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: Scientific Reports

Article Title: Circadian gene BMAL1 ameliorates renal ischaemia-reperfusion injury in diabetic mice by enhancing mitophagy via the HIF-1/BNIP3 pathway

doi: 10.1038/s41598-025-03515-5

Figure Lengend Snippet: Diabetes reduces the expression of BMAL1 and the level of mitophagy and aggravates renal I/R-induced mitochondrial damage. Kidney cortex tissues were analyzed via Western blot to assess the expression levels of BMAL1, HIF-1α, BNIP3, p62, LC3B, TOMM20 and COX IV. ( A ) Representative Western blot images are shown; ( B - H ) Statistical analysis of BMAL1, HIF-1α, BNIP3, p62, LC3B II, TOMM20, and COX IV protein levels are presented. ( I ) Black arrows denote intact mitochondria, yellow arrows indicate damaged mitochondria, blue arrows point to mitochondrial autophagosomes, and green arrows highlight autophagosomes. ( J ) Average mitochondria number, n = 6 independent experiments. ( K ) Average mitochondria area analysis, n = 6 independent experiments. ( L ) Average mitochondrial lenght analysis, n = 6 independent experiments. Scale bar = 2 μm. Results are expressed as means ± SD; n = 3 independent experiments, otherwise specified. * P < 0.05 vs. the NS group; # P < 0.05 vs. the DS group; & P < 0.05 vs. the NI/R group. Supplementary Fig. S2 includes both cropped images and full-length blots.

Article Snippet: The primary antibodies used were diluted as follows: HIF-1α (GB114936-100, Servicebio, Wuhan, China) at 1:1000, Histone H3 (ab1791, Abcam, Cambridge, FL, USA) at 1:5000, BMAL1 (14020, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, LC3B (83506, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, COX IV (4850, Cell Signaling Technology, Danvers, MA, USA) at 1:500, TOMM20 (42406, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, p62 (39749, Cell Signaling Technology, Danvers, MA, USA) at 1:500, BNIP3 (ab109362, Abcam, Cambridge, FL, USA) at 1:1000, cleaved caspase 3 (CC3) (9661, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, and β-actin (GB15001-100, Servicebio, Wuhan, China) at 1:3000. β-actin or Histone H3 was used as a loading control to ensure equal protein loading.

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Circadian gene BMAL1 ameliorates renal ischaemia-reperfusion injury in diabetic mice by enhancing mitophagy via the HIF-1/BNIP3 pathway

doi: 10.1038/s41598-025-03515-5

Figure Lengend Snippet: The mitophagy mediated by the HIF-1α/BNIP3 pathway is attenuated by the HIF-1α inhibitor PX-478 (10µM). Cells from each group were collected, and WB was performed to detect the protein expression of HIF-1α, BNIP3, p62, LC3B I, LC3B II, TOMM20, and COX IV. ( A ) WB images. ( B - H ) Statistical analysis of the expression of BMAL1, HIF-1α, BNIP3, p62, LC3B II, TOMM20, and COX IV. Results are expressed as means ± SD; n = 3 independent experiments, otherwise specified. * P < 0.05 vs. the HG + OEB group; # P < 0.05 vs. the HG + OEB + PX478 group; & P < 0.05 vs. the HG + OEB + H/R group. Supplementary Fig. S7 includes both cropped images and full-length blots.

Article Snippet: The primary antibodies used were diluted as follows: HIF-1α (GB114936-100, Servicebio, Wuhan, China) at 1:1000, Histone H3 (ab1791, Abcam, Cambridge, FL, USA) at 1:5000, BMAL1 (14020, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, LC3B (83506, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, COX IV (4850, Cell Signaling Technology, Danvers, MA, USA) at 1:500, TOMM20 (42406, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, p62 (39749, Cell Signaling Technology, Danvers, MA, USA) at 1:500, BNIP3 (ab109362, Abcam, Cambridge, FL, USA) at 1:1000, cleaved caspase 3 (CC3) (9661, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, and β-actin (GB15001-100, Servicebio, Wuhan, China) at 1:3000. β-actin or Histone H3 was used as a loading control to ensure equal protein loading.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Circadian gene BMAL1 ameliorates renal ischaemia-reperfusion injury in diabetic mice by enhancing mitophagy via the HIF-1/BNIP3 pathway

doi: 10.1038/s41598-025-03515-5

Figure Lengend Snippet: Overexpression of BMAL1 induces mitophagy mediated by the HIF-1α/BNIP3 pathway. TCMK-1 cells were transfected with BMAL1-OE lentiviral vector and BMAL1-NC empty vector to construct the BMAL1 overexpression group (OEB group) and the empty vector control group (Vector group) respectively. Cells from each group were collected, and WB was performed to detect the expression of BMAL1, HIF-1α, BNIP3, p62, LC3B I, LC3B II, TOMM20, and COX IV proteins. ( A , B ) WB image and BMAL1 expression of vector and OEB group. ( C ) WB image. ( D - J ) Statistical analysis of BMAL1, HIF-1α, BNIP3, p62, LC3B II, TOMM20, and COX IV expression. Results are expressed as means ± SD; n = 3 independent experiments, otherwise specified. * P < 0.05 vs. the HG + Vector group; # P < 0.05 vs. the HG + OEB group; & P < 0.05 vs. the HG + Vector + H/R group. Supplementary Fig. S5 includes both cropped images and full-length blots.

