Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 3. Phosphorylation of the human PER2 FASP region inhibits CK1 activity (A–D) NMR kinase assay for the WT FASP or indicated mutant peptides monitoring the reaction kinetics of priming phosphorylation at S662 by NMR. (E) Schematic of FASP alanine mutations and resulting discrete phosphostates. (F) Plot of priming rate constant (kprime) as a function of the possible successive phosphosites in the FASP. Error bars represent SEM from fits in (A)–(D). (G) ADP-Glo kinase assay with titration of FASP mutant peptides with mean and SD from 2 replicates, representative of n = 3 independent assays. Shaded area indicates 95% CI of the fit. (H) Data from (F) normalized by Vmax values calculated from the preferred kinetic model (see Figure S2; Table 1). (I) ADP-Glo kinase assay of hPER2 PAS-Degron peptide (see Figure S2B) with titration of pFASP peptides corresponding to 2 (2p) or 3 (3p) phosphoserines (see Figure S3A) with mean and SD from 2 replicates, representative of n = 3 independent assays. Shaded area indicates 95% CI of the fit. (J and K) Western blot and quantification of the phosphorylation of the FASP priming site (pS659 in mouse PER2). Full-length mouse PER2 was immunopre- cipitated from transfected HEK293T cells, dephosphorylated, and subjected to an in vitro kinase assay with 200 ng CK1 in the presence and absence of un- phosphorylated mouse PER2 FASP or human PER2 4pFASP peptides as indicated.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Phospho-proteomics, Activity Assay, Kinase Assay, Mutagenesis, Titration, Western Blot, Transfection, In Vitro

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 4. The human PER2 pFASP binds to the active site of CK1 (A) Surface representation of CK1 catalytic domain bound to ADP (PDB 5X17). Spheres, sulfate anions from the crystallization condition bound at anion binding sites as indicated. (B) Surface representation of the CK1 catalytic domain bound to a human PER2 3pFASP peptide (see Figure S3A). (C–E) Zoom of 3pFASP interactions within anion binding site 1 (C), the active site (D), and anion binding site 2 (E). (F) Structural alignment showing the main chain for the activation loop of CK1 WT (gray) and the tau (R178C) mutant (red) showing a clash of the 3pFASP (teal) with the conformation of the activation loop stabilized by the tau mutation. (G) Characterization of key interactions from molecular dynamics simulations that stabilize the 5pFASP product in the active site, site 1, site 2, and an additional anion binding site located proximal to site 2 (site 30). Histograms were computed based on distances sampled during the GaMD simulations.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Crystallization Assay, Binding Assay, Activation Assay, Mutagenesis

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 5. Kinase anchoring interactions with the PER2 CK1BD increase FASP phosphorylation and feedback inhibition of CK1 (A) Alignment of human and mouse PER1/2 proteins showing conservation of the disordered FASP region flanked by two highly conserved binding motifs (CK1BD-A/B) characterized as PONDR (predictor of natural disordered regions) minima and predicted to be a-helices by AlphaFold2. The priming site (S662, human PER2 numbering) is shown, with sequential phosphorylation sites indicated by asterisks.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Phospho-proteomics, Inhibition, Binding Assay

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 6. Circadian rhythms are shortened by small deletions in the conserved FASP domain of human PER2 (A) Schematic representation of select in-frame deletions within the human PER2 FASP region are separated into 2 classes: priming-disrupted (P2E17-50) or priming intact (P2E17-25). (B) Immunoblot of select priming-disrupted PER2::LUC mutants compared with WT PER2::LUC. Blot representative of two independent experiments. (C and D) Representative immunoblots for (C) priming-disrupted or (D) priming intact PER2 mutants. *nonspecific band. The Per2 allele in clone 17-3 has a frame- shifting mutation leading to the deletion of the untagged PER2. (E and F) Real-time bioluminescence traces of circadian rhythms from WT and mutant Per2 clones (E, priming-disrupted; F, priming intact) with quantification of mean period and SD from n = 3 cultures. Periods from mutant clones were compared with WT with an unpaired t test: ***, p < 0.001. Per2 priming intact mutants exhibited rhythms that were shortened to a lesser degree than the priming-disrupted mutants in (E), p < 0.05. In both graphs, the first peaks are aligned to show differences in period clearly. (G) Western blot and quantification of degradation of WT PER2::LUC and clone 17–50 after cycloheximide (CHX) treatment at time 0. Blot representative of two independent assays (n = 2). (H) Western blot and densitometric quantification of phosphorylation of de novo PER2 in WT PER2::LUC and clone 17–50 after protein depletion by 10 h CHX treatment and washout at time 0. Blot representative of two independent assays (n = 2).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Western Blot, Mutagenesis, Clone Assay, Phospho-proteomics

