Journal: Breast Cancer : Basic and Clinical Research

Article Title: Oscillation of Clock and Clock Controlled Genes Induced by Serum Shock in Human Breast Epithelial and Breast Cancer Cells: Regulation by Melatonin

doi: 10.4137/BCBCR.S9673

Figure Lengend Snippet: Primers of clock and clock controlled genes for qPCR.

Article Snippet: Per2 shRNA and lentiviral and control particles were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques:

Journal: Breast Cancer : Basic and Clinical Research

Article Title: Oscillation of Clock and Clock Controlled Genes Induced by Serum Shock in Human Breast Epithelial and Breast Cancer Cells: Regulation by Melatonin

doi: 10.4137/BCBCR.S9673

Figure Lengend Snippet: Per2 knockdown in MCF-10A cells alters clock gene and CCG expression. MCF-10A Per2 knockdown or control cell lines were generated by infecting MCF-10A cells with Per2 shRNA or control lentiviral particles (Santa Cruz Biotechnology) according to the vendor’s protocol. ( A ) Stably infected cell lines were tested by Western blotting to determine the level of Per2 knockdown, with GAPDH expression used as a measure of protein loading. ( B ) Control and Per2 knockdown MCF-10A cells were plated as described in Materials and Methods and cell proliferation was determined by counting the number number of cells on a heamoctyometer after 4 days. ( C ) qPCR expression of clock and CCGs was determined as control and Per2 knockdown MCF-10A cells were harvested, mRNA extracted, and the clock gene expression for Clock, Bmal1, Per1, Cry1, Cry2, Rev-erbα and clock controlled genes MT 1 , c-Myc, and Sirt1 were analyzed by qPCR. Notes: * P < 0.05 , n = 3.

Article Snippet: Per2 shRNA and lentiviral and control particles were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Expressing, Control, Generated, shRNA, Stable Transfection, Infection, Western Blot, Gene Expression

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: (A–C) C57BL/6 mice housed under intense light (IL; 10,000 lux, L:D 14:10 h) for 3,5, or 7 days were subjected to 60 min of in situ myocardial ischemia followed by 2 h reperfusion at ZT3 (9 a.m.) and compared with mice housed under standard room light (RL; 200 lux, L:D 14:10h,7 days) (mean ± SD; n = 6; ANOVA with Tukey’s multiple comparison test). (A) Infarct size measurements. (B) Parallel measurements of serum troponin-I by ELISA (mean ± SD; n = 6; ANOVA with Tukey’s multiple comparison test). (C) Representative images of infarcts. (D–F) Wheel running measurements during 7 days of RL or IL housing in C57BL/6J mice (L, light phase; D, dark phase; n = 6; Student’s t test). (D) Wheel running activity graphs. (E) Distance walked. (F) Circadian amplitude. (G) Cardiac PER2 luciferase activity indicating protein in mice after RL or IL for 7 days (mean ± SD; n = 4; all IL versus RL p < 0.05 via ANOVA with Tukey’s multiple comparison test). (H–J) Wheel running during 7 days of RL or IL housing in C57BL/6J and Per2 −/− mice (n = 5–6; ANOVA with Tukey’s multiple comparison test). (H) Distance walked. (I) Circadian amplitude. (J) Wheel running activity graphs. (K and L) Immunoblot and quantification for PER2 protein in seeing or enucleated (blind) C57BL/6J mice after 7 days of RL or IL at ZT3 (mean ± SD; n = 5; Student’s t test). (K) Immunoblot. (L) Protein quantification. (M) Troponin-I serum levels in seeing or blind C57BL/6J mice housed under RL conditions followed by 60 min ischemia and 2 h reperfusion at ZT3 or ZT15 (mean ± SD; n = 4; ANOVA with Tukey’s multiple comparison test). (N) Wheel running measurements during 7 days of RL or IL housing in blind C57BL/6J mice (mean ± SD; n = 4; Student’s t test). See also .

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: In Situ, Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase, Western Blot

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: (A) Infarct sizes in C57BL/6J mice that were housed under intense light (IL; 10,000 lux, L:D 14:10 h) for 7 days and subjected to 60 min of in situ myocardial ischemia followed by 2 h reperfusion at ZT3 or ZT15 (mean ± SD; n = 6; Student’s t test). (B–D) C57BL/6J mice exposed to voluntary wheel running for 1 versus 2 weeks. Shown are infarct sizes after 60 min of myocardial ischemia and 2 h reperfusion at ZT3 (B) or circadian amplitude (C) and distance walked measurements in relation to infarct sizes (D, mean ± SD; n = 6; Student’s t test). (E–H) Wheel running measurements during or infarct size studies after 2 weeks of wheel running at ZT3 in C57BL/6J or Per2 −/− mice (mean ± SD; n = 5; Student’s t test). (E) Distance walked. (F) Circadian amplitude. (G and H) Infarct size measurements (G) and one representative infarct size staining and one wheel running activity recording are shown (H). (I and J) Adenosine (I) or cAMP (J) levels in heart tissue from C57BL/6J or Per2 −/− mice at ZT3 after 7 days of room light (RL; 200 lux, L:D 14:10 h) or intense light (IL; 10,000 lux, L:D 14:10 h) housing (mean ± SD; n = 5; ANOVA with Tukey’s multiple comparison test). (K) Cardiac U- 13 C-glucose-1,6-bisphosphate levels at ZT3 from C57BL/6J mice that were housed under RL or IL for 7 days (mean ± SD; n = 4; Student’s t test). (L and M) Phosphofructokinase (PFK) activity in both heart tissue (L) and plasma samples (M) from C57BL/6J or Per2 −/− mice at ZT3 after 7 days of RL or IL housing (mean ± SD; n = 4–5; ANOVA with Tukey’s multiple comparison test). (N) HIF1A-hypoxia response element (HRE) binding was determined at ZT3, ZT9, ZT15, and ZT21 (mean ± SD; n = 5; *p < 0.05 for ZT21 versus ZT3 in RL- and IL-housed mice via Student’s t test). (O) C57BL/6J or Per2 −/− mice housed under IL for 7 days before 60 min myocardial ischemia and 2 h reperfusion at ZT3 (mean ± SD; n = 5; Student’s t test). (P) Representative infarct staining.

