Journal: Advanced Science

Article Title: An Automated Organotypic SCN Culture System Revealing Novel Insights into VIP Regulation of Circadian Rhythm

doi: 10.1002/advs.202511069

Figure Lengend Snippet: Spatial synchronization of amplitude and phase among SCN neurons. (a) 8‐circadian‐day time‐lapse bioluminescent images of an Vip‐/‐ SCN slice. A regular 60 µM VIP introduction, with 24 h period and 2 h duration, was applied to the BaSIC culture chamber. Each VIP introduction was associated with a clear intensity drop (the trough). (b) Spatial distribution of PER2 during the phases of the troughs (left column) and the peaks (middle column) induced by VIP stimulations at the 72nd, 96th, and 120th hour. The histograms of amplitudes of VIP stimulated oscillations (experimental data in red, and sine‐function‐fitted data in green) are also presented. (c) Spatial distribution (left: 2D, middle: 3D) of the rate of PER2 reduction induced by VIP dosing at the 72th, 96th, and 120th hour. The scatter plots (right column) show the negative correlation between PER2 decrease rate and the distance from the centroids (black dots) of SCN slices (Pixels of the left area in blue, and pixels of the right area in green). (d) Spatial distribution of the average values of rhythm baselines that present basal expression levels of PER2 protein. The baselines are estimated from the lowest frequency of the VMD model. (e) Heatmap of PER2 rhythm of each pixel of SCN slice time‐lapse images. The data are sorted by distance from centroids, and VIP pulses are marked. Synchrony index ( SI ) was calculated for peak and trough, separately. (f) 60 µM VIP pulses stimulated an Vip‐/‐ SCN slice with a 12‐h period. The grey curves depict the PER2::LUC oscillations from individual pixels of the Vip‐/‐ SCN, while the red curve represents the averaged oscillation (left column). Orange arrows indicate the times at which VIP was added. Spatial distribution (upper: 2D, lower: 3D) of the rate of PER2 reduction induced by VIP dosing at the 52th, and 64th hour (middle column). Synchrony index was calculated for troughs (right column).

Article Snippet: PER2::LUC U2OS cells (HTB‐96, ATCC) expressing TIR1‐myc served as a negative control.

Techniques: Expressing

Journal: Advanced Science

Article Title: An Automated Organotypic SCN Culture System Revealing Novel Insights into VIP Regulation of Circadian Rhythm

doi: 10.1002/advs.202511069

Figure Lengend Snippet: Rapid decrease of PER2::LUC signal and circadian resetting induced by periodic VIP pulses. (a) Circadian rhythm of Vip‐/‐ SCN slices stimulated with 60 µM VIP. VIP was applied every 24 h period with 2 h duration. Between 96 and144 h, medium pulses replaced VIP pulses to simulate the condition with only culture medium (gray arrows). The gray curve represents the PER2‐LUC rhythmic curve of Vip‐/‐ SCN with medium perfusion only. (b) Fast decrease of PER2 stimulated by 60 µM VIP dosing in multiple experiments using 4 wild‐type and 4 Vip‐/‐ SCN slices. (c) PER2 rhythm of Vip‐/‐ SCN slices stimulated by different concentrations of VIP and different elution rates. The shaded areas represent the duration of elution. VIP pulses are indicated by orange arrows. (d) PER2 rhythm of 4 Vip‐/‐ SCN slices with 24‐h‐period VIP dosing at different phases of PER2 rhythm (left panel). Time intervals between the initial troughs and VIP pulses (right panel) become small and stable after several VIP pulses, indicating that the troughs of PER2 rhythm are reset to the time of VIP pulsing. (e) Circadian rhythm of wild‐type and VIP receptor over expression Mouse Adult Fibroblasts (MAF) cells stimulated with VIP ( n = 3). 60 µM VIP was applied every 24 h. (f) PER2 rhythm of 1 wild‐type and 1 Vip‐/‐ SCN slices with 1 µg/ml PTX introduction (shaded). VIP was perfused every 24 h with 2 h duration. VIP pulses are indicated by orange dashed lines.

Article Snippet: PER2::LUC U2OS cells (HTB‐96, ATCC) expressing TIR1‐myc served as a negative control.