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 7. The phosphorylated PER-Short domain of Drosophila PER binds CK1 site 1 to inhibit kinase activity (A and B) Western blot of dPER fragment (aa1–100) cleaved from full-length dPER (A) or a fragment containing the dPER CK1BD but lacking the PAS dimerization domain (B) co-expressed with DBT. Samples collected at indicated time points after kinase induction, followed by protein extraction and TEV protease cleavage. (C) Densitometric quantification of the hypophosphorylated band (n = 4). WT and S596A compared with two-way ANOVA with Sidak multiple comparisons test: * p < 0.05. (D) ADP-Glo kinase assay of CK1 on the human PER2 PAS-Degron substrate in the presence of indicated peptides from dPER. Data are mean and SD from 2 replicates, representative of n = 3 independent assays. (E) Structure of human CK1 (gray) bound to the dPER-Short peptide with pS589 (light green). (F) Close-up view of dPER pS589 coordinated by CK1 residues in anion binding site 1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Activity Assay, Western Blot, Protein Extraction, Kinase Assay, Binding Assay

Journal: Communications Biology

Article Title: Bmal1 in the striatum influences alcohol intake in a sexually dimorphic manner

doi: 10.1038/s42003-021-02715-9

Figure Lengend Snippet: a Quantitative PCR analysis of Bmal1 mRNA levels measured at ZT1 in dorsal striatal tissue of control, Bmal1 heterozygote and knockout mice. ANOVA, significant genotype effect, F (2, 7) = 113.8, p < 0.0001, Tukey’s post-hoc test, * p < 0.005. b A representative image of BMAL1 immunofluorescence staining at ZT 1 in dorsal striatal tissue of control, Bmal1 heterozygote and knockout mice. BMAL1: red, Gpr88-Cre-GFP: green. Scale bar = 30 µm. c Quantitative PCR analysis of Per2 mRNA levels in dorsal striatal tissue of control, Bmal1 heterozygote and knockout male mice. Two-way ANOVA, significant genotype effect, F (2, 5) = 21.46, p = 0.0035, and time point effect, F (2, 8) = 51.7, p < 0.0001, significant interaction effect, F (4, 8) = 13.97, p = 0.0011. d Quantitative PCR analysis of Dbp mRNA levels in dorsal striatal tissue of control, Bmal1 heterozygote and knockout mice. Two-way ANOVA, significant genotype effect, F (2, 5) = 116.6, p < 0.0001, and time point effect F (2, 8) = 5.236, p < 0.05 effect, Tukey’s test, ** p < 0.05. e Daily alcohol consumption (left) and average alcohol consumption (right) of control, and Bmal1 knockout male mice. Two-way repeated measure ANOVA, (RM-ANOVA) significant genotype effect, F (1, 23) = 13.26, p = 0.0014, Unpaired two-tailed t-test, ** p < 0.01. f Daily alcohol consumption (left) and average alcohol consumption (right) of control, and Bmal1 knockout female mice. RM-ANOVA, significant genotype effect, F (1, 29) = 5.0, p = 0.033. Unpaired two-tailed t-test, * p < 0.05. g Daily alcohol preference (left) and average alcohol preference (right) of control, and Bmal1 knockout male mice. RM-ANOVA, significant genotype effect, F (1, 23) = 10.73, p = 0.0033. Unpaired two tailed t-test, ** p < 0.01. h Daily alcohol preference (left) and average alcohol preference (right) of control, and Bmal1 knockout female mice. RM-ANOVA, significant genotype effect, F (1, 29) =4.708, p = 0.0384. Unpaired two tailed t-test, * p < 0.05. i Daily fluid intake (left) and average fluid intake (right) of control and Bmal1 knockout male mice. RM-ANOVA, no significant effect, F (1, 23) = 0.3737, p = 0.5470. Unpaired two-tailed t-test, NS. j Daily fluid intake (left) and average fluid intake (right) of control and Bmal1 knockout female mice. RM-ANOVA, no significant effect, F (1,29) = 0.3185, p = 0.5769. Unpaired two-tailed t-test, NS. NS = no significant differences. CTR: control, HET: Bmal1 heterozygote, SKO: Bmal1 knockout. ZT: Zeitgeber time. c-j , the values express mean ± S.E.M. a – d , n = 3/genotype. e , g , i , CTR n = 12, SKO n = 13. f , h , j , CTR n = 17, SKO n = 14.