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: In Situ, Staining, Activity Assay, Comparison, Clinical Proteomics, Binding Assay

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: (A) Whole-genome array from C57BL/6J or Per2 −/− heart tissue after 7 days of intense light (IL; 10,000 lux, L:D 14:10 h) or standard room light (RL; 200 lux, L:D 14:10 h) housing at ZT3 (n = 3 per group, total of 12 arrays). Top light-regulated pathways are shown. (B) Validation of transcript levels of the top light and PER2-dependent gene (ANGPTL-4) identified by whole-genome array (mean ± SD; n = 4–5; Student’s t test). (C) Per2 mRNA transcript levels from endothelial cells isolated from endothelial-specific PER2-deficient ( Per2 loxP/loxP -VE-Cadherin-Cre) or control (VE-Cadherin-Cre) hearts (mean ± SD; n = 3; Student’s t test). (D and E) Infarct sizes (D) or serum troponin-I (E) in Per2 loxP/loxP -VE-Cadherin-Cre) or VE-Cadherin-Cre mice housed under RL or IL conditions for 7 days followed by 60 min of in situ myocardial ischemia and 2 h reperfusion at ZT3 (mean ± SD; n = 5; ANOVA with Tukey’s multiple comparison test). (F) Representative infarct staining. (G–I) Vascular leakage of Evans blue dye in C57BL/6J (G and H) or Per2 loxP/loxP -VE-Cadherin-Cre (I) after 60 min of in situ myocardial ischemia and 2 h reperfusion at ZT3 following 7 days of RL or IL housing (mean ± SD; n = 5; Student’s t test for G and ANOVA with Tukey’s multiple comparison test for I). (G) Vascular leakage quantification in C57BL/6J. (H) Representative Evans blue staining in C57BL/6J. (I) Per2loxP/loxP-VE-Cadherin-Cre. (J) Vascular leakage of Evans blue dye in Ador-a2b −/− after 60 min of in situ myocardial ischemia and 2 h reperfusion at ZT3 following 7 days of RL or IL housing (mean ± SD; n = 5; Student’s t test). (K) ChIP assay for HIF1A binding to the promoter region of Angptl4 in C57BL/6J following 7 days of RL or IL housing (mean ± SD; n = 3; Student’s t test). See also and .

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Biomarker Discovery, Isolation, Control, In Situ, Comparison, Staining, Binding Assay

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: HMEC-1 or stable lentiviral-mediated PER2KD and Scr control HMEC-1 were synchronized and exposed to 24 h of normoxia (Nx) or 1% hypoxia (Hx). In a subset of experiments, synchronized stable lentiviral-mediated HIF1AKD and Scr HMEC-1 were exposed to Nx or Hx. (A and B) Affinity purification-mass spectrometry-based proteomics screen for PER2 protein interactions in normoxic and hypoxic HMEC-1. (A) Number of PER2 proteins regulated. (B) Pathways analysis using Ingenuity. (C and D) Coimmunoprecipitation for PER2 in hypoxic or normoxic HMEC-1 against isocitrate dehydrogenase (IDH) 2, succinyl coenzyme A (CoA) ligase (SUCLG) 1, and aconitase (ACO) 2 (C), and vice versa (D). One representative blot of three is displayed. (E) Subcellular compartment analysis of PER2 during normoxia or hypoxia (C, cytoplasm; N, nucleus; M, mitochondria; compartment-specific loading controls: tubulin alpha 1a (TUBA1A) for cytoplasm, TATA-box binding protein (TBP) for nucleus, and voltage-dependent anion channel 1 (VDAC1) for mitochondria). (F) Translocation of PER2 into the mitochondria during hypoxia (scale bar, 20 μm). (G–I) TCA cycle enzyme activities of IDH (G), SUCLG (H), and ACO (I) from stable lentiviral-mediated PER2KD and Scr control HMEC-1 during hypoxia (mean ± SD; n = 3; Student’s t test). (J) Carbon dioxide evolution rate (CDER), as a surrogate for TCA cycle function, in PER2KD or Scr HMEC-1 measured by a mitochondrial stress test using a Seahorse XF24 FluxPak assay (mean ± SD; n = 5; Student’s t test). (K–M) SIRT3 transcript (K and L) or protein (M) levels from stable lentiviral-mediated PER2KD and Scr (K and M, upper panel) or stable lentiviral-mediated HIF1AKD and Scr (L and M, lower panel) control HMEC-1 (mean ± SD; n = 3; ANOVA with Tukey’s multiple comparison test). See also – .

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Control, Affinity Purification, Mass Spectrometry, Binding Assay, Translocation Assay, Comparison

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: (A–D) Oxygen consumption rates (OCRs) in PER2KD or Scr HMEC-1. Quantification of basal respiration, maximum achievable respiration, and ATP production are shown (mean ± SD; n = 5; Student’s t test). (A) Seahorse mitochondrial stress test. (B) Basal respiration. (C) Maximal respiration. (D) ATP production. (E) COX4.2 transcript levels in PER2KD or Scr HMEC-1 after 24 h of Nx or 1% Hx treatment (mean ± SD; n = 6; ANOVA with Tukey’s multiple comparison test). (F) Complex IV enzyme activity in Per2 −/− or C57BL/6 mouse hearts subjected to 45 min of ischemia (mean ± SD; n = 4; ANOVA with Tukey’s multiple comparison test). (G) Cardiac Cox42 mRNA levels at ZT3, ZT9, ZT15, and ZT21 in C57BL/6 mice after 7 days of room light (RL) or intense light (IL) housing (mean ± SD; n = 5; #p < 0.05 for ZT3 IL versus ZT3 in RL-housed mice via two-way ANOVA with Sidak’s multiple comparison test). (H) MitoTracker red CMXRos staining of PER2KD or Scr HMEC-1 at baseline. One representative image of five is shown (scale bar, 20 μm). (I) Quantification of the mitochondrial membrane potential probe JC-1 (mean ± SD; n = 6; ANOVA with Tukey’s multiple comparison test). (J–M) 13 C metabolites from supernatants of PER2KD or Scr HMEC-1 following 24 h of Nx or 1 % Hx treatment. Data are presented as the percentage of total metabolites present (mean ± SD; n = 3; ANOVA with Tukey’s multiple comparison test). (J) 13 C fructose-6-phosphate. (K) 13 C α-ketoglutarate. (L) 13 C 6-phosphogluconate. (M) 13 C palmitic acid. (N) Permeability assay in PER2KD or Scr HMEC-1 during 24 h of 1% hypoxia (mean ± SD; n = 5; two-way ANOVA with Tukey’s multiple comparison test). Note that permeability increases after prolonged hypoxia exposure of endothelial cells due to morphological changes. (O) CLDN1 (claudin-1) transcript levels in PER2KD or Scr HMEC-1 after 4 h of Nx or 1% Hx treatment (mean ± SD; n = 3; ANOVA with Tukey’s multiple comparison test). (P) Cardiac Cldn1 mRNA was determined at ZT3, ZT9, ZT15, and ZT21 in C57BL/6 mice after 7 days of RL or IL treatment (mean ± SD; n = 5; #p < 0.05 for ZT3 IL versus ZT3 in RL-housed mice via two-way ANOVA with Sidak’s multiple comparison test). See also and .

