thyroid binding globulin promoter  (Addgene inc)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: Acute RACK1 deficiency disrupts glucose homeostasis. ( A ) Western blotting analysis of multiple tissues showing liver-specific RACK1 knockout in RACK1 fl/fl mice retro-orbitally injected with AAV8-TBG-GFP (GFP) or AAV8-TBG-iCre (Cre). L, liver; K, kidney; SK, skeletal muscle; H, heart; B, brain. ( B ) Blood glucose levels in GFP- or Cre-injected RACK1 fl/fl mice measured under non-fasted (NF) conditions and after 6 or 18 hours of fasting. ∗ P < .05 compared with GFP controls; 2-tailed Student’s t -test. ( C–F ) Metabolic tolerance tests in GFP and Cre mice: ( C ) insulin tolerance test, ( D ) glucose tolerance test, ( E ) pyruvate tolerance test, and ( F ) glucagon tolerance test. Area under the curve (AUC) quantifications for ( D–F ) are shown below each graph. Group differences over time were analyzed by 2-way repeated-measures ANOVA (time × genotype) with Sidak’s post hoc tests for point-by-point comparisons (n = 6–8 per group). AUCs were compared using 2-tailed unpaired Student’s t -tests. ∗, ∗∗, ∗∗∗ P < .05, .01 and 001 vs GFP, respectively.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Western Blot, Knock-Out, Injection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: RACK1 deficiency impairs hepatic gluconeogenesis in vitro. Primary hepatocytes were isolated from RACK1 fl/fl mice injected with AAV8-TBG-GFP (GFP) or iCre (Cre) and subjected to glucose production assays. ( A ) Glucose output was measured under basal conditions or following stimulation with glucagon (Glu, 200 nM), insulin (Ins, 20 nM), or both. ∗∗, ∗∗∗ P < .01 and .001 vs corresponding basal; # P < .001 vs GFP + Glu; $ P < .05 vs Cre + Glu; & P < .01 vs GFP; 2-way ANOVA followed by Sidak’s post hoc test. ( B ) Cells were treated with vehicle (control) or the PKA inhibitors H89 (5 μM) and compound 3i (0.5 μM) in the absence or presence of glucagon (200 nM). ∗∗∗ P < .001 vs control + basal; & P < .001 vs control + Glu; 2-way ANOVA followed by Sidak’s post hoc test.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: In Vitro, Isolation, Injection, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: RACK1 deficiency attenuates hepatic PKA signaling. ( A–B ) RACK1 fl/fl mice injected with AAV8-TBG-iGFP (GFP) or AAV8-TBG-iCre (Cre) and fasted for 2 hours (basal) or treated with glucagon (100 μg/kg body weight; Glu) or insulin (1.5 U/kg body weight) for 15 minutes. Liver lysates were analyzed for cAMP levels ( A ) and by Western blotting ( B ). Quantified Western blot data are shown in the left panel. ∗∗∗ P < .001 vs corresponding basal. & P < .001 vs GFP + glucagon; 2-way ANOVA followed by Sidak’s post hoc test. ( C ) cAMP levels in primary hepatocytes stimulated with vehicle (basal) or glucagon (200 nM) for 10 minutes. ∗∗∗ P < .001 vs GFP + basal; 2-way ANOVA followed by Sidak’s post hoc test. ( D ) Western blot analysis of primary hepatocytes from GFP and Cre mice stimulated with vehicle (basal) or glucagon (200 nM) for the indicated times. Relative phosphorylation of pCREB S133 and phosphor-PKA substrates (pPKA sub) is expressed as fold change over GFP at 0 minutes, normalized to total protein ( left panel ). ∗, ∗∗∗ P < .05 and .001 vs Glu at 0 minutes, respectively; $ P < .001 vs Cre at 0 minutes; # P < .01 vs corresponding GFP; 2-way ANOVA followed by Sidak’s post hoc test. Arrows indicate pPKA sub unchanged or increased in RACK1-deficient cells. ( E ) Western blot analysis of primary hepatocytes from GFP and Cre mice stimulated with vehicle (basal), or insulin (20 nM) for the indicated times. Relative phosphorylation of pAKT S473 is expressed as fold change over GFP at 0 minutes, normalized to total protein (lower panel). ∗∗∗, # P < .001 vs corresponding Ins at 0 minutes; 2-way ANOVA followed by Sidak’s post hoc test. ( F ) qPCR analysis of PKA target genes G6PC and PCK1 in hepatocytes treated for 4 hours with vehicle, insulin (20 nM), glucagon (100 nM), glucagon + insulin, cAMP (20 μM), or cAMP + insulin. ∗∗∗ P < .001 vs corresponding basal; # P < .001 vs corresponding GFP; % P < .001 vs corresponding cAMP or glucagon; 2-way ANOVA followed by Sidak’s post hoc test. ( G ) qPCR analysis of the indicated genes in liver tissues from GFP and Cre mice following a 6-hour fast. ∗, ∗∗, ∗∗∗ P < .05, .01 and .001 vs GFP, respectively; 2-tailed Student’s t -test.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Injection, Western Blot, Phospho-proteomics

