clock  (Santa Cruz Biotechnology)

Journal: Nature Chemical Biology

Article Title: Pharmacological targeting of BMAL1 modulates circadian and immune pathways

doi: 10.1038/s41589-025-01863-x

Figure Lengend Snippet: a , Effect of CCM (100 μM) on target genes compared to BMAL1 knockdown. The RNA levels of BMAL1 target genes were quantified by RT–qPCR in unsynchronized U2OS cells. Six and 12 biological replicates were used for knockdown and CCM treatment, respectively. b , Dose-dependent effects of CCM on BMAL1 target genes in unsynchronized U2OS cells ( n = 3, biological replicates). c , The effects of CCM (100 μM) are impaired when BMAL1, CLOCK or NPAS2 is knocked down in unsynchronized U2OS cells ( n = 5, biological replicates). d , Validation of BMAL1 and CLOCK siRNAs by confirming reduction in their protein levels in U2OS cells. This experiment was performed independently two times with similar results. e , CCM dose-dependent modulation of real-time circadian rhythm observed in peritoneal macrophages directly isolated from Per2–Luc (PER2::Luc) mice (synchronized, n = 3–6). f , CCM demonstrates no significant cytotoxicity on U2OS cells ( n = 3, biological replicates) at concentrations of 100 µM and 200 µM for 48 h. The lysis buffer provided in the CyQUANT LDH Cytotoxicity Assay Kit was used as a positive control. g , Monitoring of the circadian rhythm in synchronized U2OS cells ( n = 3, biological replicates) using RT–qPCR, showing differences between DMSO vehicle (black) and 100 μM CCM treatment (red). All error bars ( a – c and e – g ) denote the mean ± s.d. Statistical tests were performed using unpaired two-tailed t -test. Amp., amplitude; Conc., concentration; NS, not significant; Veh, vehicle; RLU, relative luminescence.

Article Snippet: Lipofectamine RNAiMAX (Invitrogen, 13778-075) was used for siRNA knockdown experiments. siRNA oligos targeting BMAL1 (sc-38165), CLOCK (sc-35074) and NPAS2 (sc-38169) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Quantitative RT-PCR, Biomarker Discovery, Isolation, Lysis, CyQUANT Assay, LDH Cytotoxicity Assay, Positive Control, Two Tailed Test, Concentration Assay

Journal: Nature Chemical Biology

Article Title: Pharmacological targeting of BMAL1 modulates circadian and immune pathways

doi: 10.1038/s41589-025-01863-x

Figure Lengend Snippet: a , Co-IP studies between BMAL1 and CLOCK in U2OS cells ( n = 3, biological replicates), demonstrating minimal effect of CCM on the stability of their complex. The ratio of BMAL1/CLOCK intensity in each IP sample was calculated and then normalized to DMSO control for each independent experiment. Three independent experiments were integrated for statistical analysis. Error bars denote the mean values ± s.d., and P value was generated by unpaired two-tailed t -test. b , BMAL1 localization in the nucleus of U2OS cells remains unchanged, with no observed alterations in the distribution of cytosolic versus nuclear levels after 3 h of CCM treatment at 100 μM. This experiment was performed independently two times with similar results. c , Overall BMAL1 protein levels in U2OS cells ( n = 6, biological replicates), as detected by western blot, also remain unaltered after CCM treatment at 100 μM (left). BMAL1/β-actin intensity ratio was quantified (right). d , The PASB α-helix F and amino acids positioned on this helix, His389 and Gln379, shift positions with CCM binding. e , CCM weakens the association of histone H2A with BMAL1. Protein interactions of BMAL1 were analyzed by RIME. U2OS cells ( n = 3) were treated with 100 μM CCM or 0.1% DMSO for 3 h. f , CCM does not change the binding of BMAL1 to the promotor of corresponding genes in U2OS cells ( n = 3, biological replicates) detected by ChIP–qPCR. Error bars in a , c and f are presented as mean values ± s.d., and statistical tests were performed using unpaired two-tailed t -test. FC, fold change; RMSD, root mean square deviation.

Article Snippet: Lipofectamine RNAiMAX (Invitrogen, 13778-075) was used for siRNA knockdown experiments. siRNA oligos targeting BMAL1 (sc-38165), CLOCK (sc-35074) and NPAS2 (sc-38169) were purchased from Santa Cruz Biotechnology.

Techniques: Co-Immunoprecipitation Assay, Control, Generated, Two Tailed Test, Western Blot, Binding Assay, ChIP-qPCR

Journal: Cell reports

Article Title: Hepatocyte Period 1 dictates oxidative substrate selection independent of the core circadian clock

doi: 10.1016/j.celrep.2024.114865

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: siClock , Santa Cruz Biotechnology , Cat#sc-35075.

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, In Vivo, Sequencing, Software

Journal: Cell reports

Article Title: Hepatocyte Period 1 dictates oxidative substrate selection independent of the core circadian clock.

doi: 10.1016/j.celrep.2024.114865

Figure Lengend Snippet: Figure 4. Fasting induces Per1, Fgf21, and Pdk4 independent of the core circadian clock (A and C) Schematic of in vitro feed/starve experiment in AML12 cells treated with either adenovirus or siRNA targeting Per1, Bmal1, Clock, or Cry1. (B) Relative expression level of Per1, Fgf21, and Pdk4 in AML12 cells treated with Ad-GFP or Ad-shPer1, fed or starved; n = 5. (D) Relative expression level of Per1, Fgf21, and Pdk4 in AML12 cells treated with lipofectamine (Control), siBmal1, siClock, or siCry1, fed or starved; n = 3. (E) Schematic of ex vivo primary hepatocyte starvation experiment from Per2WT and Per2KO female mice. (F) Relative expression level of Per1, Fgf21, and Pdk4 in primary hepatocytes isolated from Per2WT or Per2KO mice, fed or starved; n = 3–4. (G) Schematic of in vivo time-course experiment in Bmal1fl/fland Bmal1cLKO (Bmal1fl/fl, Alb-Cre) mice. Mice were either fed ad libitum or fasted for 16 h, and liver tissues were harvested every 4 h in a 24-h duration; n = 3–4.

Article Snippet: Herzog Mouse: Fgf21fl/fl Jackson Laboratory Strain#022361 RRID:IMSR_JAX:022361 Mouse: C57BL/6J Jackson Laboratory Strain#000664 RRID:IMSR_JAX:000664 Mouse: Alb-Cre Jackson Laboratory Strain#003574 RRID:IMSR_JAX:003574 Oligonucleotides siBmal1 Santa Cruz Biotechnology Cat#sc-38166 siClock Santa Cruz Biotechnology Catsc-35075 siCry1 Santa Cruz Biotechnology Cat#sc-44835 siPpara Santa Cruz Biotechnology Cat#sc-36380 siEsrrg Santa Cruz Biotechnology Cat#sc-44705 siAtf1 Santa Cruz Biotechnology Cat#sc-29755 Fgf21 antisense oligonucleotide (ASO) IONIS Pharmaceuticals Cat#ION-256,617 qRT-PCR sequences Table S3 N/A Software and algorithms ImageJ software https://imagej.nih.gov/ij. https://imagej.nih.gov/ij.

Techniques: In Vitro, Expressing, Control, Ex Vivo, Isolation, In Vivo