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Comparison, Activity Assay, Staining, Membrane, Permeability

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: (A) Study design and verification of melanopsin overexpression by immunoblot. pCMV6 is the empty vector control, and OPN4-pCMV6 is the plasmid containing the gene encoding melanopsin (n = 3). (B–H) cAMP (B), pCREB levels (C), PER2 transcript (D) seahorse glycolytic stress test (E), glycolytic capacity (F), seahorse mitochondrial stress test (G), and maximum achievable respiration (H) after light-sensing cells were exposed to intense light (mean ± SD; n = 6–10; Student’s t test). (I) Schematic model.

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Control

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: (A) Protocol for intense light exposure experiments in healthy human volunteers. 20 healthy volunteers (11 female and 6 male, age range between 21 and 44 years) were exposed to intense light (10,000 lux) from 8:30–9:00 a.m. on 5 consecutive days. (B and C) PER2 protein levels from buccal tissue (B) or plasma samples (C) at 9 a.m. during 5 days of intense light exposure assessed by immunoblot or ELISA, respectively (mean ± SD; n = 6; ANOVA with Tukey’s multiple comparison test). (D) Effect of room light versus intense light on human plasma melatonin levels (mean ± SD; n = 3–6; ANOVA with Tukey’s multiple comparison test). (E) Longitudinal monitoring of human plasma melatonin levels during 5 days of intense light exposure at 9 a.m. (mean ± SD; n = 3–6; ANOVA with Tukey’s multiple comparison test). (F) Human plasma phosphofructokinase (PFK) activity during 5 days of intense light exposure at 9 a.m. (mean ± SD; n = 3–6; ANOVA with Tukey’s multiple comparison test). (G) Human plasma PFK activity after 5 days of intense light exposure at 9 p.m. (mean ± SD; n = 3; Student’s t test). (H) Human plasma triglyceride levels during 5 days of intense light exposure at 9 a.m. (mean ± SD; n = 8; ANOVA with Tukey’s multiple comparison test). (I–K) Targeted metabolomics using mass spectrometry on human plasma samples from healthy volunteers exposed to intense light therapy for 5 days. (I) Pathway analysis. Key metabolites of glycolysis (pyruvate) or the TCA cycle (succinate, K) are shown for day 3 and day 5 of intense light therapy (mean ± SD; n = 3; ANOVA with Tukey’s multiple comparison test). (L–P) Actigraphy data using a validated accelerometer (Actiwatch 2). Shown are the wake after sleep onset (WASO) episodes (L), sleep efficiency (M), day activity (N), circadian amplitude (O) (mean ± SD; n = 6; Student’s t test), and one representative actigraphy recording from one healthy volunteer (P) before and during intense light therapy (synchronized sleep phases [turquoise bar] during intense light exposure [red square]). C, control subjects before light exposure; IL, intense light. See also and .

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, Activity Assay, Mass Spectrometry, Control

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Binding Assay, Virus, Variant Assay, Recombinant, Protein Extraction, Extraction, Isolation, Cell Culture, Membrane, Transfection, Enzyme-linked Immunosorbent Assay, Reporter Assay, Activity Assay, Colorimetric Assay, Transcription Factor Assay, Bicinchoninic Acid Protein Assay, Chromatin Immunoprecipitation, Qubit Protein Assay, SYBR Green Assay, LDH Cytotoxicity Assay, Microarray, Luciferase, Generated, shRNA, Sequencing, Control, Software

Journal: Cell

Article Title: SIRT1 regulates circadian clock gene expression through PER2 deacetylation.

doi: 10.1016/j.cell.2008.06.050

Figure Lengend Snippet: Figure 5. SIRT1 Binds to CLOCK, BMAL1, and PER2 in a Circadian Manner SIRT1 was immunoprecipitated from mouse liver nuclear extracts (A) and from NIH 3T3 cells (B). The immunoprecipitated proteins were analyzed by immuno- blotting. Rabbit yeast RAP1 antibody was used as a negative control. (C) Immunostaining of SIRT1 (red) and CLOCK (green) in NIH 3T3 cells was performed with rabbit SIRT1 and CLOCK antibodies. In blue: DAPI staining. In yellow: merge of SIRT1 and CLOCK staining. (D) Mice were sacrificed at 4 hr intervals, and liver nuclear extracts were analyzed by immunoblotting. (E) CLOCK was immunoprecipitated from mouse liver nuclear extracts, and the immunoprecipitated proteins were analyzed by immunoblotting. Rabbit yeast RAP1 antibody was used as a negative control. (F) SIRT1 was immunoprecipitated from mouse liver nuclear extracts, and the immunoprecipitated proteins were analyzed by immunoblotting. (G) NIH 3T3 cells were synchronized by a dexamethasone shock, and protein extracts were prepared at 6 hr intervals, starting 24 hr after the shock. SIRT1 was immunoprecipitated from NIH 3T3 cells, and the immunoprecipitated proteins were analyzed by immunoblotting.

Article Snippet: Antibodies used were rabbit CRY1, PER2, BMAL1, and CLOCK (kindly provided by S. Brown and J. Ripperger) and rabbit SIRT1 (Upstate), human-SIRT1 (Santa Cruz), pan acetylated lysine (Cell Signaling), TAP (OPEN BIOSYSTEMS), mouse V5 (Invitrogen), and U2AF65 (Sigma).

