Journal: iScience

Article Title: Colonic inflammation modulates the intestinal circadian landscape

doi: 10.1016/j.isci.2025.113183

Figure Lengend Snippet: IEC-specific Bmal1 deletion alters the colonic transcriptome (A) Schematic showing the breeding used to generate Bmal1 fl/fl Villin Cre/+ (intestinal epithelial cell (IEC)- Bmal1 −/− ) mice. (B) Colonic IEC clock gene expression in naive mice, represented as fold change in ΔΔCt values, using naive zeitgeber (ZT)0 group as referent population and Gapdh as housekeeping gene, n = 5/genotype. (C) Wheel running periods determined during constant dark conditions, n = 4–5/genotype. (D) Temporal food intake during the day (ZT0-ZT12) and night (ZT12-ZT0) in single-housed mice, n = 4–6/genotype. (E) Body composition analysis determined by EchoMRI using mice maintained on normal chow, n = 60–66/genotype ( n = 85 males; n = 41 females). (F) Volcano plot illustrating differentially expressed (DE) colonic transcripts between naive IEC- Bmal1 −/− ( n = 16) and Bmal1 flox ( n = 15) mice, Log 2 fold change cutoff; p -value cutoff, 10e −5 . (G) Temporal expression of Cldn8 transcripts from RNASeq data. Error bars represent standard error of the mean. (H) Intestinal barrier function measured by fecal albumin ELISA after 7 days of DSS or water, n = 5/genotype. Statistics: (B, C, and E) two-tailed t test; (D and H) two-way ANOVA with multiple comparisons (Šídák); (G) two-way ANOVA and JTK_CYCLE. (B, C, D, and H) error bars represent the standard error of the mean. ∗∗, p < 0.01, ∗∗∗, p < 0.001.

Article Snippet: Mouse: Bmal1 fl/fl , The Jackson Laboratory , JAX: 007668.

Techniques: Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: Colonic inflammation modulates the intestinal circadian landscape

doi: 10.1016/j.isci.2025.113183

Figure Lengend Snippet: IEC-specific Bmal1 deletion alters the colonic rhythmic transcriptome, rhythmic microbiome, and key immune pathways (A) Differential rhythmicity analysis with compareRhythms categorizes rhythmicity in colonic transcript expression in naive IEC- Bmal1 −/− compared to Bmal1 flox mice. n = 3–4/genotype/timepoint (zeitgeber time (ZT)0, ZT6, ZT12, and ZT18). (B) Core clock gene transcript expression across time. N = 3–4/genotype/timepoint. Error bars represent standard error of the mean. (C) Functional pathways enriched (using Enrichr tool and KEGG 2019 mouse database) in gene transcripts with significantly differential expression (DE up/down) or significantly differential rhythmicity (loss/gain/change). Size of the spot represents the category of fold enrichment, color represents the significance of enrichment. (D) Spline plots showing mean normalized expression of all genes from the dataset within a selected pathway (inflammatory bowel disease, mmu05321, 54/62; intestinal immune network for IgA production, mmu04672, 37/43; and antigen processing and presentation, mmu04530, 71/87), error bars represent 95% confidence intervals. (E) The top 20 activated and inhibited upstream regulator pathways. Open circle represents the significance of enrichment by Ingenuity Pathway Analysis, and filled bars represent the fraction of downstream targets present in the dataset. (F) Beta diversity (Bray-Curtis) within 16S microbiome sequencing ( n = 30 per genotype). (G) Rhythmicity in relative abundance of OTUs, n = 5/timepoint/genotype. Abbreviations: ZT = zeitgeber time; DE = differential expression; OTU = operational taxonomic unit. Statistics: (A) compareRhythms rhythmicity analysis. (B) Vertical lines represent the genotype significance by two-way ANOVA. Colored, horizontal asterisks indicate rhythmicity analysis by JTK_CYCLE (NS = not significant). (F) PERMANOVA test. (G) Rhythmicity assessment by JTK_CYCLE; Chi squared test for comparison of rhythmic fraction between genotypes. ∗, p < 0.05; ∗∗, p < 0.01, ∗∗∗, p < 0.001.

Article Snippet: Mouse: Bmal1 fl/fl , The Jackson Laboratory , JAX: 007668.