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: Glucagon regulates the dynamic interaction and compartmentation of RACK1 with components of the PKA signaling axis. ( A ) Coimmunoprecipitation of lysates from primary hepatocytes isolated from Alb-Cre/RACK1 fl/fl mice injected with pAd-GFP or pAd-Flag-RACK1 and treated with glucagon (200 nM) for the indicated times. Representative blot showing total proteins in input lysates and immunoprecipitated proteins in pellet fractions. ( B ) Quantification of RACK1-associated proteins from ( A ) after subtraction of GFP background, shown as fold enrichment relative to 0 minutes. ∗∗, ∗∗∗ P < .01 and .001 vs 0 minutes, respectively; n = 3; 1-way ANOVA followed by Dunnett’s post hoc test. ( C–D ) Western blot analysis of plasma membrane ( C ) and nuclear ( D ) fractions from primary hepatocytes isolated from RACK1 fl/fl mice injected with AAV8-TBG-iGFP (GFP) or AAV8-TBG-iCre (Cre) and stimulated with glucagon (200 nM) for the indicated time. Glucagon-induced changes in protein localization were quantified as fold change relative to 0 minutes in GFP cells and values are indicated below each blot. Quantitative data from multiple experiments are presented in left panels. ∗, ∗∗, ∗∗∗ P < .05, .01 and .001 vs GFP at 0 minutes, respectively; # P < .001 vs corresponding GFP; 2-way ANOVA followed by Sidak’s post hoc test.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Isolation, Injection, Immunoprecipitation, Western Blot, Clinical Proteomics, Membrane

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: Rescue of glucose homeostasis defects induced by acute RACK1 deficiency via expression of constitutively active PKAcα W196R . ( A ) qPCR analysis of PKA target gene expression in livers of RACK1 fl/fl mice injected with AAV-TBG-GFP (RACK1 fl/fl /GFP), RACK1 fl/fl mice injected with AAV-TBG-Cre (RACK1 fl/fl /Cre), PKAca W196R mice injected with AAV-TBG-Cre (PKACA/Cre), or RACK1 fl/fl /PKAca W196R mice injected with AAV-TBG-Cre (RACK1 fl/fl /PKACA/Cre). ∗, ∗∗∗ P < .05 and .001 vs corresponding RACK1 fl/fl /GFP, respectively; 1-way ANOVA followed by Dunnett’s post hoc test. ( B ) Blood glucose levels in the indicated mice following an 18-hour fast. ∗, ∗∗ P < .05 and .01 vs RACK1 fl/fl /GFP, respectively; 1-way ANOVA followed by Dunnett’s post hoc test (n = 8). ( C–F ) Glucose tolerance test ( C ) with corresponding area under the curve (AUC) analysis ( D ), pyruvate tolerance test ( E ) with corresponding AUC analysis ( F ). Group differences over time were analyzed by 2-way repeated-measures ANOVA (time × genotype) with Sidak’s post hoc tests for point-by-point comparisons (n = 5–8 per group). AUCs were compared using 1-way ANOVA followed by Dunnett’s post hoc test. ∗, ∗∗, ∗∗∗ P < .05, .01, and .001 vs RACK1 fl/fl /GFP, respectively.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Expressing, Targeted Gene Expression, Injection

Journal: Nature

Article Title: Hepatic zonation determines tumorigenic potential of mutant β-catenin

doi: 10.1038/s41586-025-09733-1

Figure Lengend Snippet: a , Mouse model recombining alleles at a low clonal density in the hepatocyte epithelium. The illustrations of the mouse and adenovirus were adapted from Medical Art Servier ( https://servier.com ) under a CC BY 4.0 licence. b , Representative image of RFP IHC; R26 LSL-tdTomato liver 30 days post administration of AAV8.TBG.Cre (GC/ml = genome copy per ml), n = 3. c , Liver-to-body weight ratio (LW/BW). Biological replicates: Apc fl/fl (A) n = 6, Ctnnb1 ex3/WT (B) n = 12, Rnf43 fl/fl ; Znrf3 fl/fl (RZ) n = 6, R26 LSL-MYC (M) n = 8, Ctnnb1 ex3/WT ; R26 LSL-MYC (BM) n = 11. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. d , Tumour scoring. Biological replicates: A n = 6, B n = 12, RZ n = 6, M n = 9, BM n = 10. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. e , Whole-tissue normalised RNA-Seq read counts for Myc in indicated tissues from wild-type and Apc fl/fl mice. Box plots: centre line = median, upper (25 th percentile) and lower quartiles (75 th percentile) (box limits), and 1.5× interquartile range (whiskers) Biological replicates, moving left to right on the x-axis: n = 4,4,4,3,4,4,5,5,5,3,4,5. f , Top reactome pathways enriched in genes differentially upregulated in: AAV8.TBG.Cre-treated livers 10 days post induction (2 × 10 11 GC/ml) BM versus B; and Villin CreER Apc fl/fl versus Villin CreER (WT) intestines at day 4 post induction with tamoxifen. Dots are coloured according to their adjusted P-values, with the size of the dots representing the number of differentially expressed. Statistical significance was determined with the enrichPathway() function in R, using a two-sided hypergeometric model to determine the probability of geneset overlap occurring by chance. Multiple testing correction was applied using the Benjamini-Hochberg method. Biological replicates: liver, n = 3; intestinal, n = 16 WT and n = 15 Apc fl/fl . g - k , Ctnnb1 ex/WT ; R26 LSL-MYC liver, 60 days post AAV8.TBG.Cre (6.4 × 10 8 GC/ml). g , Representative images of CTNNB1, BrdU, p21 and p16 IHC on serial sections, n = 3. Arrowheads represent single mutant clones and dashed black lines indicate a lesion. h , Representative images of MYC IHC, black arrowheads highlight MYC pos nuclei i , Spatial transcriptomics GSEA. Normalised Enrichment Score (NES) was calculated by normalising to the mean enrichment of random samples, and two-sided permutation testing with a Benjamini-Hochberg test was applied, p-adj = p adjusted value. j - k , In situ hybridisation for Notum and IHC for peEF2, p4E-BP1 (Thr37/46), pS6(Ser235/236), pS6(Ser240/244), and CCND1. Dashed outline highlights lesion. Representative images of n = 4 per group. All black scale bars = 100 μm. All red scale bars = 20 μm.

Article Snippet: Genetic recombination was induced in both male and female mice, 2–4 months of age, with either an adeno-associated virus expressing Cre under the control of the TBG promoter (AAV8.TBG.Cre; AAV.TBG.PI.Cre.rBG was a gift from J. M. Wilson (Addgene plasmid #107787) to achieve temporal-specific and hepatocyte-specific Cre-mediated recombination of floxed alleles, or tamoxifen to activate Rosa26 CreER (whole body), Villin CreER (intestinal specific) Lgr5 CreER , Glul CreER , Cyp1a2 CreER , Igfbp2 CreER and Gls2 CreER (only male mice were used for the Gls2 CreER experiments as female mice did not recombine as efficiently as male mice when treated with tamoxifen).

Techniques: RNA Sequencing, Mutagenesis, Clone Assay, In Situ, Hybridization

Journal: Nature

Article Title: Hepatic zonation determines tumorigenic potential of mutant β-catenin

doi: 10.1038/s41586-025-09733-1

Figure Lengend Snippet: a - e , AAV8.TBG.Cre (2 × 10 11 GC/ml) treated livers sampled 4 and 10 days post administration a , LW/BW, Biological replicates: Day 4—WT n = 10, R26 LSL-MYC (M) n = 11, Ctnnb1 ex3/WT (B) n = 14, Ctnnb1 ex3/WT ; R26 LSL-MYC (BM) n = 9; Day 10—WT n = 10, M n = 11, B n = 10, BM n = 11. Bars are mean ± s.d. One-way ANOVA with Holm-Sidak’s multiple comparisons test. b , Confocal IF staining for BrdU (red), GLUL (yellow), and HNF4α (white) 10 days post induction. Nuclei counterstained with DAPI (blue), n = 6. Scale bars, 100μm. c , Quantification of GLUL pos hepatocytes. Biological replicates: Day 4—WT n = 10, M n = 11, B n = 14, BM n = 7; Day 10—WT n = 10, M n = 10, B n = 11, BM n = 7. Bars are mean ± s.d. One-way ANOVA with Holm-Sidak’s multiple comparisons test. d , Whole liver RNA-Seq, top reactome pathways enriched in downregulated and upregulated genes between day-4 and -10 BM livers. Dots are coloured according to their adjusted P-values. The enrichPathway() function in R, using a two-sided hypergeometric model determined the probability of geneset overlap occurring by chance. Multiple testing correction was applied using the Benjamini-Hochberg method. Biological replicates: Day 4, n = 5; Day 10, n = 3. e , Quantification of BrdU pos hepatocytes, stratified into GLUL-positive and -negative subsets. Two-sided Wilcoxon matched-pairs test. Biological replicates: n = 6 per condition. f , Quantification of BrdU pos hepatocytes in Day-4 vehicle- and rapamycin-treated mice. Biological replicates: M vehicle n = 5, rapamycin n = 7; B vehicle n = 4, rapamycin n = 4. Bars are mean ± s.d. One tailed Mann–Whitney test. g , Quantification of BrdU pos hepatocytes in Igfbp2 knockout ( Igfbp2 –/– ) mice. Biological replicates: M Igfbp2 +/+ n = 13, Igfbp2 –/– n = 9; B Igfbp2 +/+ n = 17, Igfbp2 –/– n = 8. Bars are mean ± s.d. One tailed t-test and Mann–Whitney test. Data from Fig. included in Igfbp2 +/+ groups. h , Acute BM activation combined with viral-vector delivery of an Igfbp2 expressing transgene. i - k , LW/BW and quantification of BrdU pos and GLUL pos hepatocytes 8 days after AAV8-vector administration. Biological replicates: n = 10. Bars are mean ± s.d. two tailed Mann–Whitney test. l , Acute BM activation combined with viral-vector delivery of a Akt transgene containing a myristoylation sequence. m - o , LW/BW and quantification of BrdU pos and GLUL pos hepatocytes 8 days after AAV8-vector administration. Biological replicates: n = 5. Bars are mean ± s.d. two tailed Mann–Whitney test. p , Acute BM activation combined with PTEN loss. The illustrations of the mouse and adenovirus in panels h , l , p were adapted from Medical Art Servier ( https://servier.com ) under a CC BY 4.0 licence. q - s , LW/BW and quantification of BrdU pos and GLUL pos hepatocytes 8 days after AAV8-vector administration. Biological replicates: BM n = 7, BMP n = 6. Bars are mean ± s.d. two tailed Mann–Whitney test.