Techniques: Immunoprecipitation, Negative Control, Immunostaining, Staining, Western Blot

Journal: Cell

Article Title: SIRT1 regulates circadian clock gene expression through PER2 deacetylation.

doi: 10.1016/j.cell.2008.06.050

Figure Lengend Snippet: Figure 6. SIRT1 Deacetylates PER2 (A) Protein extracts from nonsynchronized WT and Sirt1 KO MEFs were subjected to immunoprecipitation with rabbit pan acetyl lysine antibody, and the immu- noprecipitated proteins were analyzed by immunoblotting. (B) Purified PER2-TAP was incubated in the absence or presence of recombinant SIRT1 and NAD+ for 3 hr at 30, and samples were analyzed by immunoblotting. (C) WT and Sirt1 KO MEFs were synchronized by a dexamethasone shock, and protein extracts were prepared at 4 hr intervals, starting 24 hr after the shock. Immunoprecipitation experiments were performed with rabbit pan acetyl lysine antibody, and the immunoprecipitated proteins were analyzed by immunoblotting. (D) Mice were sacrificed at 4 hr intervals and liver nuclear extracts were prepared. PER2 was immunoprecipitated, and the immunoprecipitated proteins were analyzed by immunoblotting. Rabbit yeast RAP1 antibody was used as a negative control.

Article Snippet: Antibodies used were rabbit CRY1, PER2, BMAL1, and CLOCK (kindly provided by S. Brown and J. Ripperger) and rabbit SIRT1 (Upstate), human-SIRT1 (Santa Cruz), pan acetylated lysine (Cell Signaling), TAP (OPEN BIOSYSTEMS), mouse V5 (Invitrogen), and U2AF65 (Sigma).

Techniques: Immunoprecipitation, Western Blot, Incubation, Recombinant, Negative Control

Journal: Cell

Article Title: SIRT1 regulates circadian clock gene expression through PER2 deacetylation.

doi: 10.1016/j.cell.2008.06.050

Figure Lengend Snippet: Figure 7. SIRT1-Dependent PER2 Deacetylation Determines PER2 Protein Stability (A) WT and Sirt1 KO MEFs were synchronized by a dexamethasone shock, and 24 hr after the shock cells were untreated or treated with cycloheximide. Cells were harvested 1, 2, 3, and 4 hr following the treatment, and protein extracts were analyzed by immunoblotting. (B) The graph illustrates the quantification of PER2 by densitometry of triplicate experiments (mean ± standard error). (C) NIH 3T3 cells were transfected with PER2-TAP expression vector either alone or together with HA-FLAG-human SIRT1 expression vector. Protein extracts were analyzed by immunoblotting. (D) PER2-TAP was purified from NIH 3T3 cells transfected with PER2-TAP expression vector either alone or together with HA-FLAG-human SIRT1 expression vector and analyzed by immunoblotting. (E) NIH 3T3 cells were transfected with the V5-PER2 expression vector either alone or together with the Sirt1 siRNA expression vector. Protein extracts were prepared, and immunoprecipitation experiments were performed with mouse V5 antibody. The immunoprecipitated proteins were analyzed by immunoblotting. (F) Purified PER2-TAP was incubated for 3 hr at 30 with protein extract obtained from WT or Sirt1 KO MEFs in the absence or presence of 100 mM NAD+ or 25 mM MG132, and samples were analyzed by immunoblotting. (G) Hypothetical model showing the possible role of SIRT1 in circadian oscillator function. BMAL1-CLOCK heterodimers bind and activate transcription of the Per, Cry, Rre-Erba, and Rorg genes. Once the PER and CRY proteins accumulate to a critical level, they form complexes with BMAL1-CLOCK and thereby repress their own transcription. In addition, there is an interconnecting feedback loop in which REV-ERBa represses and RORg activates Bmal1 transcription. SIRT1 binds CLOCK-BMAL1 complexes and promotes PER2 deacetylation and degradation.

Article Snippet: Antibodies used were rabbit CRY1, PER2, BMAL1, and CLOCK (kindly provided by S. Brown and J. Ripperger) and rabbit SIRT1 (Upstate), human-SIRT1 (Santa Cruz), pan acetylated lysine (Cell Signaling), TAP (OPEN BIOSYSTEMS), mouse V5 (Invitrogen), and U2AF65 (Sigma).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Incubation

Journal: Cancer Drug Resistance

Article Title: PER2 expression and cellular localization play a critical role in tumor aggressiveness and drug resistance in an in vitro model of hepatocellular carcinoma

doi: 10.20517/cdr.2024.193

Figure Lengend Snippet: (A) In silico analysis of PER2 mRNA expression in LIHC samples (purple boxplot) from TCGA cohort (obtained from RNAseq) compared with normal liver tissues (grey boxplot); (B) In silico analysis of PER2 mRNA expression in different stages of LIHC; (C) and (D) In silico analysis of PER2 mRNA expression in HCC cell lines from CCLE based on a dataset posted in 2022; (C) A comparison of PER2 mRNA expression between HCC and hepatoblastoma cell lines; and (D) a comparison of PER2 mRNA expression in HCC cell lines derived from human primary HCC, HCC cell lines with no clear origin, and HCC cell lines derived from human HCC metastasis; (E) Kaplan-Meier curve to assess the overall survival rate of patients with HCC depending on the PER2 gene expression; (F) SOR effect on cell line proliferation. Cell proliferation evaluated by DNA assay in PLC/PRF/5 cells: parental (black bars) and SorR (gray bars). The data presented are mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001 between groups; (G) PER2 mRNA expression in PLC/PRF/5 parental, PLC/PRF/5 EveR, PLC/PRF/5 SorR, PLC/PRF/5 PER2 KD, and PLC/PRF/5 PER2 KO relative to HPRT housekeeping gene. * P < 0.05; ** P < 0.01; *** P < 0.001; (H) PER2 protein expression in PLC/PRF/5 parental, PLC/PRF/5 PER2 KD, and PLC/PRF/5 PER2 KO with related densitometry. **** P < 0.0001. PER2: Period 2; LIHC: Liver Hepatocellular Carcinoma; CCLE: Cancer Cell Line Encyclopaedia; TCGA: The Cancer Genome Atlas; HCC: hepatocellular carcinoma; SOR: sorafenib; SEM: standard error of the mean; EveR: everolimus-resistant; SorR: sorafenib-resistant; KD: knockdown; KO: knockout; HPRT: Hypoxanthine Phosphoribosyltransferase 1.

Article Snippet: The cells were co-transfected with 2 μg of human PER2 KO plasmid (sc-401089-KO-2) and 2 μg of human PER2 HDR plasmid (sc-401089-HDR-2) using 10 μL of UltraCruz Transfection Reagent (sc-395739, Santa Cruz Biotechnology, Inc.) in a final volume of 150 μL Plasmid Transfection Medium (sc-108062, Santa Cruz Biotechnology, Inc.).