Article Snippet: The following antibodies and dilutions were used: PER2 rabbit polyclonal (1:500, Novus Biologicals, # NB100-125, Littleton, CO, USA), BMAL1 rabbit polyclonal (1:500, Novus Biologicals # NB100-2288, Littleton, CO, USA), anti-rabbit secondary Alexa-647 (1:500, Life Technologies, Carlsbad, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Knock-Out, Immunofluorescence, Staining, Two Tailed Test

Journal: Communications Biology

Article Title: Bmal1 in the striatum influences alcohol intake in a sexually dimorphic manner

doi: 10.1038/s42003-021-02715-9

Figure Lengend Snippet: a Daily alcohol intake (left) and average alcohol intake (right) of control and Bmal1 heterozygote male mice. Two-way repeated measure ANOVA (RM-ANOVA), no significant effect, F (1, 26) = 2.793, p = 0.1067, Unpaired two-tailed t-test, NS. b Daily alcohol intake (left) and average alcohol intake (right) of control and Bmal1 heterozygote female mice. RM-ANOVA, no significant effect, F (1, 25) = 2.507, p = 0.1259, Unpaired two-tailed t-test, NS. c Daily alcohol preference (left) and average alcohol preference (right) of control and Bmal1 heterozygote male mice. RM-ANOVA, no significant effect, F (1, 26) = 1.326, p = 0.26, Unpaired two-tailed t-test, NS. d Daily alcohol preference (left) and average alcohol preference (right) of control and Bmal1 heterozygote female mice. RM-ANOVA, no significant effect, F (1, 25) = 3.506, p = 0.0729, Unpaired two tailed t-test, NS. e Total fluid intake (left) and average fluid intake (right) of control and Bmal1 heterozygote male mice. RM-ANOVA, no significant effect, F (91, 26) = 1.498, p = 0.2320, Unpaired two-tailed t-test, NS. f Total fluid intake (left) and average fluid intake (right) of control and Bmal1 heterozygote female mice. RM-ANOVA, no significant effect, F (1, 25) = 0.5874, p = 0.4506, Unpaired two-tailed t-test, NS. g Daily alcohol intake (left) and average alcohol intake (right) of control and Per2 heterozygote male mice. RM-ANOVA, no significant effect, F (1, 13) = 0.09317, p = 0.7650. Unpaired two-tailed t-test, NS. h Daily alcohol intake (left) and average alcohol intake (right) of control and Per2 heterozygote female mice. RM-ANOVA, no significant effect, F (1, 13) = 0.08137. p = 0.7799. Unpaired two-tailed t-test, NS. i Daily alcohol preference (left) and average alcohol preference (right) of control and Per2 heterozygote male mice. RM-ANOVA, no significant effect, F (1, 13) = 0.01314, p = 0.9105. Unpaired two-tailed t-test, NS. j Daily alcohol preference (left) and average alcohol preference (right) of control and Pe2 heterozygote female mice. RM-ANOVA, no significant effect, F (1, 13) = 0.04381, p = 0.8375. Unpaired two-tailed t-test, NS. k Total fluid intake (left) and average fluid intake (right) of control and Per2 heterozygote male mice. RM-ANOVA, no significant effect, F (1, 13) = 0.02780, p = 0.8702. Unpaired two-tailed t-test, NS. l Total fluid intake (left) and average fluid intake (right) of control and Per2 heterozygote female mice. RM-ANOVA, no significant effect, F (1, 13) = 1.833, p = 0.1988. Unpaired two tailed t-test, NS. NS = no significant differences. The values express mean ± S.E.M. a–f , CTR: control, HET: Bmal1 heterozygote. a , c , e CTR n = 12, HET n = 16. b , d , f CTR n = 17, HET n = 10. g – l CTR: control, HET: Per2 heterozygote. g , i , k CTR n = 8, HET n = 7. h , j , l CTR n = 9, HET n = 6.

