Journal: Journal of biological rhythms

Article Title: Bootstrapping and Empirical Bayes Methods Improve Rhythm Detection in Sparsely Sampled Data

doi: 10.1177/0748730418789536

Figure Lengend Snippet: Select functional annotations from the DAVID webtool ( Huang et al. 2009a ) for the 119 genes identified as rhythmic in 9 or more tissues by applying BooteJTK with a Benjamini-Hochberg adjusted p-value threshold of 0.05 to the Zhang et al. (2014) dataset. Fold Enrichment is relative to the expectation of observing the indicated functional annotation in a set of randomly selected genes. Abbreviations: BH, Benjamini-Hochberg adjusted p-value; SP-PIR, Swiss-Prot Protein Information Resource keywords; GO, Gene Ontology keywords.

Article Snippet: Thirteen genes are identified as rhythmic by BooteJTK in all 12 tissues: Arntl (Bmal), Nrld1 (Rev-erbA), Nr1d2 (Rev-erbB), Dbp, Per1, Per2, Per3, Ciart (Chrono), Bhlhe41 (Dec2), Tns2, Tsc22d3, Usp2, and Tspan4.

Techniques: Functional Assay

Journal: BMC Genomics

Article Title: Methamphetamine-induced changes in myocardial gene transcription are sex-dependent

doi: 10.1186/s12864-021-07561-x

Figure Lengend Snippet: Methamphetamine selectively regulates transcription of circadian clock-related genes in female hearts. Methamphetamine-induced changes in genes that regulate the circadian rhythm were identified by RNA sequencing and qPCR. RNA sequencing identified significant effects of methamphetamine on the number of transcripts encoding Per2 a , Per3 c , Dbp e , Bmal1 g , Npas2 i , Clock k , and Cry2 m . Two-way ANOVA of qPCR data indicated significant effects of methamphetamine on the number of transcripts encoding Per2 [F = 17 [ , ], p < 0.0005] b , Dbp [F = 47 [ , ], p < 0.0001] f , BmalI [F = 51 [ , ], p < 0.05] h , and Npas2 [F = 19 [ , ], p < 0.0005] j . There was also a significant interaction between sex and methamphetamine on Npas transcription [F = 8 [ , ], p < 0.01] j . a indicates p adj < 0.0001 compared to hearts from saline treated females. b indicates p adj < 0.00005 compared to hearts from saline treated females. c indicates p adj < 0.0005 compared to hearts from saline treated females. d indicates p adj < 0.001 compared to hearts from saline treated females. e indicates p < 0.05 vs female saline; f indicates p < 0.0005 vs female saline; g indicates p < 0.0005 vs male saline. Data represent the mean ± SEM of 5–6 animals

Article Snippet: The membrane was blocked with 5% nonfat dry milk and then blotted overnight with antibodies Dbp (Thermo Fisher Catalog # PA5–4045; Waltham, MA), BmalI (Cell Signaling Catalog # 14020; Danvers, MA), or glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling, Danvers, MA; Catalog # 2118).

Techniques: RNA Sequencing, Saline

Journal: bioRxiv

Article Title: Vutiglabridin Alleviates Cellular Senescence Process of Dysfunctional Replication, Metabolic Regulation, and Circadian Clock in Primary Human Dermal Fibroblasts

doi: 10.1101/2023.02.12.528227

Figure Lengend Snippet: (a) The effect of vutiglabridin on the rhythmic expression profile of the BMAL1 promoter linked to luciferase (Luc) was measured in HDFs for 5 days using Kronos-Dio (Atto). Representative luciferase activity in BMAL1 Luc HDFs measured from Kronos Dio Real-Time luminometer. Luciferase activity was normalized by cell counting at the endpoint. Young control represents BMAL1 Luc HDFs derived from passage 14 HDFs. Old control represents BMAL1 Luc HDFs derived from cells cultured with a medium with vector (DMSO) for 58 days. Vutiglabridin represents BMAL1 Luc HDFs derived from cells administered with vutiglabridin for 58 days. (b) The amplitude of luciferase activity graphs from stable BMAL1 Luc HDF groups was measured. 1 st amplitude was defined as the difference of luciferase activity between the 1 st peak and 1 st through. 2 nd and 3 rd amplitudes were defined similarly to the first. Data are shown as means ± SEM (four independent experiments were performed, n = 9∼10, *** p<0.001, by student’s two-tailed t-test). (c) Representative detrended luciferase activity in BMAL1 Luc HDFs measured from Kronos Dio Real-Time luminometer. (d-f) Box-whisker plots of 1st period (d), 2nd period (e), and cosinor period (f). 1st period was defined as the interval between 1st through and 2nd through. 2nd period was defined as the interval between 2nd through and 3rd through. Cosinor analyzed period was measured mathematically using the cosinor software provided by Dr. R Refinetti ( https://www.circadian.org/softwar.html ). Data are shown as means ± SEM (eight independent experiments were performed, n=20, ns., no signal. *p<0.05, *** p<0.001, by student’s two-tailed t-test).