Techniques: In Silico, Expressing, Comparison, Derivative Assay, Gene Expression, Knockdown, Knock-Out

Journal: Cancer Drug Resistance

Article Title: PER2 expression and cellular localization play a critical role in tumor aggressiveness and drug resistance in an in vitro model of hepatocellular carcinoma

doi: 10.20517/cdr.2024.193

Figure Lengend Snippet: Evaluation of E-cadherin, vimentin and ZEB1 protein expression by immunofluorescent imaging in PLC/PRF/5 parental, PLC/PRF/5 PER2 KD, PLC/PRF/5 PER2 KO, PLC/PRF/5 EveR, and PLC/PRF/5 SorR cells. The data presented in the graphs are mean ± SEM of two independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001 vs. control or among the groups. ZEB1: Zinc finger E-box binding homeobox 1; PER2: Period 2; KD: knockdown; KO: knockout; EveR: everolimus-resistant; SorR: sorafenib-resistant.

Article Snippet: The cells were co-transfected with 2 μg of human PER2 KO plasmid (sc-401089-KO-2) and 2 μg of human PER2 HDR plasmid (sc-401089-HDR-2) using 10 μL of UltraCruz Transfection Reagent (sc-395739, Santa Cruz Biotechnology, Inc.) in a final volume of 150 μL Plasmid Transfection Medium (sc-108062, Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Imaging, Control, Binding Assay, Knockdown, Knock-Out

Journal: Cancer Drug Resistance

Article Title: PER2 expression and cellular localization play a critical role in tumor aggressiveness and drug resistance in an in vitro model of hepatocellular carcinoma

doi: 10.20517/cdr.2024.193

Figure Lengend Snippet: Cell proliferation evaluated by DNA assay in PLC/PRF/5 PER2 KD (A) and PLC/PRF/5 PER2 KO (B) with and without treatment with EVE (10 -9 M) and (SOR 5 × 10 -6 M). Cell migration evaluation by analysis of wound coverage in PLC/PRF/5 PER2 KD (C) and PLC/PRF/5 PER2 KO (D) and by scratch assay (E) with and without treatment with EVE (10 -9 M) and SOR (5 × 10 -6 M) compared to parental PLC/PRF/5. The data presented in the graphs are mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. control or among the groups. PER2: Period 2; KD: knockdown; KO: knockout; EVE: everolimus; SOR: sorafenib; SEM: standard error of the mean.

Article Snippet: The cells were co-transfected with 2 μg of human PER2 KO plasmid (sc-401089-KO-2) and 2 μg of human PER2 HDR plasmid (sc-401089-HDR-2) using 10 μL of UltraCruz Transfection Reagent (sc-395739, Santa Cruz Biotechnology, Inc.) in a final volume of 150 μL Plasmid Transfection Medium (sc-108062, Santa Cruz Biotechnology, Inc.).

Techniques: Migration, Wound Healing Assay, Control, Knockdown, Knock-Out

Journal: Cancer Drug Resistance

Article Title: PER2 expression and cellular localization play a critical role in tumor aggressiveness and drug resistance in an in vitro model of hepatocellular carcinoma

doi: 10.20517/cdr.2024.193

Figure Lengend Snippet: Images and statistical assessment of colony number (A, C, E, G and I) and size (B, D, F, H, and L) evaluated by colony formation assay in parental PLC/PRF/5, PLC/PRF/5 PER2 KO, PLC/PRF/5 EveR, and SorR with and without treatment with EVE (10 -9 M) and SOR (5 × 10 -6 M). The data presented in the graphs are mean ± SEM of two independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. control or among the groups. PER2: Period 2; KO: knockout; EveR: everolimus-resistant; SorR: sorafenib-resistant; EVE: everolimus; SOR: sorafenib; SEM:standard error of the mean.

Article Snippet: The cells were co-transfected with 2 μg of human PER2 KO plasmid (sc-401089-KO-2) and 2 μg of human PER2 HDR plasmid (sc-401089-HDR-2) using 10 μL of UltraCruz Transfection Reagent (sc-395739, Santa Cruz Biotechnology, Inc.) in a final volume of 150 μL Plasmid Transfection Medium (sc-108062, Santa Cruz Biotechnology, Inc.).

Techniques: Colony Assay, Control, Knock-Out

Journal: Cancer Drug Resistance

Article Title: PER2 expression and cellular localization play a critical role in tumor aggressiveness and drug resistance in an in vitro model of hepatocellular carcinoma

doi: 10.20517/cdr.2024.193

Figure Lengend Snippet: Immunofluorescent evaluation of (A) PER2 protein expression in PLC/PRF/5 parental, PLC/PRF/5 EveR, and PLC/PRF/5 SorR, and (B) colocalization of PER2 and CK1ε in PLC/PRF/5 parental, PLC/PRF/5 EveR, and PLC/PRF/5 SorR. PER2: Period 2; CK1ε: casein kinase 1ε; EveR: everolimus-resistant; SorR: sorafenib-resistant.

Article Snippet: The cells were co-transfected with 2 μg of human PER2 KO plasmid (sc-401089-KO-2) and 2 μg of human PER2 HDR plasmid (sc-401089-HDR-2) using 10 μL of UltraCruz Transfection Reagent (sc-395739, Santa Cruz Biotechnology, Inc.) in a final volume of 150 μL Plasmid Transfection Medium (sc-108062, Santa Cruz Biotechnology, Inc.).

Techniques: Expressing

Journal: Cancer Drug Resistance

Article Title: PER2 expression and cellular localization play a critical role in tumor aggressiveness and drug resistance in an in vitro model of hepatocellular carcinoma

doi: 10.20517/cdr.2024.193

Figure Lengend Snippet: (A) PER2 and p53 protein expression in PLC/PRF/5 parental, PLC/PRF/5 PER2 KD, PLC/PRF/5 PER2 KO, PLC/PRF/5 EveR, and PLC/PRF/5 SorR cells. White arrows indicate the co-expression of PER2 and p53 proteins. The data presented in the graphs are mean ± SEM of two independent experiments. * P < 0.05, **** P < 0.0001; (B) Protein-Protein Interaction Networks Functional Enrichment Analysis using STRING software to analyze and visualize the network connection among PER2, MDM2, p53, p21, and c-MYC; (C) Western blot analysis of PER2, MDM2, p53, p21, and c-MYC parental PLC/PRF/5, PLC/PRF/5 PER2 KD, PLC/PRF/5 PER2 KO, PLC/PRF/5 EveR, and PLC/PRF/5 SorR cells. PER2: Period 2; KD: knockdown; KO: knockout; EveR: everolimus-resistant; SorR: sorafenib-resistant; SEM: standard error of the mean; MDM2: mouse double minute 2 homolog; c-MYC: cellular myelocytomatosis oncogene.