Article Snippet: The following antibodies and dilutions were used: PER2 rabbit polyclonal (1:500, Novus Biologicals, # NB100-125, Littleton, CO, USA), BMAL1 rabbit polyclonal (1:500, Novus Biologicals # NB100-2288, Littleton, CO, USA), anti-rabbit secondary Alexa-647 (1:500, Life Technologies, Carlsbad, CA, USA).

Techniques: Control, Two Tailed Test

Journal: Communications Biology

Article Title: Bmal1 in the striatum influences alcohol intake in a sexually dimorphic manner

doi: 10.1038/s42003-021-02715-9

Figure Lengend Snippet: a A representative image of PER2 immunofluorescence staining in dorsal striatal tissue of control and Per2 knockout mice. PER2: red, Gpr88-Cre-GFP: green. n = 3/genotype, scale bar = 50 µm. b A representative image of BMAL1 immunofluorescence staining in dorsal striatal tissue of control, and Per2 knockout mice. BMAL1: red, Gpr88-Cre-GFP: green. n = 3/genotype, scale bar = 50 µm. c Daily alcohol consumption (left) and average alcohol consumption (right) of control and Per2 knockout male mice. Two-way repeated measure ANOVA (RM-ANOVA), significant genotype effect, F (1, 14) = 8.332, p = 0.0120. Unpaired two-tailed t-test, * p < 0.05. d Daily alcohol consumption (left) and average alcohol consumption (right) of control and Per2 knockout female mice. RM-ANOVA, no significant effect, F (1, 17) = 0.014, p = 0.9072. Unpaired two-tailed t-test, NS. e Daily alcohol preference (left) and average alcohol preference (right) of control and Per2 knockout male mice. RM-ANOVA, significant genotype effect, F (1, 14) = 6.552, p = 0.0227. Unpaired two-tailed t-test, * p < 0.05. f Daily alcohol preference (left) and average alcohol preference (right) of control and Per2 knockout female mice. RM-ANOVA, no significant effect, F (1, 17) = 0.01779, p = 0.8955. Unpaired two-tailed t-test, NS. g Daily fluid intake (left) and average fluid intake (right) of control and Per2 knockout male mice. RM-ANOVA, no significant effect, F (1, 14) = 0.142, p = 0.7116. Unpaired two-tailed t-test, NS. h Daily fluid intake (left) and average fluid intake (right) of control and Per2 knockout female mice. RM-ANOVA, significant genotype effect, F (1, 17) = 5.665, p = 0.0293. Unpaired two-tailed t-test, * p < 0.05. NS = no significant differences. CTR: control, HET: Per2 heterozygote, SKO: Per2 knockout. c – h The values express mean ± S.E.M. a , b n = 3/genotype. c , e , g , CTR n = 8, SKO n = 8. d , f , h CTR n = 9, SKO n = 10.

Article Snippet: The following antibodies and dilutions were used: PER2 rabbit polyclonal (1:500, Novus Biologicals, # NB100-125, Littleton, CO, USA), BMAL1 rabbit polyclonal (1:500, Novus Biologicals # NB100-2288, Littleton, CO, USA), anti-rabbit secondary Alexa-647 (1:500, Life Technologies, Carlsbad, CA, USA).

Techniques: Immunofluorescence, Staining, Control, Knock-Out, Two Tailed Test

Journal: Communications Biology

Article Title: Bmal1 in the striatum influences alcohol intake in a sexually dimorphic manner

doi: 10.1038/s42003-021-02715-9

Figure Lengend Snippet: a Average daily intake (left) and preference (right) of 0.25% sucrose solution of control and Bmal1 knockout male mice. Unpaired two-tailed t-test, NS. b Average daily intake (left) and preference (right) of 0.25% sucrose solution of control and Bmal1 knockout female mice. Unpaired two-tailed t-test, NS. c Average daily intake (left) and preference (right) of 2% sucrose solution of control and Bmal1 knockout male mice. Unpaired two-tailed t-test, NS. d Average daily intake (left) and preference (right) of 2% sucrose solution of control and Bmal1 knockout female mice. Unpaired two-tailed t-test, NS. e Average daily intake (left) and preference (right) of 0.25% sucrose solution of control and Per2 knockout male mice. Unpaired two-tailed t-test, NS. f Average daily intake (left) and preference (right) of 0.25% sucrose solution of control and Per2 knockout female mice. Unpaired two-tailed t-test, NS. g Average daily intake (left) and preference (right) of 2% sucrose solution of control and Per2 knockout male mice. Unpaired two-tailed t-test, NS. h , Average daily intake (left) and preference (right) 2% sucrose solution of control and Per2 knockout female mice. Unpaired two-tailed t-test, NS. NS = no significant differences. a – h , the values express mean ± S.E.M. a – d , CTR: Bmal1 control, SKO: Bmal1 Knockout. a , c , CTR n = 9, SKO n = 9. b , d CTR n = 5, SKO n = 6. e – h CTR: Per2 control, SKO: Per2 knockout. e , g CTR n = 9, SKO n = 9. f , h CTR n = 10, SKO n = 8.