Techniques: Expressing, Functional Assay, Quantitative Proteomics, Sequencing, Comparison

Journal: iScience

Article Title: Colonic inflammation modulates the intestinal circadian landscape

doi: 10.1016/j.isci.2025.113183

Figure Lengend Snippet: IEC-specific Bmal1 deletion does not affect the severity of acute DSS colitis (A) Percentage weight change of mice treated with 2.5% DSS or standard water, normalized to day 0 weight, n = 8–10/treatment/genotype. (B) Daily severity score, n = 10/treatment/genotype. (C) Colon length measured from distal colon to anus, n = 8–10/genotype/treatment. (D) Mouse colitis histology index scored on mid-colon samples at day 7, n = 5–9/genotype/treatment. (E) Log fold change in paired serum cytokine concentration quantified by bio-plex immunoassay pre- and post-DSS. (F) qPCR data from colon samples collected at day 7, represented as fold change in ΔΔCt values, using naive zeitgeber time (ZT)0 as referent population and Bactin as housekeeping gene. n = 3–5/treatment/genotype/timepoint. Statistics: (A) Three-way ANOVA with multiple comparisons (Tukey). (B) Mixed effects analysis. (C and D) One-way ANOVA with multiple comparisons (Šídák) (E) two-way ANOVA with multiple comparisons (Šídák) (Asterisk). # represents a significant 95% confidence interval (does not include 1.0). (F) Three-way ANOVA with multiple comparisons (Šídák). (D, E, and F) error bars represent the standard error of the mean. ∗, p < 0.05; ∗∗, p < 0.01, ∗∗∗, p < 0.001.

Article Snippet: Mouse: Bmal1 fl/fl , The Jackson Laboratory , JAX: 007668.

Techniques: Concentration Assay

Journal: iScience

Article Title: Colonic inflammation modulates the intestinal circadian landscape

doi: 10.1016/j.isci.2025.113183

Figure Lengend Snippet: Regulatory T cells exhibit treatment-dependent and tissue-dependent rhythmicity (A) Number of colonic lamina propria (LP) T cells (live CD45 + CD3 + ), LP CD4 + T cells (live CD45 + CD3 + CD4 + ), and LP Tregs (live CD45 + CD3 + CD4 + FoxP3 + ) determined by flow cytometry in wildtype mice ( n = 5/timepoint/treatment/genotype). (B) Number of colonic LP leukocytes (live CD45 + ) and LP Tregs (live CD45 + CD3 + CD4 + FoxP3 + ) in IEC- Bmal1 −/− and Bmal1 flox mice determined by flow cytometry. N = 3–5/timepoint/treatment/genotype. (C) Number and proportion of colonic LP Helios + Tregs (live CD45 + CD3 + CD4 + FoxP3 + Helios + ) in Bmal1 flox mice, n = 4–5/treatment/timepoint. (D) Markers of function and proliferation on colonic lamina propria Tregs determined by flow cytometry, n = 5/timepoint/treatment in C57BL/6 mice. (E) Clock gene expression in Tregs isolated from LP and mesenteric lymph nodes (MLN) of naive DEREG mice (n = 4–6/timepoint), represented as fold change in ΔΔCt values, using naive zeitgeber time (ZT)0 LP group as referent population and Gapdh as housekeeping gene. Statistics: (A, C, D, and E) two-way ANOVA with multiple comparisons (Šídák). (B) Three-way ANOVA with multiple comparisons (Šídák). (B, C, D, and E) error bars represent the standard error of the mean. (A) Nonlinear regression to compare whether best fit is given by horizontal line or sine wave with nonzero baseline, constraints: wavelength = 24 h; amplitude >0. p value for sine wave displayed in colored text on plots, where significant. ∗, p < 0.05; ∗∗, p < 0.01, ∗∗∗, p < 0.001.

Article Snippet: Mouse: Bmal1 fl/fl , The Jackson Laboratory , JAX: 007668.

Techniques: Flow Cytometry, Gene Expression, Isolation

Journal: STAR Protocols

Article Title: Protocol to evaluate fasting metabolism and its relationship to the core circadian clock in mice

doi: 10.1016/j.xpro.2025.103660

Figure Lengend Snippet:

Article Snippet: Mouse : Bmal1 fl/fl ; 8 weeks; male , Jackson Laboratory , Strain#007668 RRID:IMSR_JAX:007668.