Article Snippet: The cells were co-transfected with 2 μg of human PER2 KO plasmid (sc-401089-KO-2) and 2 μg of human PER2 HDR plasmid (sc-401089-HDR-2) using 10 μL of UltraCruz Transfection Reagent (sc-395739, Santa Cruz Biotechnology, Inc.) in a final volume of 150 μL Plasmid Transfection Medium (sc-108062, Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Functional Assay, Software, Western Blot, Knockdown, Knock-Out

Journal: Frontiers in Endocrinology

Article Title: Identification of angiotensin II-responsive circadian clock gene expression in adrenal zona glomerulosa cells and human adrenocortical H295R cells

doi: 10.3389/fendo.2025.1525844

Figure Lengend Snippet: Circadian oscillations exhibited by adrenal ZG cells. (A) Time-lapse images of circadian PER2::LUC bioluminescence obtained from Per2 Luc / + and Per2 Luc / + : Clock Δ19 / Δ19 mouse adrenal slices. Intensity was traced from a region of the adrenal cortex outer layer containing ZG cells (white boxes) over 80 h. Bioluminescence intensity is represented in pseudo-color scale. Scale bars, 200 μm. (B) Representative long-term bioluminescence recording of the ZG in Per2 Luc / + adrenal from five independent experiments. Data were detrended by 24-h moving average. The maximum bioluminescence was set to 100%.

Article Snippet: A luciferase reporter (Luc2P) driven by a mouse or human Per2 promoter sequence (positions –1670 to +53 for mouse; –1840 to +108 for human) was inserted between the inverted terminal repeat (ITR) sequences in pAAV-MCS2 plasmid (Addgene, Plasmid #46954) to obtain pAAV- mPer2 - Luc2P or pAAV- hPER2 - Luc2P .

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Identification of angiotensin II-responsive circadian clock gene expression in adrenal zona glomerulosa cells and human adrenocortical H295R cells

doi: 10.3389/fendo.2025.1525844

Figure Lengend Snippet: Circadian oscillations displayed by dispersed cell culture of ZG cells and human H295R adrenocortical cells. (A) Representative Per2 - dluc bioluminescence recording of dissociated rat primary ZG cells. ZG cells were entrained by 24-h interval medium changes and then released into constant conditions with no medium change. The data were detrended by 24-h moving average and plotted from the last medium change. Immunocytochemistry for CYP11B2 confirmed the isolation of ZG cells from rat adrenal glands. Scale bar, 10 μm. Periods were determined from three measurements. (B) Circadian oscillation of clock genes in human adrenocortical H295R cells. Cells pre-synchronized to 37°C/33°C temperature cycles were harvested under constant 37°C temperature conditions. The data were normalized to RPLP0 . The peak mRNA values of each gene were set to 1. n = 3 biological replicates per timepoint. Values are means ± SEM.

Article Snippet: A luciferase reporter (Luc2P) driven by a mouse or human Per2 promoter sequence (positions –1670 to +53 for mouse; –1840 to +108 for human) was inserted between the inverted terminal repeat (ITR) sequences in pAAV-MCS2 plasmid (Addgene, Plasmid #46954) to obtain pAAV- mPer2 - Luc2P or pAAV- hPER2 - Luc2P .

Techniques: Cell Culture, Immunocytochemistry, Isolation

Journal: Frontiers in Endocrinology

Article Title: Identification of angiotensin II-responsive circadian clock gene expression in adrenal zona glomerulosa cells and human adrenocortical H295R cells

doi: 10.3389/fendo.2025.1525844

Figure Lengend Snippet: Ang II elicits phase-dependent phase shifts of the adrenal ZG clock. (A) Autoradio-graphs showing expression of Agtr1a , Agtr1b , and Cyp11b2 in the mouse adrenal section. (B) Phase shifts of PER2::LUC rhythm after Ang II treatment in adrenal slices. Arrows indicate the time of Ang II or vehicle administration. Luminescence of ZG was traced. The peak and trough values were adjusted to 100 and 0, respectively. (C) Quantification of the magnitude of phase shifts shown in (B) . Phase delays and advances are plotted as negative and positive values, respectively. n = 3–4 slices per condition. Values are means ± SEM. * P < 0.05, ** P < 0.01, unpaired two-sided Student’s t test.

Article Snippet: A luciferase reporter (Luc2P) driven by a mouse or human Per2 promoter sequence (positions –1670 to +53 for mouse; –1840 to +108 for human) was inserted between the inverted terminal repeat (ITR) sequences in pAAV-MCS2 plasmid (Addgene, Plasmid #46954) to obtain pAAV- mPer2 - Luc2P or pAAV- hPER2 - Luc2P .

Techniques: Expressing

Journal: Frontiers in Endocrinology

Article Title: Identification of angiotensin II-responsive circadian clock gene expression in adrenal zona glomerulosa cells and human adrenocortical H295R cells

doi: 10.3389/fendo.2025.1525844

Figure Lengend Snippet: Ang II resets circadian rhythms in H295R cells. (A) Circadian expression profiles of representative core clock genes and clock-controlled genes in H295R cells. Cells were treated with Ang II at Time 0 and were harvested at 0, 2, and every 4 hour over a 68-h period. Values are means ± SEM from n = 3 biological replicates per timepoint. Results of cosinor analysis of the clock gene expression profiles are available in Supplementary Table S1 . (B) A cartoon for viral infection to H295R cells and luminescence traces of cells harboring a luciferase reporter under the control of human or mouse Per2 promoter. Arrows indicate the time of Ang II or vehicle administration. Bar graphs illustrate the amplitude of the first surge and second cycle of luminescence following Ang II administration. Data are the means ± SD from n = 4 biologically independent experiments. (C) Single-cell bioluminescence tracing in H295R cells expressing mouse Per2-Luc reporter. Heat maps show individual cellular luminescence, where magenta corresponds to peak bioluminescence and green to trough. Rayleigh plot shows phase distribution of acrophase of individual cells before and after Ang II treatment. n = 37 cells. Statistics in (B) , unpaired two-sided Student’s t test; in (C) , Rayleigh’s uniformity test. **** P < 0.0001.

Article Snippet: A luciferase reporter (Luc2P) driven by a mouse or human Per2 promoter sequence (positions –1670 to +53 for mouse; –1840 to +108 for human) was inserted between the inverted terminal repeat (ITR) sequences in pAAV-MCS2 plasmid (Addgene, Plasmid #46954) to obtain pAAV- mPer2 - Luc2P or pAAV- hPER2 - Luc2P .

