000) |
| Antibody | GAPDH Monoclonal antibody | Proteintech | Cat# 60004–1-Ig; RRID:AB_2107436 | WB (1:5000) |
| Chemical compound, drug | Acetaminophen(APAP) | Sigma | Cat# A7085 | |
| Chemical compound, drug | Paraformaldehyde, (CH2O)n | Sangon Biotech | Cat# A500684-0500 | |
| Chemical compound, drug | Phosphate Buffered Saline | VivaCell | Cat# C3580-0500 | |
| Chemical compound, drug | 10% Neutral Formalin Fix Solution | Sangon Biotech | Cat# E672001-0500 | |
| Chemical compound, drug | Sucrose, C12H22O11 | Sangon Biotech | Cat# A502792-0005 | |
| Chemical compound, drug | High effect paraffinceresin | Shanghai Hualing Rehabilitation Equipment Manufacturing Plant. | Cat# N/A | |
| Chemical compound, drug | Xylene | Tianjin Zhiyuan Chemical Reagents Co., Ltd. | Cat# N/A | |
| Chemical compound, drug | Ethanol | Tianjin Zhiyuan Chemical Reagents Co., Ltd. | Cat# N/A | |
| Chemical compound, drug | Citrate Antigen Retrieval Solution (Powder) | Sangon Biotech | Cat# E673002-0020 | |
| Chemical compound, drug | Tris | Solarbio | Cat# T8060 | |
| Chemical compound, drug | Disodium salt dihydrate, C10H14N2O8Na2·2H2O(EDTA) | Sangon Biotech | Cat# A500838-0500 | |
| Chemical compound, drug | Hydrogen peroxide | Cat# N/A | ||
| Chemical compound, drug | Triton X-100 | Sangon Biotech | Cat# A600198-0500 | |
| Chemical compound, drug | Goat serum | VivaCell | Cat# C2530-0100 | |
| Chemical compound, drug | Hematoxylin | Sangon Biotech | Cat# A600701-0050 | |
| Chemical compound, drug | Eosin Y(water soluble) | Aladdin | Cat# E141405 | |
| Chemical compound, drug | Neutral balsam | Solarbio | Cat# G8590 | |
| Chemical compound, drug | Tissue-tek OCT compound | SAKURA | Cat# REF:4583 | |
| Chemical compound, drug | Acetone | Chron Chemicals | Cat# N/A | |
| Chemical compound, drug | Tween20 | Sangon Biotech | Cat# A600560-0500 | |
| Chemical compound, drug | DAPI Staining Solution | Beyotime | Cat# C1006 | |
| Chemical compound, drug | Isopentane | Aladdin | Cat# M108171 | |
| Chemical compound, drug | Methanol | Tianjin Zhiyuan Chemical Reagents Co., Ltd. | Cat# N/A | |
| Chemical compound, drug | 20 X TBS buffer | Sangon Biotech | Cat# B548105-0500 | |
| Chemical compound, drug | UltraPure DNase/RNase-Free Distilled Water | Invitrogen | Cat# 10977015 | |
| Chemical compound, drug | Trichloromethane | Chron Chemicals | Cat# N/A | |
| Chemical compound, drug | FBS | VivaCell | Cat# C04001-500 | |
| Chemical compound, drug | DMEM(High glucose) | VivaCell | Cat# C3113-0500 | |
| Chemical compound, drug | Penicillin-Streptomycin Solution | VivaCell | Cat# C3421-0100 | |
| Chemical compound, drug | Trypsin 1:300 from Porcine pancreas | Sangon Biotech | Cat# A100260-0050 | |
| Chemical compound, drug | PEI | Polysciences | Cat# 23966–2 | |
| Chemical compound, drug | Opti-MEM | Gibco | Cat# 11058021 | |
| Chemical compound, drug | PEG-8000 | Sangon Biotech | Cat# A600433-0500 | |
| Chemical compound, drug | MgCl2 | Ghtech | Cat# N/A | |
| Chemical compound, drug | 1 M HEPES | Solarbio | Cat# H1095 | |
| Chemical compound, drug | 37% formaldehyde | Sigma | Cat# F8775 | |
| Chemical compound, drug | 10% SDS Solution | Sangon Biotech | Cat# B548118-0100 | |
| Chemical compound, drug | Glycine | Sangon Biotech | Cat# A502065-0005 | |
| Chemical compound, drug | Pierce Protease inhibitor tablets, EDTA free | Thermo | Cat# A32965 | |
| Chemical compound, drug | DNA extract buffer | Solarbio | Cat# P1012 | |
| Chemical compound, drug | 3 M Sodium acetate | Invitrogen | Cat# AM9740 | |
| Chemical compound, drug | Glycogen | Thermo | Cat# R0561 | |
| Chemical compound, drug | SSC buffer 20 X concentrate | Sigma | Cat# S6639 | |
| Chemical compound, drug | PowerUp SYBR Green Master Mix | Applied biosystems | Cat# A25742 | |
| Chemical compound, drug | Proteinase K Solution (20 mg/ml) | Sangon Biotech | Cat# B600169-0002 | |
| Chemical compound, drug | DNaseⅠ,RNase-free | Thermo | Cat# EN0521 | |
| Chemical compound, drug | Rnase A | Thermo | Cat# R1253 | |
| Chemical compound, drug | Agencourt AMPure XP magnetic beads | Beckman Coulter | Cat# A63880 | |
| Chemical compound, drug | Ethyl alcohol, Pure (200 proof, molecular biologygrade) | Sigma-Aldrich | Cat# E7023-500ML | |
| Chemical compound, drug | DEPC-treated water | Biosharp | Cat# 701062 | |
| Commercial assay or kit | Visium Spatial Gene Expression Slide & Reagent Kit, 4 rxns | 10 X Genomics | Cat# PN-1000187 | |
| Commercial assay or kit | Visium Accessory Kit | 10 X Genomics | Cat# PN-1000194 | |