Article Snippet: 25 μl of lipofectamine 3000 (Invitrogen, Waltham, Massachusetts) was added to one tube, and 9 μg of psPAX2 (Addgene), 6 μg of psPAX2 (Addgene), and 15 μg of pLV6- BMAL1-Luc vector (Addgene), and 25 μg of P3000 reagent was added.

Techniques: Expressing, Luciferase, Activity Assay, Cell Counting, Derivative Assay, Cell Culture, Plasmid Preparation, Two Tailed Test, Whisker Assay, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The role of the circadian clock in cancer hallmark acquisition and immune-based cancer therapeutics

doi: 10.1186/s13046-021-01919-5

Figure Lengend Snippet: Core circadian clock markers and their demonstrated links to cancer hallmarks and immune function

Article Snippet: REV-ERBA/B (NR1D1/2) , Represses rhythmic expression of BMAL1 and BMAL2 , 2. Agonist suppresses cyclin A expression in vitro [ ] .

Techniques: Knockdown, Expressing, In Vitro, Knock-Out, In Vivo, Cell Differentiation, Over Expression, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Activity Assay, Cell Function Assay

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet: HIV-1 replication is rhythmic (A) Cartoon of the HIV-1 NL4.3 R-E-luc (NL4.3-luc) reporter, encoding HIV genes with flanking long terminal repeats (LTR), including defective envelope ( Δenv ), regulator of expression of virion proteins ( Δrev ) and negative regulator factor ( Δnef ) and the luciferase ( luc ) gene which is the readout for viral replication. (B) U-2 OS cells were infected with HIV-1 NL4.3-luc VSV-G for 24 h followed by serum shock synchronization for 1 h. 24 h later, luciferase activity was measured at 30 min intervals or cells harvested at 4 h intervals for RNA extraction for a total of 48 h. (C) U-2 OS cells were infected with HIV-1 NL4.3-luc VSV-G, synchronized and HIV-1 replication measured by luciferase activity (mean ± S.E.M., n = 6) or cells were harvested at 4 h intervals for RNA extraction and HIV-1 Gag transcripts measured relative to a B2M housekeeper by qPCR (mean ± S.E.M., n = 4). Analysis of luciferase data: eJTK cycle p < 0.00001, period = 24.7 h, peak expression = 12.2 h (FFT-NLLS analysis, BioDare2). (D) U-2 OS cells stably expressing luciferase under control of the Bmal1 promoter (Bmal1-luc) were synchronized and promoter activity measured at 30 min intervals (mean ± S.E.M., n = 7). Wild-type U-2 OS cells were synchronized, harvested at 4 h intervals, followed by RNA extraction and qPCR detection of Bmal1 RNA relative to a B2M housekeeper (mean ± S.E.M., n = 4). All data are normalized to peak expression.

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Expressing, Luciferase, Infection, Activity Assay, RNA Extraction, Stable Transfection

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet: BMAL1 regulates HIV-1 replication (A) U-2 OS parental control (ctrl) cells or U-2 OS Bmal1 knockdown (KD) cells generated by shRNA-mediated silencing were infected with HIV-1 NL4.3-luc VSV-G. 24 h later, infected cells were synchronized by serum shock, and viral replication measured by luciferase readout (mean ± S.E.M., n = 3, normalized to peak, raw data in Figure S9 A). BMAL1 and β-actin protein levels were assessed by western blotting (representative of n = 2). (B) Jurkat cells were infected with HIV-1 NL4.3-luc VSV-G for 24 h and Bmal1 KD or Bmal1/Clock over-expression (OE) was generated by siRNA-mediated silencing (scrambled siRNA as ctrl) or transfection of plasmids (pcDNA3.1 as ctrl). 48 h post transfection, viral replication was measured by luciferase expression (mean ± S.E.M., n = 4, Mann-Whitney test, normalized to ctrl) and BMAL1 and β-actin protein levels assessed by western blotting (representative of n = 2). (C) Cartoon of the 5′ HIV-long terminal repeat (LTR) which encodes four E-box motifs (E-box 1: CAGCTG, E-box 2: CAGATG, E-box 3: CACATG, E-box 4: CAGTTG) in the NL4.3 strain; base pairs (bp) are shown relative to the transcriptional start site (TSS). (D) Binding of BMAL1 to the HIV-LTR was assessed by chromatin immunoprecipitation (ChIP). Jurkat cells were infected with NL4.3-luc VSV-G, and chromatin extracts immunoprecipitated with BMAL1 antibody or rabbit IgG as a negative control. Fold enrichment of binding to either the E-boxes in the HIV-LTR or to the Per1 promoter as a positive control was quantified by qPCR and is shown compared to non-specific binding of IgG (mean ± S.E.M., n = 4, Mann-Whitney test).

