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: Nature Communications

Article Title: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock

doi: 10.1038/ncomms12696

Figure Lengend Snippet: ( a ) Immunoblotting analysis of endogenous Bmal1, Clock, Per1 and Cry1 proteins in nuclear, cytosolic and total extracts of HepG2 cells treated with insulin (INS, 50 nM) or control vehicle for 30 min. ( b ) Immunostaining analysis of endogenous Bmal1 localization in primary hepatocytes in the presence or absence of LMB (20 ng ml −1 , 5 h pretreatment) or insulin (INS, 50 nM, 30 min). Bmal1 staining (left), DAPI staining (middle) to visualize nuclei, and merged together (right). A short purplish red line indicates a scale bar=50 μm. ( c ) Immunoblotting analysis of endogenous Bmal1 protein in nuclear, cytosolic and total extracts of primary hepatocytes treated with LMB (20 ng ml −1 , 5 h pretreatment) or insulin (INS, 50 nM, 30 min). Mice were fasted from ZT0 and injected intraperitoneally with insulin (INS, 2 U kg −1 ) or normal saline at ZT6, and then animals were killed at indicated intervals ( d – f , n= 3). ( d ) Immunoblotting analysis of endogenous Bmal1 protein in pooled nuclear, cytosolic and total extracts of livers (top), and corresponding densitometry analysis of relative Bmal1 protein amounts were shown at the bottom; ( e ) ChIP analysis of the occupancy of Bmal1 on Dbp and Nr1d1 promoters in livers from mice collected at ZT6.5 or ZT12 (0.5 or 6 h post-injection, respectively); ( f ) quantitative PCR analysis of hepatic mRNA levels of Dbp and Nr1d1 . ( g ) Quantitative PCR analysis of mRNA levels of Dbp in insulin-treated (INS, 50 nM, 1 h) primary hepatocytes from WT (+/+) or Bmal1 liver-specific knock-out (−/−) mice ( n= 3). Data are represented as mean ±s.e.m, statistical analyses were performed with a two-tailed unpaired Student's t -test, * P <0.05, ** P <0.01, # no significant difference.

Article Snippet: The Akt2 − / − mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1 fl/fl mouse line was purchased from Jackson Laboratories.

Techniques: Western Blot, Control, Immunostaining, Staining, Injection, Saline, Real-time Polymerase Chain Reaction, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock

doi: 10.1038/ncomms12696

Figure Lengend Snippet: Immunoblotting analysis of proteins recognized by phospho-Ser/Thr-Akt substrate antiserum in FLAG immunoprecipitates (IP) prepared from ( a ) primary hepatocytes infected with Ad-FLAG-Bmal1 or Ad-GFP, ( b ) primary WT or Akt2 null ( Akt2 KO) hepatocytes infected with Ad-FLAG-Bmal1 in the presence or absence of insulin (INS, 50 nM, 30 min). ( c ) Immunoblotting analysis of endogenous Bmal1 proteins co-immunoprecipitated by anti-FLAG antibody from lysates of HEK 293T cells transfected with FLAG-Akt2-CA (top), the corresponding gel was silver-stained (bottom). ( d ) Immunoblotting analysis of FLAG-Bmal1 proteins recognized by FLAG-HRP antiserum in phospho-Ser/Thr-Akt substrate immunoprecipitates prepared from HEK 293T cells transfected with indicated plasmids. ( e ) Immunoblotting analysis of pSer42-Bmal1 protein amounts in liver homogenates from mice fasted from ZT0, injected intraperitoneally with insulin (INS, 2 U kg −1 ) at ZT6 and then killed at ZT7. ( f ) Immunoblotting analysis of pSer42-Bmal1 protein amounts in primary WT or Akt2 null ( Akt2 KO) hepatocytes exposed to insulin (INS, 50 nM) for indicated times. ( g ) In vitro kinase assay of Bmal1 protein phosphorylated by active recombinant Akt in immunoprecipitates of WT or Ser42Ala mutant (S42A) FLAG-Bmal1 from HEK 293T cell lysates. Ser42-phosphorylated Bmal1 (pSer42-Bmal1) was detected by phospho (Ser42) specific Bmal1 antiserum, the corresponding gel was Coomassie Brilliant Blue-stained (CBB, bottom). ( h ) Immunoblotting analysis of Ser42-phosphorylated Bmal1 (pSer42-Bmal1) protein amounts in Ad-GFP, Ad-FLAG-tagged WT or S42A mutant Bmal1 virus-infected primary hepatocytes treated with insulin (50 nM, 30 min) in the presence or absence of rapamycin (100 nM, 1 h pretreatment).