Techniques: Recombinant, Injection, Software

Journal: iScience

Article Title: Core circadian transcription factor Bmal1 mediates β cell response and recovery from pro-inflammatory injury

doi: 10.1016/j.isci.2024.111179

Figure Lengend Snippet:

Article Snippet: (Cg)- Arntl tm1Weit /J ( Bmal1 fl/fl ) , Jackson Lab , B6.129S4; RRID:IMSR_JAX:007668.

Techniques: Purification, Recombinant, Plasmid Preparation, In Situ, Enzyme-linked Immunosorbent Assay, Software, Expressing, Microscopy

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: ( A ) Experimental design of shotgun microbiome sequencing performed on feces from WT, Bmal1 −/− , Apc +/− , and Apc +/− ; Bmal1 −/− mice ( n = 9 for WT, n = 8 for Bmal1 −/− and Apc +/− , and n = 10 for Apc +/− ; Bmal1 −/− mice). Comparisons of α-diversity in microbiome sequencing at the genus level using Shannon ( B ) and Chao ( C ) indices. Data are expressed as a box plot including the means ± the minimum and maximum values. Statistical significance was determined by the Wilcoxon signed-rank test, and P values from significant multiple comparisons are shown on the graph with * < 0.05. ( D ) β-Diversity as determined by principle covariant analysis (PCoA) using Bray-Curtis distances. Ellipsoids show the 95% confidence region. ( E ) Analysis of similarities (ANOSIM) among genotypes expressed as a box plot. Effect size ( R ) value indicates the degree of difference between groups (0, no difference; 1, greatest difference), and P value indicates the significance. Genotypes are indicated as follows: WT (W), Bmal1 −/− (B), Apc +/− (A), and Apc +/− ; Bmal1 −/− (AB).

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Sequencing

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: ( A ) Relative abundance of microbial phyla and genera between WT, Bmal1 −/− , Apc +/− , and Apc +/− ; Bmal1 −/− mice ( n = 9 for WT, n = 8 for Bmal1 −/− and Apc +/− , and n = 10 for Apc +/− ; Bmal1 −/− mice). Genotypes are indicated as follows: WT (W), Bmal1 −/− (B), Apc +/− (A), and Apc +/− ; Bmal1 −/− (AB). Relative abundance of bacteria altered in fecal samples from clock mutants ( B ), cancer mutants ( C ), and in both clock and cancer mutants ( D ). Error bars represent SEM, significance was determined using MaAsLin2, and P values of pathways with significant q values ( q > 0.25) are shown on the graph with * < 0.05, ** < 0.01, and *** < 0.001.

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Bacteria

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: Linear discriminant analysis effect size (LEfSe) of microbiome sequencing data from all four genotypes ( n = 9 for WT, n = 8 for Bmal1 −/− and Apc +/− , and n = 10 for Apc +/− ; Bmal1 −/− mice). Cladograms show comparisons between WT and Bmal1 −/− ( A ), WT and Apc +/− ( B ), Bmal1 −/− and Apc +/− ; Bmal1 −/− ( C ), and Apc +/− and Apc +/− ; Bmal1 −/− ( D ). Bacterial species corresponding to each letter are shown in the key below, grouped by phylum. Blocks of purple (WT), green ( Bmal1 −/− ), blue ( Apc +/− ), or red ( Apc +/− ; Bmal1 −/− ) in the figure show a significant enrichment in the microbiota of that genotype has been identified at the order, family, genus, or species level. Circles in the same colors reflect where there is a significant difference at the genus or species level. Species are indicated by an alphanumeric key with full species names below the diagrams, grouped by phylum.

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Sequencing

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: Microbial functional pathways were profiled with HUMAnN using microbiome sequencing data from all four genotypes ( n = 9 for WT, n = 8 for Bmal1 −/− and Apc +/− , and n = 10 for Apc +/− ; Bmal1 −/− mice). ( A ) Significant changes as determined by MaAsLin2 of microbial pathways altered in Bmal1 −/− , Apc +/− , and Apc +/− ; Bmal1 −/− mice relative to WT. Pathways are colored by effect size adjusted by q value, with signs indicating the direction of change. Relative abundance in all genotypes of the top pathways most increased ( B ) or decreased ( C ) in Apc +/− ; Bmal1 −/− relative to WT. Additional pathways are shown in fig. S4 (A and B). Error bars represent SEM, significance was determined using MaAsLin2, and P values of pathways with significant q values ( q > 0.25) are shown on the graph with ** < 0.01, *** < 0.001, and **** < 0.0001.