Article Snippet: The primary antibodies used were diluted as follows: HIF-1α (GB114936-100, Servicebio, Wuhan, China) at 1:1000, Histone H3 (ab1791, Abcam, Cambridge, FL, USA) at 1:5000, BMAL1 (14020, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, LC3B (83506, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, COX IV (4850, Cell Signaling Technology, Danvers, MA, USA) at 1:500, TOMM20 (42406, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, p62 (39749, Cell Signaling Technology, Danvers, MA, USA) at 1:500, BNIP3 (ab109362, Abcam, Cambridge, FL, USA) at 1:1000, cleaved caspase 3 (CC3) (9661, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, and β-actin (GB15001-100, Servicebio, Wuhan, China) at 1:3000. β-actin or Histone H3 was used as a loading control to ensure equal protein loading.

Techniques: Over Expression, Transfection, Plasmid Preparation, Construct, Control, Expressing

Journal: Scientific Reports

Article Title: Circadian gene BMAL1 ameliorates renal ischaemia-reperfusion injury in diabetic mice by enhancing mitophagy via the HIF-1/BNIP3 pathway

doi: 10.1038/s41598-025-03515-5

Figure Lengend Snippet: Overexpression of BMAL1 alleviates TCMK-1 cell injury and mitochondrial dysfunction induced by HG and H/R. ( A , B ) WB analysis of CC3 protein expression levels in kidney tissue. ( C ) Cell viability detected by CCK8 assay, n = 6 independent experiments. ( D ) ATP content, n = 6 independent experiments. ( E , F ) ROS levels detected by DCFH-DA probe. ( G , H ) Mitochondrial ROS levels detected by MitoSox Red. ( I ) JC-1 staining observation of mitochondrial membrane potential. ( J ) JC-1 staining analysis of the ratio of green/red fluorescence intensity, reflecting MMP levels. ( K ) Representative fluorescent images of lysophagy in each group. ( L ) Quantification of K. Scale bar = 50 μm. Results are expressed as means ± SD; n = 3 independent experiments, otherwise specified. * P < 0.05 vs. the HG + Vector group; # P < 0.05 vs. the HG + OEB group; & P < 0.05 vs. the HG + Vector + H/R group. Supplementary Fig. S6 includes both cropped images and full-length blots.

Article Snippet: The primary antibodies used were diluted as follows: HIF-1α (GB114936-100, Servicebio, Wuhan, China) at 1:1000, Histone H3 (ab1791, Abcam, Cambridge, FL, USA) at 1:5000, BMAL1 (14020, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, LC3B (83506, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, COX IV (4850, Cell Signaling Technology, Danvers, MA, USA) at 1:500, TOMM20 (42406, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, p62 (39749, Cell Signaling Technology, Danvers, MA, USA) at 1:500, BNIP3 (ab109362, Abcam, Cambridge, FL, USA) at 1:1000, cleaved caspase 3 (CC3) (9661, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, and β-actin (GB15001-100, Servicebio, Wuhan, China) at 1:3000. β-actin or Histone H3 was used as a loading control to ensure equal protein loading.

Techniques: Over Expression, Expressing, CCK-8 Assay, Staining, Membrane, Fluorescence, Plasmid Preparation

Journal: Scientific Reports

Article Title: Circadian gene BMAL1 ameliorates renal ischaemia-reperfusion injury in diabetic mice by enhancing mitophagy via the HIF-1/BNIP3 pathway

doi: 10.1038/s41598-025-03515-5

Figure Lengend Snippet: The protective effect of BMAL1 on cell damage and mitochondrial function is attenuated by the HIF-1α inhibitor PX-478. ( A , B ) WB analysis of CC3 protein expression levels in kidney tissue. ( C ) Cell viability detected by CCK8 assay, n = 6 independent experiments. ( D ) ATP content, n = 6 independent experiments. ( E , F ) ROS levels detected by DCFH-DA probe. ( G , H ) Mitochondrial ROS levels detected by MitoSox Red. ( I ) JC-1 staining observation of mitochondrial membrane potential. ( J ) JC-1 staining analysis of the ratio of green/red fluorescence intensity, reflecting MMP levels. ( K ) Representative fluorescent images of lysophagy in each group. ( L ) Quantification of K. Scale bar = 50 μm. Results are expressed as means ± SD; n = 3 independent experiments, otherwise specified. * P < 0.05 vs. the HG + OEB group; # P < 0.05 vs. the HG + OEB + PX478 group; & P < 0.05 vs. the HG + OEB + H/R group. Supplementary Fig. S8 includes both cropped images and full-length blots.

Article Snippet: The primary antibodies used were diluted as follows: HIF-1α (GB114936-100, Servicebio, Wuhan, China) at 1:1000, Histone H3 (ab1791, Abcam, Cambridge, FL, USA) at 1:5000, BMAL1 (14020, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, LC3B (83506, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, COX IV (4850, Cell Signaling Technology, Danvers, MA, USA) at 1:500, TOMM20 (42406, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, p62 (39749, Cell Signaling Technology, Danvers, MA, USA) at 1:500, BNIP3 (ab109362, Abcam, Cambridge, FL, USA) at 1:1000, cleaved caspase 3 (CC3) (9661, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, and β-actin (GB15001-100, Servicebio, Wuhan, China) at 1:3000. β-actin or Histone H3 was used as a loading control to ensure equal protein loading.

Techniques: Expressing, CCK-8 Assay, Staining, Membrane, Fluorescence