Article Snippet: Genetic recombination was induced in both male and female mice, 2–4 months of age, with either an adeno-associated virus expressing Cre under the control of the TBG promoter (AAV8.TBG.Cre; AAV.TBG.PI.Cre.rBG was a gift from J. M. Wilson (Addgene plasmid #107787) to achieve temporal-specific and hepatocyte-specific Cre-mediated recombination of floxed alleles, or tamoxifen to activate Rosa26 CreER (whole body), Villin CreER (intestinal specific) Lgr5 CreER , Glul CreER , Cyp1a2 CreER , Igfbp2 CreER and Gls2 CreER (only male mice were used for the Gls2 CreER experiments as female mice did not recombine as efficiently as male mice when treated with tamoxifen).

Techniques: Staining, RNA Sequencing, One-tailed Test, MANN-WHITNEY, Knock-Out, Activation Assay, Plasmid Preparation, Expressing, Two Tailed Test, Sequencing

Journal: Nature

Article Title: Hepatic zonation determines tumorigenic potential of mutant β-catenin

doi: 10.1038/s41586-025-09733-1

Figure Lengend Snippet: a , Representative images of cPARP immunohistochemistry, n = 7. b , c , Quantification of apoptotic cells by IHC for cleaved PARP (cPARP) and cleaved caspase-3 (CC3) 10 days post AAV8.TBG.Cre induction. b , Biological replicates: WT n = 8, R26 LSL-MYC ( M ) n = 10, Ctnnb1 ex3/WT ( B ) n = 7, Ctnnb1 ex3/WT ; R26 LSL-MYC ( BM ) n = 11. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. c , Biological replicates: WT n = 9, B n = 11, M n = 10, BM n = 9. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. d , Apoptosis gene set enrichment in differentially expressed genes among TCGA-LIHC binned by CTNNB1 mutation status and MYC expression. A two sided, pairwise Wilcoxon test was used to compare data to the MYChigh/CTNNB1:WT group. Boxplots represent the median (central line) and 25th and 75th percentiles of the data (box). Whiskers represent the maximum and minimum of non-outlier values within 1.5× the interquartile range. Data beyond the whiskers are outliers. N = 372. e - g , NanoString GeoMx digital spatial profiling of Ctnnb1 ex3/WT day-4 liver. e , Representative image of immunofluorescence masks used to define the regions selected for spatial transcriptomics. BrdU (red), GLUL (yellow), DNA (blue). PN, portal node, adj., adjacent. f , Principal component analysis (PCA) of spatial transcriptomics data. ROI per Biological replicate: n = 8. g , Gene set variance analysis (GSVA) scores for liver-zonation gene sets Lobule layers (L1–9), CV, layer 1 to PN, layer 9 according to Halpern et al. . Biological replicates n = 4. h , Representative CTNNB1 IHC in AAV8.TBG.Cre-treated WT and Ctnnb1 ex3/WT liver at day 4 post induction, n = 3. Panels 1 - 4 are higher magnification images of the dashed areas in the left panel, highlighting hepatocytes near the PN and the CV. All scale bars, 100 μm.

Article Snippet: Genetic recombination was induced in both male and female mice, 2–4 months of age, with either an adeno-associated virus expressing Cre under the control of the TBG promoter (AAV8.TBG.Cre; AAV.TBG.PI.Cre.rBG was a gift from J. M. Wilson (Addgene plasmid #107787) to achieve temporal-specific and hepatocyte-specific Cre-mediated recombination of floxed alleles, or tamoxifen to activate Rosa26 CreER (whole body), Villin CreER (intestinal specific) Lgr5 CreER , Glul CreER , Cyp1a2 CreER , Igfbp2 CreER and Gls2 CreER (only male mice were used for the Gls2 CreER experiments as female mice did not recombine as efficiently as male mice when treated with tamoxifen).