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Generated, shRNA, Infection, Luciferase, Western Blot, Over Expression, Transfection, Expressing, MANN-WHITNEY, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Positive Control

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet: ROR inhibition modulates host circadian clock factors and HIV-1 replication (A) U-2 OS cells stably expressing luciferase under control of the Bmal1 promoter (Bmal1-luc) were synchronized followed by treatment with GSK805. Luminescence was measured at 30 min intervals (mean ± S.E.M., n = 3, raw data in Figure S9 B). Amplitude reduction compared to UT: 36.1% for 3 μM, 63.9% for 10 μM (FFT-NLLS analysis, BioDare2). Synchronized parental U-2 OS cells were treated with GSK805 (10 μM) and harvested at 4 h intervals, followed by RNA extraction and qPCR detection of Bmal1 transcripts relative to a B2M housekeeper (mean ± S.E.M., n = 3). (B) Jurkat cells stably expressing Bmal1-luc were treated with GSK805 for 24 h, and luciferase activity quantified (mean ± S.E.M., n = 3, Kruskal-Wallis ANOVA). Jurkat cells were treated with GSK805 (20 μM), with BMAL1 and β-actin protein expression assessed by western blotting (representative of n = 3). (C) CD8-depleted PBMCs were activated for 3 days with anti-CD3/CD28, treated with GSK805 (2.5 μM) for 7 days, followed by RNA extraction, and qPCR detection of Bmal1, Rev-erbα, Per1, or Cry2 RNA levels relative to a B2M housekeeper (mean ± S.E.M., n = 4–5, Mann-Whitney test). (D) U-2 OS cells infected with NL4.3-luc VSV-G were synchronized, treated with GSK805 (10 μM) and luciferase activity measured at 30 min intervals (mean ± S.E.M., n = 3, normalized to peak) or cells harvested at 4 h intervals, followed by qPCR detection of HIV-1 Gag RNA relative to a B2M housekeeper (mean ± S.E.M., n = 3, raw data in Figure S9 C). (E) Jurkat cells or activated CD8-depleted PBMCs were infected with NL4.3-luc VSV-G, treated with GSK805 for 24 h and luciferase quantified as a readout of HIV-1 replication (mean ± S.E.M, n = 3–4, Kruskal-Wallis ANOVA). (F) Activated CD8-depleted PBMCs were spinoculated for 2 h with patient-derived HIV-1 (transmitted founder virus clone CH185) and cultured with GSK805 for 7 days, followed by qPCR detection of HIV-1 Gag RNA relative to B2M housekeeper (mean ± S.E.M., n = 3, Kruskal-Wallis ANOVA). All data are expressed relative to UT control.

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Inhibition, Stable Transfection, Expressing, Luciferase, RNA Extraction, Activity Assay, Western Blot, MANN-WHITNEY, Infection, Derivative Assay, Cell Culture

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet: An independent ROR inhibitor reduces Bmal1 expression and HIV-1 replication (A) U-2 OS cells stably expressing luciferase under control of the Bmal1 promoter (Bmal1-luc) were synchronized, treated with GSK2981278 and luciferase activity measured at 30 min intervals (mean ± S.E.M., n = 4, raw data in Figure S9 D). Amplitude reduction compared to UT: 63.9% for 20 μM, 88.9% for 40 μM (FFT-NLLS analysis, BioDare2). (B) Jurkat cells stably expressing Bmal1-luc were treated with indicated concentrations of GSK2981278 for 24 h and luciferase activity quantified (mean ± S.E.M., n = 3, Kruskal-Wallis ANOVA). Wild-type Jurkat cells were treated with GSK2981278 (40 μM), and BMAL1 and β-actin protein expression assessed by western blotting (representative of n = 3). (C) CD8-depleted PBMCs were activated for 3 days with anti-CD3/CD28, cultured in the presence of GSK2981278 (20 μM) for 7 days, followed by RNA extraction and qPCR detection of Bmal1 RNA relative to a B2M housekeeper (mean ± S.E.M., n = 4, Mann-Whitney test). (D) Jurkat cells (mean ± S.E.M., n = 3, Kruskal-Wallis ANOVA) or activated CD8-depleted PBMCs (mean ± S.E.M., n = 6, Kruskal-Wallis ANOVA) were infected with NL4.3-luc VSV-G, treated with different doses of GSK2981278 for 24 h and luciferase quantified as readout of HIV-1 replication. (E) Activated CD8-depleted PBMCs were spinoculated for 2 h with patient-derived HIV-1 (transmitted founder virus clone CH185), cultured in medium with GSK2981278 (20 μM) for 7 days, followed by RNA extraction and qPCR detection of HIV-1 Gag RNA relative to a B2M housekeeper (mean ± S.E.M., n = 3). All data are expressed relative to UT control.

