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