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Functional Assay, Sequencing

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: ( A ) Expression of the top four most highly expressed mucin genes as determined by RNA sequencing (RNA-seq) of small intestinal organoids from all four genotypes ( n = 3 organoid lines derived from independent mice). ( B ) Expression of mucin genes in IECs from WT mice relative to zeitgeber time, as determined by quantitative polymerase chain reaction (qPCR; n = 5 independent mice per time point). Average circadian period is shown when the rhythmicity P value was less than 0.01. All values are shown in table S2. ( C ) Expression of mucin genes in WT and Bmal1 −/− IECs collected from n = 3 independent mice at ZT4 and ZT16. ( D ) Expression of mucin genes in WT IECs and Apc +/− ; Bmal1 −/− tumors collected from n = 3 independent mice at ZT4 and ZT16. ( E ) Periodic acid–Schiff (PAS) staining on formalin-fixed paraffin-embedded small intestinal sections from WT, Bmal1 −/− , Apc +/− , and Apc +/− ; Bmal1 −/− mice. Tumor-bearing genotypes are divided into predominantly normal (surrounding) or predominantly tumor containing (polyp) areas. Scale bars, 200 μm. Error bars represent SEM, and statistical significance was determined by DEseq2 for (A) and by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons for (C) and (D). Asterisks represent false discovery rate (FDR) or P values from multiple comparisons with * < 0.05, ** < 0.01, and **** < 0.0001. Comparisons without labels are not significant.

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Expressing, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Staining, Formalin-fixed Paraffin-Embedded

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: ( A ) Expression of five key tight junction genes as determined by RNA-seq of small intestinal organoids from all four genotypes ( n = 3 organoid lines derived from independent mice). ( B ) Expression of tight junction genes in IECs from WT mice relative to zeitgeber time, as determined by qPCR (n = 5 independent mice per time point). Average circadian period is shown when the rhythmicity P value was less than 0.01. All values are shown in table S2. ( C ) Expression of tight junction genes in WT and Bmal1 −/− IECs collected from n = 3 independent mice at ZT4 and ZT16. ( D ) Expression of tight junction genes in WT IECs and Apc +/− ; Bmal1 −/− tumors collected from n = 3 independent mice at ZT4 and ZT16. Error bars represent SEM, and statistical significance was determined by DEseq2 for (A) and by one-way ANOVA with Tukey’s multiple comparisons for (C) and (D). Asterisks represent FDR or P values from multiple comparisons with * < 0.05, ** < 0.01, and **** < 0.0001. Comparisons without labels are not significant.

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Expressing, RNA Sequencing Assay, Derivative Assay

Journal: Science Advances

Article Title: Disruption of the intestinal clock drives dysbiosis and impaired barrier function in colorectal cancer

doi: 10.1126/sciadv.ado1458

Figure Lengend Snippet: Expression of core clock ( A ), mucin ( B ), and tight junction ( C ) genes in DEX synchronized Caco-2 cells ( n = 3 independent experiments). ( D ) Monolayer permeability of DEX synchronized Caco-2 cells as determined by 4-kDa FITC-dextran transfer in a transwell assay ( n = 3 independent experiments). ( E ) Intestinal permeability from WT, Bmal1 , Apc +/− , and Apc +/− ; Bmal1 −/− mice as determined by gavage of 4-kDa FITC-dextran (600 mg/kg) at ZT23 and serum collection at ZT0 ( n = 3 to 8 independent mice). Statistical significance was determined by Student’s unpaired t test for (A) to (D) and by one-way ANOVA with Tukey’s multiple comparisons for (E). Asterisks represent P values from unpaired t-test or multiple comparisons with * < 0.05, ** < 0.01, and *** < 0.001. Comparisons without labels are not significant.

Article Snippet: Mice containing flox sites flanking one allele of Apc exons 1 to 15 ( Apc +/ Δ ex1-15 ) (the Jackson Laboratory, strain 009045) ( ) were crossed with mice with flox sites flanking both alleles of exon 8 of Bmal1 ( Bmal1 fl/fl ) (the Jackson Laboratory, strain 007668) ( ).

Techniques: Expressing, Permeability, Transwell Assay