Article Snippet: The Akt2 − / − mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1 fl/fl mouse line was purchased from Jackson Laboratories.

Techniques: Western Blot, Infection, Immunoprecipitation, Transfection, Staining, Injection, In Vitro, Kinase Assay, Recombinant, Mutagenesis, Virus

Journal: Nature Communications

Article Title: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock

doi: 10.1038/ncomms12696

Figure Lengend Snippet: ( a ) Immunoblotting analysis of Bmal1 protein in pooled nuclear, cytosolic and total extracts of livers from ad libitum fed WT or Akt2 KO mice killed at 4 h intervals around the clock ( n= 3). ( b ) Immunoblotting analysis of FLAG-Bmal1 (F-Bmal1) in nuclear and total extracts of primary hepatocytes infected with adenoviruses expressing either GFP control or FLAG-Akt2-CA in the presence or absence of insulin treatment (INS, 50 nM) for indicated times. ( c ) Immunoblotting analysis of 14-3-3β protein amounts co-immunoprecipitated by the anti-Bmal1 antibody from control or lambda phosphatase treated lysates of primary hepatocytes in the presence or absence of insulin (50 nM, 30 min). ( d ) Immunoblotting analysis of 14-3-3β protein amounts co-immunoprecipitated by the anti-Bmal1 antibody from pooled lysates of mice fasted from ZT0, injected intraperitoneally with insulin (2 U kg −1 ) at ZT2 and then killed at ZT4 ( n= 3). ( e ) Immunoblotting analysis of 14-3-3β protein amounts co-immunoprecipitated by the anti-FLAG beads from lysates of insulin (50 nM, 30 min) treated primary hepatocytes infected with Ad-FLAG-tagged WT or S42A mutant Bmal1 adenoviruses. ( f ) Immunostaining analysis of FLAG-Bmal1-WT or S42A protein localization in primary hepatocytes infected with corresponding adenoviruses in the presence or absence of insulin (INS, 50 nM, 30 min). FLAG-Bmal1 staining (F-Bmal1, left), DAPI staining (middle) to visualize nuclei, and merged together (right). Scale bar=50 μm. ( g ) Immunoblotting analysis of FLAG-Bmal1-WT or S42A protein amounts in nuclear, cytosolic and total extracts of primary hepatocytes infected with adenoviruses of GFP, FLAG-Akt2-CA, FLAG-Bmal1-WT or S42A in the presence or absence of insulin treatment (INS, 50 nM, 30 min).

Article Snippet: The Akt2 − / − mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1 fl/fl mouse line was purchased from Jackson Laboratories.

Techniques: Western Blot, Infection, Expressing, Control, Immunoprecipitation, Injection, Mutagenesis, Immunostaining, Staining

Journal: Nature Communications

Article Title: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock

doi: 10.1038/ncomms12696

Figure Lengend Snippet: ( a ) ChIP analysis of the occupancy of Ad-FLAG-WT-Bmal1 or Ad-FLAG-S42A-Bmal1 on the promoters of Dbp and Nr1d1 in primary hepatocytes co-infected with Ad-Clock ( n= 3, left). Immunoblotting analysis of relative FLAG-Bmal1 (FLAG) protein amounts in experiments with the corresponding conditions (right). ( b ) Transient luciferase reporter assay of Per1 -Luc activity in HEK 293T cells co-transfected with Clock together with control vector, WT or indicated Bmal1 mutants (top, n= 3). The amounts of nuclear FLAG-Bmal1 proteins in experiments with the corresponding conditions were shown by immunoblotting analysis at the bottom. ( c ) Quantitative PCR analysis of mRNA levels of Dbp and Nr1d1 in insulin-treated (50 nM, 1h) primary hepatocytes infected with Ad-FLAG-WT-Bmal1 or Ad-FLAG-S42A-Bmal1 adenoviruses ( n= 3). ( d ) Immunoblotting analysis of Dbp and Nr1d1 protein amounts in liver homogenates from mice tail-vein injected with Ad-FLAG-WT-Bmal1 or Ad-FLAG-S42A-Bmal1 and injected intraperitoneally with insulin (2 U kg −1 ) at ZT6 and then killed at ZT8, and corresponding densitometry analysis of relative Dbp, Nr1d1 and FLAG-Bmal1 protein amounts were shown on the right ( n= 3). Data are represented as mean ±s.e.m, statistical analyses were performed with a two-tailed unpaired Student's t -test, * P <0.05, ** P <0.01, # no significant difference.

Article Snippet: The Akt2 − / − mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1 fl/fl mouse line was purchased from Jackson Laboratories.

Techniques: Infection, Western Blot, Luciferase, Reporter Assay, Activity Assay, Transfection, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock

doi: 10.1038/ncomms12696

Figure Lengend Snippet: Mice were killed every 4 h over the first 72 h under either AF or RF initiated at Day1-ZT12, black arrows indicated food availability; plasma, total RNA, pooled liver total and nuclear protein extracts were prepared ( a , c , d – f ; n= 3). ( a ) Immunoblotting analysis of pooled nuclear and total Bmal1 protein levels. ( b ) Immunoblotting analysis of pooled nuclear and total Bmal1 protein levels in livers from WT or Akt2 KO mice under RF during Day3 ( n= 3). ( c ) Quantitative PCR analysis of Dbp , Nr1d1 and Bmal1 mRNA levels. ( d ) Measurement of plasma insulin levels by ELISA. ( e ) Pictures of mouse stomachs from AF and RF mice as indicated. ( f ) Measurement of plasma leptin levels. Leptin levels presented in Day2 and 3 were replicated from those of Day1 (AF rep, black squares and dash lines). Data are represented as mean ±s.e.m, statistical analyses were performed with a two-tailed unpaired Student's t -test, * P <0.05, ** P <0.01 between groups at each time point.

Article Snippet: The Akt2 − / − mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1 fl/fl mouse line was purchased from Jackson Laboratories.

Techniques: Clinical Proteomics, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock

doi: 10.1038/ncomms12696

Figure Lengend Snippet: ( a ) Quantitative PCR analysis of Dbp and Nr1d1 mRNA levels in livers from AF mice tail-vein injected with FLAG-WT- or -S42A-Bmal1 adenoviruses and killed around ZT20 and ZT4 ( n= 4). ( b ) Immunoblotting analysis of pooled nuclear and total FLAG-Bmal1 protein levels ( n= 3) in livers from Day1-restrictedly fed (RF-Day1) mice tail-vein injected with FLAG-WT- or -S42A-Bmal1 adenoviruses. ( c ) Quantitative PCR analysis of hepatic Dbp and Nr1d1 mRNA levels in the same samples as described in b . Data are represented as mean ±s.e.m, statistical analyses were performed with a two-tailed unpaired Student's t -test, * P <0.05. ( d ) Hypothetical model showing the mechanism of insulin signal regulates Bmal1 in mouse liver. Postprandially, insulin inhibits Bmal1 transcriptional activity by promoting Akt-mediated Ser42 phosphorylation, which may not only dissociate Bmal1 protein from DNA to bind with 14-3-3, and followed by nuclear exportation, but also trap Bmal1 in the cytosol by masking its nuclear localization sequences.

Article Snippet: The Akt2 − / − mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1 fl/fl mouse line was purchased from Jackson Laboratories.

Techniques: Real-time Polymerase Chain Reaction, Injection, Western Blot, Two Tailed Test, Activity Assay, Phospho-proteomics