Techniques: Immunohistochemistry, Mutagenesis, Expressing, Immunofluorescence

Journal: Nature

Article Title: Hepatic zonation determines tumorigenic potential of mutant β-catenin

doi: 10.1038/s41586-025-09733-1

Figure Lengend Snippet: AAV8.TBG.Cre-treated wild-type ( WT ), R26 LSL-MYC ( M ), Ctnnb1 ex3/WT ( B ), and Ctnnb1 ex3/WT ; R26 LSL-MYC ( BM ) livers at days 4 and 10 post induction. a , Images of in situ hybridisation for Igfbp2 . Dashed boxes indicate zoomed in regions. PN, Portal Node; CV, Central Vein. Scale bars, 100 μm. b , c , Quantification of Igfbp2 RNAscope probe copies in livers of indicated genotypes on day 4 ( b ) and 10 ( c ) post induction. Biological replicates: day 4: WT n = 2, M n = 2, B n = 2, BM n = 6; day 10: WT n = 6, M n = 5, B n = 6, BM n = 6. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. d , Images of IGFBP2 immunohistochemistry. Dashed boxes indicate zoomed in regions. PN, Portal Node; CV, Central Vein. Scale bars, 100 μm. e , f , Quantification of IGFBP2 pos cells in livers of indicated genotypes on day 4 ( e ) and 10 ( f ) post induction. 10 Circular regions with a Radius of 190 µm at the portal node (PN) and central vein (CV) were quantified per mouse. Biological replicates: Day 4: WT n = 10, M n = 15, B n = 16, BM n = 12; Day 10: WT n = 10, M n = 10, B n = 14, BM n = 12. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Genetic recombination was induced in both male and female mice, 2–4 months of age, with either an adeno-associated virus expressing Cre under the control of the TBG promoter (AAV8.TBG.Cre; AAV.TBG.PI.Cre.rBG was a gift from J. M. Wilson (Addgene plasmid #107787) to achieve temporal-specific and hepatocyte-specific Cre-mediated recombination of floxed alleles, or tamoxifen to activate Rosa26 CreER (whole body), Villin CreER (intestinal specific) Lgr5 CreER , Glul CreER , Cyp1a2 CreER , Igfbp2 CreER and Gls2 CreER (only male mice were used for the Gls2 CreER experiments as female mice did not recombine as efficiently as male mice when treated with tamoxifen).

Techniques: In Situ, Hybridization, RNAscope, Immunohistochemistry

Journal: Nature

Article Title: Hepatic zonation determines tumorigenic potential of mutant β-catenin

doi: 10.1038/s41586-025-09733-1

Figure Lengend Snippet: Ribosome profiling analysis comparing AAV8.TBG.Cre-treated wild-type (WT), Ctnnb1 ex3/WT (B), R26 LSL-MYC (M), and Ctnnb1 ex3/WT ; R26 LSL-MYC (BM) livers 4 and 10 days post induction. a - e , Ribosome sequencing (Ribo-Seq) data. Biological replicates n = 3 per condition (time point and genotype). a , Principal component analysis (PCA) of Ribo-Seq data. Totals represents all sequenced cytoplasmic RNA, RPFs = ribosome protected fragments and represents sequenced RNA protected by ribosomes. b , c , Scatter plot presenting translational efficiency (TE) changes, colour scheme represents mRNAs translationally up-regulated (TE up, purple dots, padj<0.1 and TE log2FC > 0), down regulated (TE down red dots, padj<0.1 and TE log2FC > 0) WT livers were compared to either Ctnnb1 ex3/WT (c) or R26 LSL-MYC (d) livers at day 4 and 10. P values were calculated in DESeq2 using a two-sided Wald test, then multiple testing correction was applied using the Benjamini-Hochberg method. d , e , Scatter plot presenting translational efficiency (TE) changes and violin plots comparing day-4 and day-10 R26 LSL-MYC (M) livers and Ctnnb1 ex3/WT ; R26 LSL-MYC (BM) livers, coloured by TE groups from day 4 in panel c and day 4 in Fig. . Boxplots represent the median (central line) and 25th and 75th percentiles of the data (box). Whiskers represent the maximum and minimum of non-outlier values within 1.5× the interquartile range. Data beyond the whiskers are outliers. f , Polysome profiles. Line and shaded areas represent mean and SD. Biological replicates n = 2.

Article Snippet: Genetic recombination was induced in both male and female mice, 2–4 months of age, with either an adeno-associated virus expressing Cre under the control of the TBG promoter (AAV8.TBG.Cre; AAV.TBG.PI.Cre.rBG was a gift from J. M. Wilson (Addgene plasmid #107787) to achieve temporal-specific and hepatocyte-specific Cre-mediated recombination of floxed alleles, or tamoxifen to activate Rosa26 CreER (whole body), Villin CreER (intestinal specific) Lgr5 CreER , Glul CreER , Cyp1a2 CreER , Igfbp2 CreER and Gls2 CreER (only male mice were used for the Gls2 CreER experiments as female mice did not recombine as efficiently as male mice when treated with tamoxifen).

Techniques: Sequencing

Journal: Nature

Article Title: Hepatic zonation determines tumorigenic potential of mutant β-catenin