| Commercial assay or kit | Dual Index TT Set APN | 10 X Genomics | Cat# PN-1000215 | |
| Commercial assay or kit | Alanine aminotransferase assay kit | Nanjing Jiancheng Bioengineering Institute | Cat# c009-2-1 | |
| Commercial assay or kit | Aspartate aminotransferase Assay Kit | Nanjing Jiancheng Bioengineering Institute | Cat# C010-2-1 | |
| Commercial assay or kit | CUT&RUN Assay Kit | Cell Signalling Technology | Cat# 86652 | |
| Commercial assay or kit | NEBNext Ultra II DNA Library Prep Kit for Illumina | New England Biolabs | Cat# 7645 S | |
| Commercial assay or kit | NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) | New England Biolabs | Cat# 7335 L | |
| Commercial assay or kit | Qubit dsDNA HS Assay Kit | Thermo Fisher Scientific | Cat# Q32851 | |
| Commercial assay or kit | 2×Taq Master Mix (Dye Plus) | Vazyme | Cat# P112-01 | |
| Commercial assay or kit | 2×Rapid Taq Master Mix | Vazyme | Cat# P222-01 | |
| Cell line (Homo-sapiens) | HEK293T | Cell Bank of the Chinese Academy of Sciences | ATCC# CRL-3216; RRID:CVCL_0063 | |
| Recombinant DNA reagent | Plasmid AAV8-TBG-EGFP | This paper | pAAV2/8-based EGFP overexpression plasmid | |
| Recombinant DNA reagent | Plasmid AAV8-TBG-Atf4 | This paper | pAAV2/8-based Atf4 overexpression plasmid | |
| Recombinant DNA reagent | Plasmid AAV8-TBG-Ddit3 | This paper | pAAV2/8-based Ddit3 overexpression plasmid | |
| Recombinant DNA reagent | Plasmid AAV8-TBG-Btg2 | This paper | pAAV2/8-based Btg2 overexpression plasmid | |
| Recombinant DNA reagent | Plasmid IP479-EFS-sgEGFP | This paper | an EFS promoter-driven sgRNA expression plasmid targeting EGFP | |
| Recombinant DNA reagent | Plasmid IP479-EFS-sgBtg2 | This paper | an EFS promoter-driven sgRNA expression plasmid targeting Btg2 | |
| Strain, strain background (Mus musculus, male) | C57BL/6 J | GemPharmate ch Co., Ltd. | RRID:IMSR_GPT:N000013 | |
| Software and algorithms | GraphPad Prism | GraphPad Software | RRID:SCR002798 | https://www.graphpad.com |
| Software and algorithms | SPSS | IBM SPSS software | RRID:SCR_002865 | https://www.ibm.com/ |
| Software and algorithms | ImageJ | National Institutes of Health | RRID:SCR_003070 | https://imagej.nih.gov/ij/ |
| Software and algorithms | 10 x Genomics Space Ranger (v1.1.0) | 10 x Genomics | RRID:SCR_025848 | https://10xgenomics.com |
| Software and algorithms | ClusterProfiler R package (v4.1.4) | Yu et al. | RRID:SCR_016884 | https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html |
| Software and algorithms | Seurat R package (v4.2.3) | Stuart et al. | RRID:SCR_016341 | https://satijalab.org/seurat/ |
| Software and algorithms | Bowtie2 (v2.2.5) | Langmead et al. | RRID:SCR_016368 | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml |
| Software and algorithms | Deeptools (v3.5.1) | Ramirez et al. | RRID:SCR_016366 | https://deeptools.readthedocs.io/ |
| Software and algorithms | Integrative Genomics Viewer (IGV) | Robinson et al. | RRID:SCR_011793 | https://igv.org/ |
| Software and algorithms | R (v4.0.5)and Rstudio | R Consortium | RRID:SCR_000432 | https://www.rstudio.com/ |
| Software and algorithms | Python(v3.7.1) | Python | RRID:SCR_008394 | https://www.python.org/ |
| Software and algorithms | GSVA R package (v1.38.2) | Hanzelmann et al. | RRID:SCR_021058 | https://www.bioconductor.org/packages/release/bioc/html/GSVA.html |
| Software and algorithms | Limma R package (v3.46.0) | Ritchie et al. | RRID:SCR_010943 | https://www.bioconductor.org/packages/release/bioc/html/limma.html |
| Software and algorithms | Pheatmap R package (v.1.0.12) | N/A | RRID:SCR_016418 | https://ggplot2.tidyverse.org |
| Software and algorithms | Ggplot2 R package | N/A | RRID:SCR_014601 | https://ggplot2.tidyverse.org |
| Software and algorithms | Scanpy (python package) | Wolf et al. | RRID:SCR_018139 | https://scanpy.readthedocs.io |
| Software and algorithms | pySCENIC (version 0.12.1) | Van de Sande B et al. | RRID:SCR_025802 | https://scenic.aertslab.org |
| Other | Spatial transcriptomics data | This paper | GSE272564 | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272564 |
| Other | Cut&Run data | This paper | GSE272565 | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272565 |
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Mid-zone hepatocytes trade proliferation for survival via Atf4-Chop axis in early acute liver injury
eLife 14:RP108646.
https://doi.org/10.7554/eLife.108646.4