Techniques: Expressing, Gene Expression, Infection, Luciferase, Control

Journal: Frontiers in Endocrinology

Article Title: Identification of angiotensin II-responsive circadian clock gene expression in adrenal zona glomerulosa cells and human adrenocortical H295R cells

doi: 10.3389/fendo.2025.1525844

Figure Lengend Snippet: Ang II-induced clock resetting through a mechanism involving upregulation of PER1 and E4BP4 . (A, B) Effects of increasing concentrations of CV on Ang II-induced circadian luminescence in H295R cells. Cells were transduced with a luciferase reporter under the control of mouse Per2 promoter. Arrows indicate the time of Ang II/CV treatment. Bar graphs in (A) illustrate the amplitude of the first surge and second cycle of luminescence following Ang II administration with different doses of CV. Plots in (B) show the first trough and second peak phase of the luminescence rhythm following Ang II treatment. n = 3 biological replicates for each CV concentration. Traces in (A) are expressed as means ± SD. (C, D) Effects of CV on Ang II-induced PER1 and E4BP4 mRNA expression in H295R cells. n = 3 biological replicates per datapoint. (E) Sequence alignment of CRE located in the promoter of PER1 . The sequences of CRE are compared among mammalian species along with the consensus CRE motif (5′-TGACGTCA-3′). Genomic positions, relative to the transcription start site (+1), are indicated along with the conservation scores obtained from the UCSC Genome Browser ( https://genome.ucsc.edu/ ). (F) Relative reporter activities of CRE×3-Luc2CP and its mutant (Mut, T C AC A T A A). Cells received vehicle, Ang II, or Ang II plus CV (1 µM). n = 3 biological replicates. (G) Dose-dependent effects of CV on CRE reporter activity after Ang II stimulation. n = 3 biological replicates for each CV concentration. (H) Sequence alignment of two candidate NRBEs, both located in the intron 1 of E4BP4 . The sequences of NBRE-like ( left ) and NBRE-consensus ( right ) are aligned among species with the consensus NGFI-B binding motif (5′-TGACCTTT-3′ or 5′-AAAGGTCA-3′). E4BP4 consists of two exons. (I) Immunoblots showing the protein expression profiles of NGFI-B and E4BP4 after Ang II stimulation. Bar graph shows protein quantification data ( n = 3 biological replicates). β-Actin serves as a loading control. Asterisks indicate nonspecific bands. Uncropped blots are available in Supplementary Figure S5 . (J) Relative reporter activities of NBRE-like×3-Luc2CP, NBRE-consensus×3-Luc2CP, and its mutant (Mut, TGA AT T C T). n = 4 biological replicates. (K) Dose-dependent effect of CV on NBRE-consensus reporter activity after Ang II stimulation. n = 3 biological replicates for each CV concentration. Statistics in (A, B, G, K) were one-way ANOVA followed by Tukey’s multiple comparisons test; in (F) and (J) , two-way ANOVA followed by Sidak’s multiple comparison test. Values are means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: A luciferase reporter (Luc2P) driven by a mouse or human Per2 promoter sequence (positions –1670 to +53 for mouse; –1840 to +108 for human) was inserted between the inverted terminal repeat (ITR) sequences in pAAV-MCS2 plasmid (Addgene, Plasmid #46954) to obtain pAAV- mPer2 - Luc2P or pAAV- hPER2 - Luc2P .

Techniques: Transduction, Luciferase, Control, Concentration Assay, Expressing, Sequencing, Mutagenesis, Activity Assay, Binding Assay, Western Blot, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

doi: 10.1073/pnas.1607984113

Figure Lengend Snippet: Distinct p53 and Per2 phases characterize their time-dependent subcellular distribution. (A, Upper) Extracts from circadian synchronized HCT116 cells were analyzed for the expression of endogenous Per2, p53, Mdm2, and Cry1 by immunoblotting. Asterisks indicate nonspecific bands. (Lower) Bands were quantified using ImageJ, and values were normalized to tubulin levels. Data are in arbitrary units (a.u.). (B) Samples from A were processed for qRT-PCR as described in SI Materials and Methods. Data for PER2 and TP53 gene expression are shown as the mean ± SEM from three independent experiments performed in triplicate. Bar graphs are fold increase normalized to the level of expression at t = 0 h. Inset indicates level of Per2 expression within the first 4 h. (C) HCT116 extracts from various times postcircadian synchronization (t = 0–36 h) were immunoprecipitated using α-Per2. Bound proteins were identified by immunoblotting and quantified as described in A. Relative amounts of Per2 and total Per2:p53 complex were plotted in arbitrary units relative to t = 0. (D) Nuclear and cytoplasmic fractions from circadian synchronized HCT116 cells were enriched for endogenous Per2 and p53 by immunoprecipitation and blotted using α-Per2 or -p53 antibodies, respectively. Tubulin and lamin A/C were used as controls. Bands were quantified and plotted as in A. In A, C, and D, immunoblot data were originated from a single experiment that was repeated three times with similar results. Error bars represent mean ± SEM.

Article Snippet: The human colorectal carcinoma-116 (HCT116) ( TP53 +/+ and PER2 +/+ ) cell line was purchased from the American Type Culture Collection (ATCC) and maintained according to manufacturer’s recommendations.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Gene Expression, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

doi: 10.1073/pnas.1607984113

Figure Lengend Snippet: Expression of clock genes in circadian synchronized cells. (A) HCT116 cells were circadian synchronized by serum shock [50% serum for 2 h (t = −2–0)] after which they were maintained in serum-free media throughout the time course analyzed. Samples were collected at various times (t = 0–36 h) and processed for qRT-PCR analysis as described in Materials and Methods. Data for NR1D1 (encodes for Rev-erbα), circadian locomotor output cycles kaput (CLOCK), and CRY1 gene expression are shown as the mean ± SEM from three independent experiments performed in triplicate. The expression of TBP was monitored as a control of a noncircadian regulated gene. Bar graphs are fold increase normalized to the level of expression at t = 0 h. Primer sequences are shown in Table S4. (B) HeLa cells were circadian synchronized by serum shock and samples collected as described in the legend of Fig. 1A. Total extracts (5–10 μg) were analyzed for the expression of endogenous Per2 and p53 by immunoblotting using specific antibodies. Bands were quantified using Image Lab software/Gel Doc XR+ system, and values were normalized to tubulin levels. Data [in arbitrary units (a.u.)] are represented as the mean ± SEM. (C) HCT116 cells were circadian synchronized with dexamethasone [100 ng/mL for 2 h (t = −2–0) (42)], after which they were maintained in serum-free media throughout the time course analyzed. Samples were collected at various times and total extracts (10–100 μg) analyzed for expression of endogenous Per2, p53, and tubulin by immunoblotting using specific antibodies. (D) HCT116 extracts (1 mg) collected at various times postcircadian synchronization (t = 12–28 h) were immunoprecipitated using either α-Per2 antibody or rabbit IgG and protein A-beads, and bound proteins were identified by immunoblotting as indicated. For input, total cell extracts (50–100 μg) were loaded. Tubulin was used as a loading control. (E) HCT116 extracts from Fig. 1C were immunoprecipitated using α-Per2 antibody and protein A-beads, and Cry1 was identified by immunoblotting as indicated.