doi: 10.1038/s41586-025-09733-1

Figure Lengend Snippet: Modelling CTNNB1 mutations using trinucleotide mutational spectrum. a , Mutation frequency of CTNNB1, APC, RNF43, ZNRF3 , and AXIN1 in HCC. In total 1,189 HCC samples were analysed by combining multiple cohorts (Materials and Methods). For CTNNB1 , activating mutations are defined as missense mutations or in-frame indels at hotspots. For tumour suppressor genes, inactivating mutations are defined as nonsense mutations, splice-site mutations, and frame-shift indels. b , Observed CTNNB1 mutation frequencies were compared to the predicted mutation frequencies at different protein positions. Prediction is based on the trinucleotide mutational spectrum in HCC (Materials and Methods). Only missense single-nucleotide variants (SNVs) were considered. c , The lack of correlation between observed and predicted mutation frequencies indicates that the hotspot CTNNB1 mutations are mainly driven by selection advantage, rather than the underlying mutagenic processes. A two-sided p-value is associated with the Pearson correlation coefficient. d , GLUL and IGFBP2 IHC in uninduced livers (no administration of AAV8.TBG.Cre). e , Quantification of GLUL pos hepatocytes in uninduced mice n = 6 per genotype. Bars are mean ± s.d. Two-tailed Mann–Whitney test. f , Schematic of liver-specific mouse model to acutely recombine hypomorphic- Apc fl/fl and R26 LSL-MYC . The illustrations of the mouse and adenovirus were adapted from Medical Art Servier ( https://servier.com ) under a CC BY 4.0 licence. g , Quantification of BrdU pos hepatocytes. Bars are mean ± s.d. One-way ANOVA and Holm-Sidak’s multiple comparisons. Biological replicates: uninduced: n = 6; Day 4: AM n = 8, AhM n = 11; Day 8: AM n = 7, AhM n = 8. h , Fold change in liver-to-body weight ratios. Bars are mean ± s.d. One-way ANOVA and Tukey’s multiple comparisons test. Biological replicates: Day 4: AM n = 11, AhM n = 14; Day 8: AM n = 8, AhM n = 10. i – j , Aged uninduced Apc fl/fl -hypomorphic liver. Dashed line, tumour boundary. GLUL, CTNNB1, BrdU and IGFBP2 immunohistochemistry, and haematoxylin and eosin (H&E) stain, n = 7. All scale bars, 100 μm.

Article Snippet: Genetic recombination was induced in both male and female mice, 2–4 months of age, with either an adeno-associated virus expressing Cre under the control of the TBG promoter (AAV8.TBG.Cre; AAV.TBG.PI.Cre.rBG was a gift from J. M. Wilson (Addgene plasmid #107787) to achieve temporal-specific and hepatocyte-specific Cre-mediated recombination of floxed alleles, or tamoxifen to activate Rosa26 CreER (whole body), Villin CreER (intestinal specific) Lgr5 CreER , Glul CreER , Cyp1a2 CreER , Igfbp2 CreER and Gls2 CreER (only male mice were used for the Gls2 CreER experiments as female mice did not recombine as efficiently as male mice when treated with tamoxifen).

Techniques: Mutagenesis, Selection, Two Tailed Test, MANN-WHITNEY, Immunohistochemistry, Staining

Journal: Cell

Article Title: Perturb-Multimodal: a platform for pooled genetic screens with sequencing and imaging in intact mammalian tissue

doi: 10.1016/j.cell.2025.05.022

Figure Lengend Snippet: A. Liver lobule organization showing pericentral to periportal axis (BioRender). B. Integrated UMAP of RCA-MERFISH and Flex scRNA-seq data (Left: both; Top right: RCA-MERFISH; Bottom right: Flex) showing liver cell types, as determined by unsupervised clustering. C. Spatial distribution of periportal (left) and pericentral (right) scores in hepatocytes based on marker gene expression . D. Periportal vs pericentral gene expression scores across hepatocyte subtypes. E. Spatial organization of hepatocyte subtypes (left) and non-hepatocyte cells (right). F. Spatial map of hepatocyte zone marker expression radially organized around a central vein. Yellow-boxed region from (E) with cell types (top) and imputed gene expression are separately scaled for each gene (bottom). PP: Periportal; PC: Pericentral. G. Morphology panel showing 4 abundant RNA species and 14 proteins (top) with zoomed details of a subset of targets (bottom). H. Deep learning autoencoder diagram reducing protein morphologies to 512-dimensional embeddings using the VQ-VAE model with auxiliary tasks of discriminating cell types, cell states, or conditions. I. UMAP of subcellular morphology image embeddings colored by channel (target protein and abundant RNA) identity. J. Similarity of subcellular morphology channel embedding quantified by Kullback-Leibler (KL) divergence. K. Correlation heatmap of high-signal features across image embeddings, ordered by hierarchical clustering to reveal nine feature classes (see ). L. Cells displaying high weight scores from selected feature classes, including (ii) double nucleus, (iii) membrane enrichment, (iv) diffuse expression, and (viii) punctate patterns. M. Tissue-scale spatial organization of morphological embedding features. Left: Albumin mRNA feature 431; Right: Perilipin feature 203 N. Confusion matrix of hepatocyte subtype classification accuracy on held-out cells using MERFISH transcriptomic data of 209 genes. O. Confusion matrix of hepatocyte subtype classification accuracy on held-out cells using morphological feature embeddings from 14 proteins and 4 abundant RNAs. P. Heatmap of mutual information between hepatocyte subtypes Hep1 and Hep6 for individual morphological channels, quantified by quantified by KL divergence. Q. UMAP of anti-Perilipin morphological embeddings of single-cell images, colored by hepatocyte subtype. R. UMAP of anti-Perilipin morphological embeddings of single-cell images, colored by Leiden cluster. S. Sampling of hepatocytes from Perilipin embedding clusters 2 (Hep 6-enriched) and 6 (Hep 1-enriched). T. Diet experiment diagram. U. scRNA-seq UMAP from mice under ad lib, overnight fasting, or high-fat diet (HFD) conditions. V. Heatmap of mutual information between ad lib and fasted hepatocytes for individual morphological embedding features, quantified by quantified by KL divergence. W. Sampling of hepatocytes from anti-p-S6 RP embedding cluster 7 (from ad lib condition) and cluster 0 (from fasted condition). Cluster 7 is most enriched in the ad lib condition and cluster 0 is most enriched in the fasted condition. X. Same as (V), but for morphological channel embeddings between ad lib and HFD hepatocytes. Y. Same as (W) but for anti-perilipin embedding cluster 0 (from ad lib condition) and cluster 10 (from HFD condition). See also - and - .

Article Snippet: We then allowed the mice to grow to adulthood (>P30) and induced Cas9 through the retro-orbital injection of AAV8 with Cre driven by a hepatocyte promoter (Addgene 107787-AAV8; ~5 × 10 11 genome copies per animal).

Techniques: Marker, Gene Expression, Expressing, Membrane, Sampling

Journal: Cell

Article Title: Perturb-Multimodal: a platform for pooled genetic screens with sequencing and imaging in intact mammalian tissue

doi: 10.1016/j.cell.2025.05.022

Figure Lengend Snippet: A. Lentiviral CROP-seq vector for dual-mode mosaic screens with mU6-driven sgRNA expression and hepatocyte promoter driving expression of mTurquoise transcripts with perturbation-specific barcode in the 3’ UTR. B. CRISPR experiment workflow: LSL-Cas9 pups was injected with sgRNA library, followed by Cas9 activation in young adults via AAV8 TBG-CRE and perfusion-fixation of livers for RCA-MERFISH or Perturb-seq. C. Fluorescence micrograph of PFA-perfused, lentivirus- and AAV-transduced liver tissue showing Cas9-EGFP (green) and sgRNA-mTurquoise (purple) expression. D. Multimodal readout of 209 endogenous mRNAs and 456 perturbation barcodes via RCA-MERFISH and 14 proteins and 4 abundant RNAs via sequential imaging. E. Representative fluorescence micrograph showing the first three (of 21 total) bits of RCA-MERFISH perturbation imaging. F. Distribution of barcode calls per sgRNA-harboring cell: 85.3% with one barcode, 14.7% with two or more. Only single-barcode cells were analyzed. G. Fluorescence micrograph of a hepatocyte dissociated from fixed liver (Blue: DAPI ; Red: phalloidin). H. Flow cytometry of dissociated, PFA-perfused, lentivirus- and AAV-transduced liver tissue and mTurquoise+ and GFP+ cells are selected to enrich for cell containing sgRNA and active Cas9. I. Histogram of Alb_0 sgRNA counts per cell. J. Barcode calls per sgRNA-harboring cell in Perturb-seq: 85.7% with one barcode, 14.3% with two or more. K. Albumin mRNA expression histograms comparing cells receiving control vs. Albumin -targeting sgRNAs in Perturb-seq data. L. Fraction of sgRNAs causing significant Perturb-seq phenotypes: 109/406 targeting sgRNAs (27%) vs. 0/50 non-targeting sgRNAs (0%) by Holm-Šídák-corrected energy distance test (p<0.05). M. Histogram of Pearson correlations of pseudobulk Perturb-seq phenotypes between active sgRNA pairs targeting same gene, versus control sgRNA pairs. N. Knockouts ranked by energy distance between cells that received active targeting sgRNA vs cells that received control sgRNA. Energy distance is calculated using the top 20 PCs of Z-normalized Perturb-seq gene expression. O. Unbiased sampling of cells with control sgRNAs and sgRNAs targeting Albumin showing Albumin mRNA and polyA signals.. P. Histogram comparing Albumin mRNA signal between cells receiving control and Albumin -targeting sgRNAs, from the imaging data. Q. Histogram of Pearson correlations of pseudobulk imaging intensity phenotypes between active sgRNA pairs targeting same gene, versus control sgRNA pairs. R. Venn diagram of genes with significant knockout effects in imaging and sequencing phenotypes. Phenotype significance is measured by a Holm-Šídák-corrected energy distance permutation tests (p < 0.05). There is significant overlap in the two sets of genes (hypergeometric p < 10 −13 ). See also and - .

Article Snippet: We then allowed the mice to grow to adulthood (>P30) and induced Cas9 through the retro-orbital injection of AAV8 with Cre driven by a hepatocyte promoter (Addgene 107787-AAV8; ~5 × 10 11 genome copies per animal).