Article Snippet: The following antibodies and dilutions were used: PER2 rabbit polyclonal (1:500, Novus Biologicals, # NB100-125, Littleton, CO, USA), BMAL1 rabbit polyclonal (1:500, Novus Biologicals # NB100-2288, Littleton, CO, USA), anti-rabbit secondary Alexa-647 (1:500, Life Technologies, Carlsbad, CA, USA).

Techniques: Control, Knock-Out, Two Tailed Test

Journal: Communications Biology

Article Title: Bmal1 in the striatum influences alcohol intake in a sexually dimorphic manner

doi: 10.1038/s42003-021-02715-9

Figure Lengend Snippet: a Representative double-plotted actograms illustrating the daily pattern of running-wheel activity of control, Bmal1 heterozygote and knockout female (top), and male (bottom) mice. b Representative double-plotted actograms illustrating the daily pattern of running-wheel activity of control, Per2 heterozygote and knockout female (top) and male (bottom) mice. The vertical marks indicate periods of activity of at least 10-wheel revolutions per 10 min. Each horizontal line plots 48 h, and sequential days are arranged from top to bottom. The empty and gray shaded areas in each actogram illustrate the light and dark phases, respectively. c–v Circadian analysis of locomotor activity of Bmal1 and Per2 control, heterozygote and knockout male and female mice. One way-ANOVA. No significant differences were observed between the different genotypes in any of the parameters analyzed. c – f Amplitude of the locomotor activity rhythm. g – j , time to entrain to a 6-h phase advance. k – n Time to entrain to a 6-h phase delay. o – r Free running period in constant dark (DD). s-v Free running period in constant light (LL). a , b LD, 12:12 h light dark; +6 h, 6-h phase advance; −6h, 6-h phase delay; DD, constant dark; LL, constant light. Bmal1CTR: control, Bmal1HET: Bmal1 heterozygote, Bmal1SKO: Bmal1 knockout. Per2CTR: control, Per2HET: Per2 heterozygote, Per2SKO: Per2 knockout. Bars on the graphs represent the arithmetic mean ± S.E.M.

Article Snippet: The following antibodies and dilutions were used: PER2 rabbit polyclonal (1:500, Novus Biologicals, # NB100-125, Littleton, CO, USA), BMAL1 rabbit polyclonal (1:500, Novus Biologicals # NB100-2288, Littleton, CO, USA), anti-rabbit secondary Alexa-647 (1:500, Life Technologies, Carlsbad, CA, USA).

Techniques: Activity Assay, Control, Knock-Out

Journal: Experimental & molecular medicine

Article Title: microRNA-25 as a novel modulator of circadian Period2 gene oscillation.

doi: 10.1038/s12276-020-00496-5

Figure Lengend Snippet: Fig. 1 Candidate microRNAs targeting the 3′-untranslated (UTR) region of Period2 (Per2). a Conserved miR-24-3p and miR-25-3p binding sites on the 3′-UTR of Per2 in several vertebrates (indicated with red outlines). b Predicted binding sites of miR-24-3p (red) and miR-25-3p (blue) are illustrated on the 3′-UTR of Per2, and the 3′-UTR targeting sequences of miR-24-3p and miR-25-3p are indicated. c Schematic of the constructed pGL3 vectors with binding sites on the 3′-UTR of Per2 for the full-length or truncated miR-24-3p and miR-25-3p. Predicted binding sites of miR-24-3p (red bar) and miR-25-3p (blue bar) are illustrated on the 3′-UTR of Per2. d NIH3T3 fibroblasts were cotransfected with pRL-TK and either a constructed pGL3 vector carrying a miR control oligomer (50 nM), miR-24-3p mimic (50 nM, red), or miR-25-3p mimic (50 nM, blue). Data are presented as the means ± SE (n = 4), and significance was assessed by Student’s t test (*p < 0.01).