Article Snippet: The human colorectal carcinoma-116 (HCT116) ( TP53 +/+ and PER2 +/+ ) cell line was purchased from the American Type Culture Collection (ATCC) and maintained according to manufacturer’s recommendations.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control, Western Blot, Software, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

doi: 10.1073/pnas.1607984113

Figure Lengend Snippet: The Per2 and p53 phase relationship remains unaltered despite inhibition of CK1δ/ε. HCT116 cells were circadian synchronized by serum shock after which they were collected (t = 0) or maintained in serum-free media with DMSO (control, 0.2%) or PF670462 (1 µM) throughout the time course analyzed (t = 6–24 h). Cells were collected at the indicated times and lysates were subjected to subcellular fractionation as indicated in Materials and Methods. Levels of endogenous proteins were detected in total (1.2 × 105 cells), cytoplasmic (0.63 × 105 cells), and nuclear (3.15 × 105 cells) extracts by immunoblotting using α-Per2, -p53, -Mdm2, and -CK1ε antibodies. Tubulin and lamin A/C were used as a loading and purity control for the cytoplasm and nuclear fractions, respectively. Immunoblot data originated from a single experiment that was repeated three times with similar results. (B) Protein bands from A were quantified using Image Lab software/Gel Doc XR+ system, and values were normalized to tubulin or lamin A/C levels (loading controls), and data [in arbitrary units (a.u.)] were represented as the mean ± SEM from three independent experiments. Plots were grouped by the protein’s class and expression in its various compartment in cells treated with either DMSO (Left) or PF670462 (Right) treatment. (C) Data from B was replotted to better show the conservation of the phase relationship between Per2 and p53 in the indicated compartments.

Article Snippet: The human colorectal carcinoma-116 (HCT116) ( TP53 +/+ and PER2 +/+ ) cell line was purchased from the American Type Culture Collection (ATCC) and maintained according to manufacturer’s recommendations.

Techniques: Inhibition, Control, Fractionation, Western Blot, Software, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

doi: 10.1073/pnas.1607984113

Figure Lengend Snippet: The half-life of p53, but not Per2, differs in nuclear and cytoplasmic compartments. (A) Samples were obtained from HCT116 cells in the absence (t = 0) or presence of CHX as indicated in SI Materials and Methods section. Extracts from cytoplasmic and nuclear fractions were analyzed by immunoblotting using α-Per2 and -p53 antibodies. Tubulin and lamin A/C were used as controls. (B) Protein levels were quantified as described in SI Materials and Methods section, values were normalized to tubulin or lamin A/C levels, and p53 and Per2 half-life (t1/2) calculated using Excel. Error bars represent mean ± SEM of three independent experiments.

Article Snippet: The human colorectal carcinoma-116 (HCT116) ( TP53 +/+ and PER2 +/+ ) cell line was purchased from the American Type Culture Collection (ATCC) and maintained according to manufacturer’s recommendations.

Techniques: Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

doi: 10.1073/pnas.1607984113

Figure Lengend Snippet: Half-life of p53 and Per2 in total cell extracts. (Left) Samples (t = 1–4 h) were obtained from HCT116 cells maintained in complete medium in the absence (t = 0) or presence of cycloheximide (CHX) (100 μg/mL). Total extracts were analyzed by immunoblotting using α-Per2 and -p53 antibodies. Tubulin was used as a loading control. The immunoblot originated from a single experiment that was repeated three times with similar results. (Right) Protein levels were quantified using Image Lab software/Gel Doc XR+ system, and values were normalized to tubulin levels. Graphs indicate the level of remaining protein in arbitrary units (a.u.) plotted as a function of time. Curves were fitted and p53 and Per2 half-life (t1/2) calculated using Excel software. Error bars represent mean ± SEM of three independent experiments.

Article Snippet: The human colorectal carcinoma-116 (HCT116) ( TP53 +/+ and PER2 +/+ ) cell line was purchased from the American Type Culture Collection (ATCC) and maintained according to manufacturer’s recommendations.

Techniques: Western Blot, Control, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

doi: 10.1073/pnas.1607984113

Figure Lengend Snippet: Per2 enhances p53 shuttling to the nucleus. (A) HCT116 cells were transfected with either myc-Per2 (0.25 or 0.5 μg) or empty vector (0) for 24 h before the addition of MG132 and/or leptomycin B (LMB). Lysates were subjected to subcellular fractionation, and total extract (Left), cytoplasmic (Middle), and nuclear (Right) fractions were analyzed for the presence of Per2, myc-Per2, and endogenous p53 by immunoblotting. Tubulin and lamin A/C were used as controls. (B) Endogenous p53 was quantified using Image Lab software/Gel Doc XR+ system and values normalized to tubulin or lamin A/C levels. (C) HCT116 cells were transfected with either scrambled siRNA (mock) or Per2 siRNA (siRNA) for 48 h before the addition of MG132 and/or LMB. Endogenous proteins were detected as indicated in A and quantified as in B. In B and D, data are represented as fold increase of p53 (in arbitrary units) compared with similar treatment in either vector-transfected or mock samples, respectively. Values are the mean ± SEM from three independent experiments. Statistical significance was determined by t test. *P ≤ 0.05; **P ≤ 0.005, ***P < 0.001.

Article Snippet: The human colorectal carcinoma-116 (HCT116) ( TP53 +/+ and PER2 +/+ ) cell line was purchased from the American Type Culture Collection (ATCC) and maintained according to manufacturer’s recommendations.

Techniques: Transfection, Plasmid Preparation, Fractionation, Western Blot, Software