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Expressing, Stable Transfection, Luciferase, Activity Assay, Western Blot, Cell Culture, RNA Extraction, MANN-WHITNEY, Infection, Derivative Assay

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet: Differential binding of REV-ERBα and RORC to the HIV-LTR at different circadian times (A) Consensus plot showing the conservation of nucleotides in position 121 to 126 of 1266 HIV-1 sequences deposited in the Los Alamos Database (coordinates are from the HXB2 reference). Bases of coding strand are displayed which correspond to the nucleotides ‘AGGTCA’ on the antisense strand, thereby representing the ROR response element (RORE) ‘RGGTCA’. Conservation is reflected by the height of the bases (y axis 0–100%). (B) Jurkat cells were transfected with reporter constructs of HIV-1 subtypes A-G, followed by GSK2981278 treatment (40 μM) and LTR activity measured by quantifying luciferase activity 24h later (mean ± S.E.M., n = 4, Mann-Whitney test). (C) Location of the RORE in the 5′ HIV-LTR of NL4.3 strain; base pairs (bp) are shown relative to the transcriptional start site (TSS). Model where ROR and REV-ERBα compete for binding to the HIV-LTR which can be tested by chromatin immunoprecipitation (ChIP). RORC binding to the HIV-LTR was shown by Wiche Salinas et al. (D) Jurkat cells were infected with NL4.3-luc VSV-G for 24 h, and chromatin extracts immunoprecipitated with anti-REV-ERBα or rabbit IgG as a negative control. Fold enrichment of binding to the RORE in either the HIV-LTR or the Bmal1 promoter was quantified by qPCR and is shown compared to the IgG control (mean ± S.E.M., n = 4, Mann-Whitney test). (E) Jurkat cells were infected with NL4.3-luc VSV-G followed by treatment with GSK805 (10 μM) for 24 h. Fold enrichment of REV-ERBα binding to the HIV-LTR RORE or the Bmal1 promoter was compared between untreated and GSK805-treated cells (mean ± S.E.M., n = 4–6, Mann-Whitney test). (F) U-2 OS cells were infected with HIV-1 NL4.3-luc VSV-G for 24 h followed by serum shock synchronization for 1 h. 24 h (CT0), and 36 h (CT12) post-synchronization cells were harvested, and chromatin extracts immunoprecipitated with REV-ERBα antibody, RORC antibody, or rabbit IgG as a negative control. Fold enrichment of binding to either the RORE in the HIV-LTR or to the Bmal1 promoter as a positive control was quantified by qPCR, normalized to non-specific IgG binding and shown as fold change of binding (mean ± S.E.M., n = 4, Mann-Whitney test). (G) Model where RORC binding to the HIV-LTR promotes HIV-1 replication, and REV-ERBα binding supresses viral replication at different circadian times resulting in rhythmic HIV-1 replication. See related Figure S7 . All data are expressed relative to the control cells.

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Binding Assay, Transfection, Construct, Activity Assay, Luciferase, MANN-WHITNEY, Chromatin Immunoprecipitation, Infection, Immunoprecipitation, Negative Control, Positive Control

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet: HIV-1 host factors are regulated by the circadian clock (A) Expression of 90 HIV-1 host proteins was analyzed using the Circa database, and 43% of genes identified as cycling in humans. (B) BMAL1-regulated genes, REV-ERBα-regulated genes, and RORC-regulated genes were compared with host factors known to alter HIV-1 replication. (C) HOMER (Hypergeometric Optimization of Motif EnRichment tool ) was used to analyze -1kb promoter regions of HIV-1 host factors and identified gene promoters encoding E-box motifs or ROR response elements (ROREs). (D) Jurkat cells were treated with GSK2981278 (40 μM) or GSK805 (20 μM) for 24 h, cells were lysed, RNA was extracted, and gene expression analyzed via qPCR (mean ± S.E.M., n = 4, Mann-Whitney test). (E) Gene ontology (GO) cellular components analysis where each node represents an enriched GO term. Related GO terms are connected by lines, where thickness reflects the percentage of overlapping genes. See related Figure S8 .

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Expressing, MANN-WHITNEY

Journal: iScience

Article Title: Molecular components of the circadian clock regulate HIV-1 replication

doi: 10.1016/j.isci.2023.107007

Figure Lengend Snippet:

Article Snippet: Bmal1 and Clock expression plasmids, or a pcDNA3.1 control, were delivered into Jurkat cells via transfection (ViaFect, Promega).

Techniques: Recombinant, Staining, Cytotoxicity Assay, Luciferase, Construct, Expressing, Plasmid Preparation, Software