Techniques: Plasmid Preparation, Expressing, CRISPR, Injection, Activation Assay, Fluorescence, Imaging, Flow Cytometry, Control, Gene Expression, Sampling, Knock-Out, Sequencing

Journal: Cell

Article Title: Perturb-Multimodal: a platform for pooled genetic screens with sequencing and imaging in intact mammalian tissue

doi: 10.1016/j.cell.2025.05.022

Figure Lengend Snippet: A. Spatial distribution of sgRNAs in the imaging dataset showing proliferation of infected cells. Cells are colored by cell type (left; as in ) or by sgRNA barcode identity (right and zoom). B. A UMAP generated from transcriptome profiles of cells with sgRNAs targeting Hnf4a and from a random sub-sampling of cells with control sgRNAs, colored by sgRNA identity (left) or by Apoa1 expression (right). C. Heat map representation of pseudobulk transcriptional changes (log2-fold change measured by sequencing, left) and staining protein and RNA level changes (Z-normalized changes measured by imaging, right) associated with each sgRNA, relative to cells with control sgRNAs. The colormaps are clipped for visual emphasis. D. Perturbation-perturbation correlation of RNA and protein changes associated with active sgRNAs (left) and zoom-in of the color boxed regions (right). Colors in the heatmap represent Pearson correlation of perturbed gene-level pseudobulk phenotypes measured by sequencing (below diagonal) or imaging (above diagonal). Genetic perturbations are ordered by hierarchical clustering of joint sequencing and imaging phenotype vectors. E. Minimal distortion embedding. Each dot represents an mRNA expressed in hepatocytes. mRNAs that are co-varying in expression across the perturbations are placed in proximity. F. Heat map of the correlation between the expression levels of indicated proteins/RNAs across perturbations, in the imaging dataset. Imaging channels are ordered by hierarchical clustering.

Article Snippet: We then allowed the mice to grow to adulthood (>P30) and induced Cas9 through the retro-orbital injection of AAV8 with Cre driven by a hepatocyte promoter (Addgene 107787-AAV8; ~5 × 10 11 genome copies per animal).

Techniques: Imaging, Infection, Generated, Sampling, Control, Expressing, Sequencing, Staining

Journal: Cell

Article Title: Perturb-Multimodal: a platform for pooled genetic screens with sequencing and imaging in intact mammalian tissue

doi: 10.1016/j.cell.2025.05.022

Figure Lengend Snippet: A. Kernel density estimate plots showing the distribution of zonation gene expression in cells with control sgRNAs, sgRNAs targeting Ctnnb1 , and sgRNAs targeting APC . The single-cell zonation scores reflect the expression of periportal genes like Cyp2f2 and Hal and pericentral genes like Glul and Cyp2e1 . Periportal and pericentral genes contribute positively and negatively to zonation score, respectively. B. Ranking of perturbed genes by their average impact on zonal gene expression score. C. Heatmap summarizing categories of genes whose perturbation has a large impact on zonated gene expression. Here, the periportal and pericentral expression scores are shown separately. D. Perturbation-perturbation correlation heatmap showing Pearson coefficients of pseudobulk transcriptional changes between indicated sgRNA perturbations. E. Schematic of data-driven zonal segmentation. The proportion of each hepatocyte subtype is calculated in 50-μm x 50-μm bins. The bins are then grouped into two zones based on the local cell-type distribution and the enrichment of cells with each perturbation in the two zones is quantified. F. Cell types from RCA-MERFISH (left) and resulting periportal/pericentral zonal segmentation (right). G. Barplot of the fraction of cells in periportal and pericentral zones (as defined above), for the indicated perturbations. The white line represents the fraction of cells with control sgRNAs.

Article Snippet: We then allowed the mice to grow to adulthood (>P30) and induced Cas9 through the retro-orbital injection of AAV8 with Cre driven by a hepatocyte promoter (Addgene 107787-AAV8; ~5 × 10 11 genome copies per animal).

Techniques: Gene Expression, Control, Expressing

Journal: Molecular Metabolism

Article Title: Membrane-associated ring–CH–type finger 2 protects against metabolic dysfunction-associated fatty liver disease by targeting fatty acid synthase

doi: 10.1016/j.molmet.2025.102137

Figure Lengend Snippet: Overexpression of hepatic MARCH2 ameliorates MAFLD in ob/ob mice. (A) The rAAV8 with TBG promoter-driven MARCH2 (AAV-MARCH2) or GFP (AAV-GFP) were delivered by tail vein injection to 8-week-old ob/ob mice and fed with a chow diet for another 7 weeks. This schema diagram was created by Figdraw (authorization code: TTPIO86bdd). Body weight (B), morphology of the mice and liver (C), GTT (D), ITT (E), area under curve(AUC) of GTT(F), AUC of ITT(G), liver weight (H), ratios of liver weight to body weight (I), liver HE staining (J), liver Oil Red O staining (K), liver TG contents (L), liver MARCH2 mRNA expression (M), and FASN protein levels (N) of AAV-MARCH2 and AAV-GFP mice were detected. n = 6–8 mice per group, and the data were expressed as Mean ± SEM. Two-tailed unpaired student’s t -test were applied.

Article Snippet: The rAAV8 with thyroxine binding globulin (TBG) promoter-driven MARCH2 coding sequence (AAV-MARCH2) or GFP control (AAV-GFP) were generated by GenePharma (Shanghai, China).

Techniques: Over Expression, Injection, Staining, Expressing, Two Tailed Test