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

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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

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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

Journal: The Journal of Clinical Investigation

Article Title: Gut microbes and the liver circadian clock partition glucose and lipid metabolism

doi: 10.1172/JCI162515

Figure Lengend Snippet: Model figure. In SPF conditions, the liver circadian clock drives normal GNG and FA β-Ox, fecal microbial abundance oscillations, and hepatic transcriptome oscillations. Following hepatic Bmal1 deletion, GNG and FA β-Ox are reduced, oscillating microbiota increase, and oscillating hepatic transcripts are not enriched for metabolic pathways, including GNG and FA β-Ox. In GF conditions, GNG is reduced, and oscillating hepatic transcripts are not enriched for GNG and FA β-Ox metabolic pathways regardless of liver clock functionality. Green indicates upregulation, and red indicates downregulation. Solid arrows indicate intact communication, and dashed arrows indicate broken communication. The figure created using BioRender.

Article Snippet: SPF Bmal1 fl/fl and Albumin-Cre male and female mice were purchased from The Jackson Laboratory and bred in The University of Chicago vivarium as previously described ( ).

Techniques: