Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Intrinsic hippocampal molecular circadian clock function does not influence the daily rhythm of hippocampal ISF Aβ. (A) Schematic depicting tissue-specific AAV-Cre–mediated excision of Bmal1 within the hippocampus of Bmal1 f/f ; APPPS1-21 transgenic mice. (B) Diagram depicting Bmal1 deletion abrogating circadian clock function. In addition to Per and Cry genes, BMAL1 regulates transcription of many other clock-controlled genes in a tissue-specific manner. (C) Schematic illustrating the in vivo microdialysis method used to collect soluble Aβ in the hippocampus subsequent to viral injections. This technique was performed in awake, behaving mice. (D) Representative images of microdialysis probe placement (dashed lines) within the hippocampus of mice injected with either AAV-GFP (labeled Cre − ; APP, left) with normal BMAL1 expression (bottom left) or injected with AAV-Cre–IRES-GFP (labeled Cre + ; APP, right), resulting in a loss of local hippocampal BMAL1 expression (bottom right). Bar, 1 mm. (E) Graph showing that hourly ISF Aβ levels are not influenced by the targeted hippocampal excision of Bmal1 (green, AAV-Cre–labeled Cre + ; APP) compared with APP mice with normal BMAL1 expression (black, AAV-GFP–labeled Cre − ; APP). Dots represent normalized mean hourly ISF Aβ levels in 12-h:12-h L:D conditions (shaded gray) ± SEM ( n = 4–5 mice per genotype), and curves represent cosinor method fits. (F) Bar graph showing the effect of targeted hippocampal Bmal1 deletion on the rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes. Means were NS by two-tailed t test (P = 0.23). Error bars indicate SEM.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Transgenic Assay, In Vivo, Injection, Labeling, Expressing, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Bmal1 deletion throughout in the brain but sparing the SCN does not markedly affect Aβ levels or hippocampal amyloid pathology. (A) Representative images showing normal BMAL1 expression in the hippocampus and SCN regions from a control mouse ( Cre − ). (B) Representative raw tracing of core body temperature recordings over 7 d from a Cre − mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a control Cre − mouse ( n = 3). (C) Representative images of a Nestin-Cre + ;Bmal1 f/f ( Nes-Bmal1 KO ) mouse showing loss of hippocampal but not SCN BMAL1 expression. OC, optic chiasm. (D) Representative raw tracing of core body temperature recordings over 7 d from a Nes-Bmal1 KO mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a Nes-Bmal1 KO mouse ( n = 3). (E) Graph showing hourly hippocampal ISF-soluble Aβ levels in Cre − ;Bmal1 f/f ;APPPS1-21 (black) and Nestin-Cre + ; Bmal1 f/f ;APPPS1-21 ( Nes-Bmal1 KO;APP, orange) mice. Squares represent the normalized mean hourly ISF Aβ level in 12h:12–h L:D conditions (shaded gray) ± SEM ( n = 5 mice per genotype), and curves represent cosinor method fits. (F) Bar graph showing rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes in Cre − ;APPPS1-21 (black) and Nes-Bmal1 KO;APP (orange) mice. Means were NS by two-tailed t test (P = 0.26). (G) Representative images showing X34 staining of fibrillar Aβ plaques in the hippocampus of 4-mo Cre − ;APPPS1-21 (top) and Nes-Bmal1 KO;APP mice (bottom). The hippocampus is outlined by dashed gray lines. Bars: (A and C, top, and G) 500 µm; (A and C, bottom) 250 µm. (H) Bar graph showing quantification of immunoreactive Aβ (HJ3.4 antibody staining, left) and fibrillar Aβ (X34 staining, right) plaque burden within the hippocampus of Cre − APPPS1-21 (black) and Nes-Bmal1 KO;APP (orange) mice ( n = 9–14 mice per genotype). Bars represent means ± SEM (two-tailed t test; P = 0.88 for the immunoreactive Aβ comparison, and P = 0.064 for the fibrillar Aβ comparison).

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Control, Two Tailed Test, Staining, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Global Bmal1 deletion disrupts Aβ rhythmicity. (A) Representative images showing decreased BMAL1 expression in the hippocampus and SCN regions from a global Bmal1 KO mouse. (B) Representative raw tracing of core body temperature recordings over 6 d from a Bmal1 KO mouse. Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a loss of the dominant 24-h rhythm in a global Bmal1 KO mouse ( n = 3). (C) Graph showing hippocampal ISF-soluble Aβ levels in WT (labeled control, black) and global Bmal1 KO mice (red). Squares represent the normalized mean murine ISF Aβ level pooled every 3 h in 12h:12–h L:D conditions (shaded gray) ± SEM ( n = 7 mice per genotype), and curves represent cosinor method fits. (D) Bar graph showing rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes in WT (labeled control, black) and Bmal1 KO (red) mice. *, P = 0.002 by two-tailed t test. (E) Bar graph depicting daily mean hippocampal ISF Aβ levels in WT (black) and Bmal1 KO mice (red). Means were NS by two-tailed t test (P = 0.71). Means + SEM are shown; n = 7 mice/group.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Labeling, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Global Bmal1 deletion accelerates amyloid plaque deposition. (A) Representative images showing normal BMAL1 expression in the hippocampus and SCN regions from a tamoxifen-treated Cre − mouse. (B) Representative raw tracing of core body temperature recordings over 7 d from a Cre − mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a Cre- control mouse ( n = 3). (C) Representative images of a tamoxifen-treated CAG-Cre ERT2+ ;Bmal1 f/f ( Bmal1 iKO ) mouse showing a loss of both hippocampal and SCN BMAL1 expression. OC, optic chiasm. (D) Representative raw tracing of core body temperature recordings over 7 d from an inducible Bmal1 KO mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D are arrhythmic (bottom right), with a χ 2 periodogram (bottom left) showing a loss of the dominant 24-h rhythm ( n = 3). (E) Representative images showing X34 staining of fibrillar Aβ plaques in the hippocampus of 4-mo Cre − ;Bmal1 f/f ;APP ( Cre - ;APP , top) and Bmal1 iKO ; APP (red) mice (bottom). The hippocampus is outlined by dashed gray lines. Bars: (A and C, top, and E) 500 µm; (A and C, bottom) 300 µm. (F) Bar graph showing quantification of immunoreactive Aβ (HJ3.4 antibody staining, left) and fibrillar Aβ (X34 staining, right) plaque burden within the hippocampus of Cre − ;APP (black) and Bmal1 iKO ; APP (red) mice ( n = 8–13 mice per genotype). Bars represent means ± SEM (two-tailed t test; P = 0.001 for the immunoreactive Aβ comparison, and P = 0.001 for the fibrillar Aβ comparison). *, P < 0.05.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Control, Staining, Two Tailed Test, Comparison

Journal: iScience

Article Title: PKA-mediated BMAL1 phosphorylation promotes β 1 -adrenoceptor autoantibody-induced cardiomyocyte death

doi: 10.1016/j.isci.2025.112786

Figure Lengend Snippet: β 1 -AA increases Ser42 phosphorylation of BMAL1 in cardiomyocytes (A and C) Expression of Ser42-phosphorylated BMAL1 (pSer42-BMAL1) in myocardial tissues from control and β 1 -AA groups at different time points. ∗ p < 0.05 vs. control; ∗∗ p < 0.01 vs. control; n = 6. (B and D) pSer42-BMAL1 expression in H9c2 cells in the presence or absence of β 1 -AA at different time points. ∗ p < 0.05 vs. control; n = 5. (E) Expression levels of pSer42-BMAL1 in nuclear and cytosolic fractions of H9c2 cells at CT8 were detected by western blot, with GAPDH (cytoplasm) and histone H3 (nucleus) serving as markers. Data are represented as mean ± SEM.

Article Snippet: Phospho-BMAL1 (Ser42) , Cell Signaling Technology , Cat#13936; RRID: AB_2798348.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot

Journal: iScience

Article Title: PKA-mediated BMAL1 phosphorylation promotes β 1 -adrenoceptor autoantibody-induced cardiomyocyte death

doi: 10.1016/j.isci.2025.112786

Figure Lengend Snippet: Ser42 phosphorylation mediates β 1 -AA-induced cytoplasmic accumulation of BMAL1 (A) BMAL1 expression levels in cytoplasmic and nuclear fractions of phosphomimetic (S42D) and dephosphomimetic (S42A) mutant cells were analyzed by western blot. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (B) mRNA expression levels of Per2 and Nr1d1 in WT, S42D, and S42A groups were measured by real-time PCR. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. (C) BMAL1 expression levels in cytoplasmic and nuclear fractions of the S42A mutant cells after β 1 -AA treatment were analyzed by western blot. ∗ p < 0.05; ∗∗ p < 0.01; ns: no significant difference. (D) mRNA expression levels of Per2 and Nr1d1 in the S42A mutant cells after β 1 -AA treatment were measured by real-time PCR. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns: no significant difference. (E) Cell viability of the S42A mutant cells in the presence or absence of β 1 -AA. ∗∗∗∗ p < 0.0001; ns: no significant differerce. Data are represented as mean ± SEM.

Article Snippet: Phospho-BMAL1 (Ser42) , Cell Signaling Technology , Cat#13936; RRID: AB_2798348.

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: PKA-mediated BMAL1 phosphorylation promotes β 1 -adrenoceptor autoantibody-induced cardiomyocyte death

doi: 10.1016/j.isci.2025.112786

Figure Lengend Snippet: β 1 -AA promotes BMAL1 phosphorylation by enhancing PKA activity (A) Immunoprecipitation revealed the interaction between BMAL1 and PKA in cardiomyocytes, and PKA increased BMAL1 phosphorylation in the presence of β 1 -AA. The PKA substrate motif antibody was employed to enrich proteins phosphorylated by PKA, followed by immunoblotting with BMAL1-specific antibodies to verify BMAL1 in the complexes. (B) pSer42-BMAL1 expression levels were detected by western blot in control group, β 1 -AA group, and β 1 -AA+H89 group. ∗∗ p < 0.01. (C) pSer42-BMAL1 and PRKACA expression levels were detected by western blot in si- NC group, si -Prkaca -1 group, and si -Prkaca -2 group in the presence or absence of β 1 -AA. ∗∗∗ p < 0.001 vs. si- NC . (D) Schematic illustration of the proposed mechanism: β 1 -AA enhances BMAL1 phosphorylation at Ser42 by activating PKA, leading to cytoplasmic accumulation of phosphorylated BMAL1, which suppresses the transcription of downstream target genes Per2 and Nr1d1 and promotes cardiomyocyte death. Data are represented as mean ± SEM.

Article Snippet: Phospho-BMAL1 (Ser42) , Cell Signaling Technology , Cat#13936; RRID: AB_2798348.

Techniques: Phospho-proteomics, Activity Assay, Immunoprecipitation, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Circadian regulators PER1 and PER2 regulate osteoclastogenesis by balancing competing activities of innate immunity genes

doi: 10.64898/2026.01.11.698889

Figure Lengend Snippet: A. IGV images displaying BMAL1 peaks based on three pairs of ChIP-seq data with mouse liver downloaded from the GEO database. Arrows indicate the peaks studied in the current work. B. Relative expression levels of the indicated genes in synchronized osteoclasts comparing Cont and dKO osteoclasts. The value of Cont cells at 24 h was defined as 1.0 in each graph. The period (h) and amplitude of each genotype are shown above each graph. ** p < 0.01, * p < 0.05, ns for not significant, and ND for not detected with the Cosinor analysis of circadian rhythmicity above each graph. C. Enlarged BMAL1 peaks at Saa3 and Mx2 genes shown in ( A ) and at the Per2 gene. The locations of the BMAL1-binding motifs, their nucleotide sequences, and the regions amplified by qPCR are indicated below each image. D. Western blotting demonstrating BMAL1 at four time points of synchronized osteoclasts. Histone H2B was used as loading control. E. ChI P-qPCR of BMAL1 in each region in synchronized osteoclasts. ** p < 0.01, * p < 0.05, and ns with unpaired two-tailed t-test. The statistical analysis indicated in green and purple are based on the comparison with P-Region-1 (negative control) at each time point. Male osteoclasts were used in all data. Mean ± SEM based on biological triplicates with technical triplicates are shown in ( B ) and ( E ).

Article Snippet: Chromatin was sheared to 200–600 bp with a Bioruptor 300 (Diagenode) and incubated with 2 μg BMAL1 antibody described above or normal mouse IgG (Santa Cruz Biotechnology, sc-2025) as control, 2 μl Dynabeads Protein G (Thermo Fisher Scientific, 10004D), and 400 μl dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and cOmplete protease inhibitor cocktail) for 16 h at 4°C.

Techniques: ChIP-sequencing, Expressing, Binding Assay, Amplification, Western Blot, Control, Two Tailed Test, Comparison, Negative Control

Journal: bioRxiv

Article Title: Circadian regulators PER1 and PER2 regulate osteoclastogenesis by balancing competing activities of innate immunity genes

doi: 10.64898/2026.01.11.698889

Figure Lengend Snippet: A and B. Sequences of the BMAL1-binding motifs in the wild type (WT) and mutants (Mut) sequences Saa3 ( A ) and Mx2 ( B ). Conserved (light blue) and mutated (red) nucleotides are indicated in color. mm9 numbers indicate the nucleotide numbers based on NCBI37/mm9. The minus numbers above the initial C in each motif represent the nucleotide numbers counted from the transcription start site. C and D. Relative F-Luc/R-Luc ratios after transfection of the Saa3 ( C ) and Mx2 ( D ) reporter plasmids into 293FT cells. Results with EV (empty vector), BC ( Bmal1 and Clock ), BCP1 (BC with Per1 ), BCP2 (BC with Per2 ) are shown with the value with WT and EV defined as 1.0 in each graph. E. Relative expression levels of Saa3 and Mx2 upon transfection of the indicated gene combinations into RAW264.7 cells. The value with EV was defined as 1.0 in each graph. Mean ± SEM based on biological triplicates with technical triplicates are shown in ( C )–( E ). ** p < 0.01, * p < 0.05, and ns for not significant with unpaired two-tailed t-test in comparison to WT in each gene combination as color-coded in ( C ) and ( D ) or in comparison to EV in ( E ). Additional comparisons are shown with black lines in ( C ) and ( D ).

Article Snippet: Chromatin was sheared to 200–600 bp with a Bioruptor 300 (Diagenode) and incubated with 2 μg BMAL1 antibody described above or normal mouse IgG (Santa Cruz Biotechnology, sc-2025) as control, 2 μl Dynabeads Protein G (Thermo Fisher Scientific, 10004D), and 400 μl dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and cOmplete protease inhibitor cocktail) for 16 h at 4°C.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Two Tailed Test, Comparison

Journal: Journal of Lipid Research

Article Title: KSRP is critical in governing hepatic lipid metabolism through controlling Per2 expression

doi: 10.1194/jlr.m050724

Figure Lengend Snippet: Fig. 2. KSRP ablation upregulates Per2 and causes a phase shift in oscillations of some circadian clock genes in Ksrp / livers, but not eWAT. A: Circadian oscillations of Per2 , Per1 , Clock , Rev-erba , Baml1 , Cry1 , Cry2 , and Dbp in wild-type and Ksrp / livers. RNA was isolated from livers of wild-type (n = 6) and Ksrp / (n = 6) mice at different ZTs (ZT0: lights on; ZT12: lights off), and gene expression was analyzed by qPCR. The ZT0 values were plotted twice as ZT0 and ZT24. Data are mean ± SEM. * P < 0.05 between groups at each time point. B: Liver extracts were immunoprecipitated (IP) with anti-PER2 antibodies, and the immunoprecipitates were analyzed by immunoblotting (IB) using anti-PER2 antibodies. Pooled liver extracts of two mice were analyzed for each time point. Two independent IP/IB analyses using different mice are shown. -Tubulin levels in the inputs of IP were used as a loading control. Quantifi cation of PER2 levels is indicated. Data are the mean of two IP/IB experiments. C: Liver extracts of two mice for each time point were analyzed by immunoblotting using anti- CLOCK, anti-BMAL1, anti-KSRP, and anti- -tubulin. D: Circadian oscillations of Per2 , Per1 , Clock , Baml1 , and Cry1 in wild-type and Ksrp / eWAT (n = 4). Data are mean ± SEM. * P < 0.05 and ** P < 0.01 between groups at each time point.

Article Snippet: For immunoblotting, proteins were separated on a 7–10% SDS-PAGE, transferred to a polyvinylidene difl uoride membrane, and probed with following antibodies: anti-CLOCK (Cell Signaling), anti-BMAL1 (Bethyl Laboratories Inc.), antiKSRP ( 47 ), anti-FLAG (Sigma), anti- -tubulin (Sigma), and antiMyc (Roche Diagnostics ).

Techniques: Isolation, Gene Expression, Immunoprecipitation, Western Blot, Control