Article Snippet: Immunoblot analyses were carried out with rabbit anti-PER2 polyclonal antibody (Santa Cruz Biotechnology, USA) and anti-beta-ACTIN-HRP (Santa Cruz Biotechnology).

Techniques: Binding Assay, Construct, Plasmid Preparation, Control

Journal: Experimental & molecular medicine

Article Title: microRNA-25 as a novel modulator of circadian Period2 gene oscillation.

doi: 10.1038/s12276-020-00496-5

Figure Lengend Snippet: Fig. 4 miR-24-3p and miR-25-3p functioned in a dose-dependent manner and bind to their specific sites on the 3′-UTR of Per2. Real-time bioluminescence recordings of PER2::LUC oscillation in Per2::Luc KI MEFs that were transfected with a, b miR-24-3p-overexpressing vectors or d, e miR-25-3p-overexpressing vectors in a dose-dependent manner. c, f The area under the curve was calculated from the raw data, which is presented in bar graphs. g Schematic for designed site-directed mutations of the miR-24-3p- and/or miR-25-3p binding sites on the 3′-UTR of Per2 in the pGL3- LUC vector driven by the Per2 promoter. h Results of the real-time bioluminescence recordings of the transfected site-directed mutated luciferase vectors in the NIH3T3 wild-type fibroblasts and i the area under curve was calculated, which is presented in bar graphs. Data are presented as the means ± SE (n = 3), and significance was assessed by one-way ANOVA, *p < 0.05 compared to the control group.

Article Snippet: Immunoblot analyses were carried out with rabbit anti-PER2 polyclonal antibody (Santa Cruz Biotechnology, USA) and anti-beta-ACTIN-HRP (Santa Cruz Biotechnology).

Techniques: Transfection, Binding Assay, Plasmid Preparation, Luciferase, Control

Journal: Experimental & molecular medicine

Article Title: microRNA-25 as a novel modulator of circadian Period2 gene oscillation.

doi: 10.1038/s12276-020-00496-5

Figure Lengend Snippet: Fig. 5 Transduction of lentivirus overexpressing miR-24-3p and/or miR-25-3p dampens the PER2::LUC rhythm in the neonatal SCN slice cultures obtained from the PER2::LUC knock in transgenic mice. a Experimental design for transducing CMV-promoter driven lentivirus-miR- control/miR-24-3p/miR-25-3p-GFP in neonatal suprachiasmatic nucleus (SCN) slice cultures. b Representative results of PER2::LUC oscillation measured by a real-time bioluminescence recording device. The bioluminescence patterns were aligned to the first nadir of data acquired to compare changes in the expression patterns of PER2::LUC in the pre- and postlentiviral transduced SCN slice cultures (experiments were performed three independent times, n = 3). c Changes in PER2 amplitudes and periods are presented as the means ± SE. Significance was assessed by Student’s t test, *p < 0.01 compared to the prelentiviral transduction conditions.

Article Snippet: Immunoblot analyses were carried out with rabbit anti-PER2 polyclonal antibody (Santa Cruz Biotechnology, USA) and anti-beta-ACTIN-HRP (Santa Cruz Biotechnology).

Techniques: Transduction, Knock-In, Transgenic Assay, Control, Expressing

Journal: Experimental & molecular medicine

Article Title: microRNA-25 as a novel modulator of circadian Period2 gene oscillation.

doi: 10.1038/s12276-020-00496-5

Figure Lengend Snippet: Fig. 6 Expression levels of miR-24-3p and miR-25-3p vary in brain regions and peripheral organs. WT mice housed under constant dark conditions for seven days were sacrificed at CT04, 08, 12, 16, 20, and 24 h for measurements of the indicated brain and peripheral tissues. Expression profiles of the circadian clock genes (Per2 and Bmal1 mRNAs), miR-24-3p, and miR-25-3p in the a SCN and b hippocampal brain tissues were obtained by real-time qPCR with the relative quantification method. Expression levels of c miR-24-3p and d miR-25-3p in various brain and peripheral tissues were examined. CT12 tissue samples were used and normalized to the SCN miR-24-3p or miR-25-3p level. Data for the Per2 and Bmal1 mRNAs were normalized by the TATA-box binding protein (Tbp) housekeeping gene, while the miR-24-3p and miR-25-3p data were normalized by small nucleolar RNA, C/D Box 95 (SnoRD95). Error bars represent the means ± SE of A.U. for each time point measured in three independent measurements. Significance was assessed by one-way ANOVA, *p < 0.05 compared to the control group.

