Journal: Cell cycle (Georgetown, Tex.)

Article Title: Effect of circadian clock mutations on DNA damage response in mammalian cells.

doi: 10.4161/cc.21771

Figure Lengend Snippet: Figure 5. Mouse cells deficient in clock genes are not sensitive to mitomycin C. Cells were plated at a low density, allowed to attach and then treated with mitomy- cin C (MMC); after colony formation, cells were stained with Giemsa, and percent survival was determined as described in the text. Each data point represents the average of at least three independent experiments and bars signify the standard deviation. (A) Mouse embryonic fibroblasts were derived from mice with the following genotypes: wild-type (squares), Clock-/- (circles) and Bmal1-/- (triangles). (B) Mouse skin fibroblasts were derived from mice with the following genotypes: wild-type (squares), Cry1-/-Cry2-/- (circles) and Per1-/-Per2-/- (triangles).

Article Snippet: For siRNA transfections, exponentially growing cells were transfected either with human Per1, Per2, mouse Per1, Per2, Cry1, Cry2, Clock and Bmal1, and Cyclophilin B siRNAs (Santa Cruz) or non-target siRNA (Dharmacon) using the Lipofectamine RNAiMax (Invitrogen) transfection reagent for 48 h according to the manufacturer’s directions before UV or IR irradiation. pCDNA3 or V5- tagged mouse Per1 genes were transfected using the Lipofectamine 2000 (Invitrogen) transfection reagent for 48 h according to the manufacturer’s directions as described previously.40 Clonogenic UV survival assays.

Techniques: Staining, Standard Deviation, Derivative Assay

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: 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: Scientific Reports

Article Title: Circadian rhythms in pediatric high-grade gliomas may contribute to treatment efficacy

doi: 10.1038/s41598-025-17461-9

Figure Lengend Snippet: Circadian gene expression varies between pHGGs and pLGGs. ( A ) Interleaved scatter plots represent bulk RNA sequencing (rsem FPKM) analysis comparing circadian gene expression across pediatric low-grade glioma (pLGG, n = 426, blue) and pediatric high-grade glioma (pHGG, n = 211, red) samples from the Pediatric Brain Tumor Atlas (PBTA, Provisional). Data are expressed as mean ± SEM, and expression values are log₂-transformed FPKM counts. Statistical analysis was performed using a two-way ANOVA with Benjamini and Hochberg false discovery rate (FDR) to determine differences in gene expression between pHGGs and pLGGs. Each dot represents an individual tumor sample. Asterisks indicate statistically significant differences (* P < 0.05), while “NS” denotes non-significant comparisons. ( B ) Western blots of four representative pediatric high-grade glioma tissue samples (left) and four pediatric low-grade glioma tissue samples (right) comparing BMAL1, CLOCK, PER2, and REV-ERBα protein expression (sections of separate blots displayed; full blots are shown in Supplementary Figure S3). Of note, tissue sample HGG 3 is derived from the same patient as the STN-49 cell line. ( C ) Normalization to β-ACTIN control reveals BMAL1 ( p < 0.05, 1.57 vs. 0.98) and CLOCK ( p < 0.05, 0.98 vs. 0.39) protein expression is higher in high-grade gliomas compared to low-grade gliomas, REV-ERBα is lower ( p < 0.05, 0.12 vs. 0.55), and there are no significant differences in PER2 protein expression. Data are expressed as mean ± SEM.

Article Snippet: The following antibodies were used for immunoblotting: BMAL1 Rabbit mAb (A4714; ABclonal Technology, Woburn, MA), NR1D1 mAb (MA5-20772; Invitrogen), CLOCK anti-Rabbit (NB100-126; Novus Biologicals), PER2 anti-rabbit (NBP2-93587; Novus Biologicals), Beta-actin-HRP (MA5-15739-HRP; Invitrogen), anti-rabbit IgG HRP (7074; Cell Signaling Technology), and Goat anti-Mouse IgG Secondary Antibody, HRP (62-6520; Invitrogen).

Techniques: Gene Expression, RNA Sequencing, Expressing, Transformation Assay, Western Blot, Derivative Assay, Control

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: To knock out PER2 gene expression, Per2 CRISPR/CAS9 knockout (KO) Plasmid (sc-401089-KO-2, Santa Cruz Biotechonology, Inc.) was used.

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: To knock out PER2 gene expression, Per2 CRISPR/CAS9 knockout (KO) Plasmid (sc-401089-KO-2, Santa Cruz Biotechonology, Inc.) was used.

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: To knock out PER2 gene expression, Per2 CRISPR/CAS9 knockout (KO) Plasmid (sc-401089-KO-2, Santa Cruz Biotechonology, Inc.) was used.

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: To knock out PER2 gene expression, Per2 CRISPR/CAS9 knockout (KO) Plasmid (sc-401089-KO-2, Santa Cruz Biotechonology, Inc.) was used.

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: To knock out PER2 gene expression, Per2 CRISPR/CAS9 knockout (KO) Plasmid (sc-401089-KO-2, Santa Cruz Biotechonology, Inc.) was used.

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: To knock out PER2 gene expression, Per2 CRISPR/CAS9 knockout (KO) Plasmid (sc-401089-KO-2, Santa Cruz Biotechonology, Inc.) was used.

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