Article Snippet: Immunoblot analyses were carried out with rabbit anti-PER2 polyclonal antibody (Santa Cruz Biotechnology, USA) and anti-beta-ACTIN-HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Constant Light Dysregulates Cochlear Circadian Clock and Exacerbates Noise-Induced Hearing Loss

doi: 10.3390/ijms21207535

Figure Lengend Snippet: p -value of F test to detect the circadian rhythmicity of mRNA transcripts of circadian clock genes in the cochlea by CircWave software.

Article Snippet: The sections were then immunostained with primary rabbit anti-PER2 IgG1 polyclonal antibody at 1:100 (PER21-A, Alpha Diagnostic, San Antonio, TX, USA), and incubated at 4 °C overnight, which was co-labeled with donkey anti-mouse AlexaFluor 488-conjugated secondary antibody (Abcam) 1:1000 at 37 °C for 1 h in the dark.

Techniques: Software

Journal: Nature Communications

Article Title: Hippocampal clock regulates memory retrieval via Dopamine and PKA-induced GluA1 phosphorylation

doi: 10.1038/s41467-019-13554-y

Figure Lengend Snippet: a dnBMAL1 mRNA expression in dnBMAL1 mice. Dox-dependent dnBMAL1 mRNA expression in hippocampus (RT-PCR, left). dnBMAL1 mRNA expression in hippocampus (HPC, middle) but not SCN (right) (in situ hybridization). AVP mRNA expression as a marker of SCN. DAPI (nuclear stain, blue), dnBMAL1 (green), AVP (red). Scale bar, 200 μm (HPC) and 100 μm (SCN). b , c PER2 and expression levels (BMAL1 target genes) are reduced in hippocampal CA1 ( b ) but not in SCN ( c ), in dnBMAL1 mice at both ZT4 and 10. The graph represents fold changes compared to the expression levels in WT at ZT4. d dnBMAL1 blocks the CLOCK binding to Dbp promoter in the hippocampus of dnBMAL1 mice at ZT10. Anti-CLOCK antibody, but not anti-IgG, precipitated DBP promoter although DNA regions not containing E-box ( clock gene exon 6) are comparably precipitated by anti-CLOCK antibody and anti-IgG. e Normal circadian locomotor rhythm in dnBMAL1 mice. Mice were housed in a 12 h light:12 h dark (LD) cycle then in constant darkness (DD). (Left) Representative activity records are double-plotted with each horizontal line representing 48 h. Circadian period (Middle) and daily locomotor activity (Right) under DD. All values are mean ± SEM. Individual data points are displayed as dots. * p < 0.05 as determined by two-way ( b , c ) or one-way ( d , e ) ANOVA with post hoc test. The results of the statistical analyses are presented in Supplementary Table . Source data are provided as a source data file.

Article Snippet: Sections were incubated overnight with the primary antibody, including rabbit polyclonal anti-c-fos (ABE457, 1:1000; Merck Millipore), rabbit polyclonal anti-PER2 (PER21-A, 1:1000; Alpha Diagnostic International, San Antonio, TX, USA), rabbit polyclonal anti-DBP (LS-B3479, 1:1000; Lifespan Bioscience, Seattle, WA, USA), or mouse anti-NeuN (MAB377, 1:500; Merck Millipore).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Marker, Staining, Binding Assay, Activity Assay

Journal: eLife

Article Title: Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock

doi: 10.7554/eLife.50925

Figure Lengend Snippet:

Article Snippet: Antibody , anti-PER2-1 (Rabbit polyclonal) , Alpha Diagnostic Lot # 869900A1.2-L , Cat. #: PER21-A RRID: AB_2236875 : , 1:200 (IF) 1:50 (IP) 1:500/1:1000 (WB).

Techniques: Mutagenesis, CRISPR, Transfection, Construct, Diagnostic Assay, Recombinant, Cloning, Protease Inhibitor, Software, Fluorescence