Techniques: Over Expression

Journal: Advanced Science

Article Title: An Automated Organotypic SCN Culture System Revealing Novel Insights into VIP Regulation of Circadian Rhythm

doi: 10.1002/advs.202511069

Figure Lengend Snippet: VIP induced PER2 reduction, and related phase and amplitude modulation with cellular assay. (a) HEK293T cells overexpressing VIPR2 were treated with 60 µM VIP, and PER2 protein levels were quantified by western blot. The results are presented as mean ± SE ( n = 4). (b) PER2‐LUC U2OS cells were co‐transfected with CMV‐PER2‐AID and CMV‐TIR, and treated with 500 µM IAA at different circadian times. Bioluminescence was continuously recorded following IAA addition. Control cells were PER2‐LUC U2OS cells transfected with CMV‐TIR alone, which received 500 µM IAA at either CT0 or CT12. The first bioluminescence trough after recording onset was defined as circadian time zero (CT0). IAA was administered at 3‐h intervals from CT0 to CT21. Data are presented as mean ± SEM ( n = 3 per time point; control groups n = 6, with n = 3 for CT0 and CT12). The timing of IAA application is indicated by arrows in the figure. (c) Phase shift of PER2 rhythm of cells with different IAA adding time compared with a regular rhythm. Phase shift data are presented as mean ± SEM.

Article Snippet: PER2::LUC U2OS cells (HTB‐96, ATCC) expressing TIR1‐myc served as a negative control.

Techniques: Western Blot, Transfection, Control

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: PLoS ONE

Article Title: Time-Dependent Effects of Localized Inflammation on Peripheral Clock Gene Expression in Rats

doi: 10.1371/journal.pone.0059808

Figure Lengend Snippet: Animals were treated with either saline or TURP at ZT2 (0 h) and sacrificed at the 6 different time points, ZT4, ZT8, ZT12, ZT16, ZT20, and ZT0 (time of sacrifice, TOS), which correspond to 2, 6, 10, 14, 18, and 22 h after injection (HAI), respectively. Tissues were harvested upon sacrifice and RNA extracted. The relative expression of the clock genes Per1, Per2 and Rev-erbα in the liver, heart, kidney and spleen is represented. Gene expression is relative to one sample in the ZT2 sacrifice group. Closed boxes represent saline-treated animals and open boxes represent TURP-treated animals. Two-way ANOVA Time×Condition p <0.05 for Per1 in liver and heart and Per2 in liver, p >0.05 for all others (p = 0.0532 for Per1 in the heart); Tukey post hoc tests between saline and TURP-treated groups at each time point (done for graphs where there was a Time×Condition interaction) * p <0.05; n = 4. Body temperature and cytokine data for this experiment can be found in and .

Article Snippet: For clock gene expression, the following probe sets were used: Per1 (H200242988_m1), Per2 (Hs00256143_m1), Rev-erbα (Hs00253876_m1), Serum amyloid A2 (Hs00605928_g1) and Haptoglobin (Hs01667582_m1).

Techniques: Saline, Injection, Expressing, Gene Expression

Journal: PLoS ONE

Article Title: Time-Dependent Effects of Localized Inflammation on Peripheral Clock Gene Expression in Rats

doi: 10.1371/journal.pone.0059808

Figure Lengend Snippet: Animals were treated with either TURP or saline at ZT4, ZT20, ZT2 and ZT8 (time of injection, TOI) and sacrificed 10 h later at ZT0, ZT6, ZT12 and ZT18 (time of sacrifice, TOS), respectively. Tissues were harvested upon sacrifice and RNA extracted. The relative expression of the clock genes Per1, Per2 and Rev-erbα in the liver, heart, kidney and spleen is represented. Gene expression is relative to one arbitrarily chosen sample in the ZT0 TOS group. Closed boxes represent saline-treated animals and open boxes represent TURP-treated animals. Two-way ANOVA Time×Condition p <0.05 for Per1 in liver and spleen, Per2 in liver, heart and kidney, Rev-erbα in liver, kidney and spleen, p >0.05 for the others (p = 0.0862 for Per1 in the kidney); Tukey post hoc tests between saline and TURP-treated groups at each time point (done for graphs where there was a Time×Condition interaction) * p <0.05; n = 5. Body temperature and cytokine data for this experiment can be found in and .

Article Snippet: For clock gene expression, the following probe sets were used: Per1 (H200242988_m1), Per2 (Hs00256143_m1), Rev-erbα (Hs00253876_m1), Serum amyloid A2 (Hs00605928_g1) and Haptoglobin (Hs01667582_m1).

Techniques: Saline, Injection, Expressing, Gene Expression

Journal: PLoS ONE

Article Title: Time-Dependent Effects of Localized Inflammation on Peripheral Clock Gene Expression in Rats

doi: 10.1371/journal.pone.0059808

Figure Lengend Snippet: All animals were treated with either saline or TURP at ZT2 and sacrificed at either (A) ZT12 or (B) ZT16 with IL-1Ra treatment 0, 4, 8, and 12 h after the TURP treatment (12 h IL-1Ra treatment not done in the group of rats sacrificed at ZT12). At sacrifice, the liver was harvested and relative expression of the clock gene Per1, Per2 and Rev-erbα was determined. One-way ANOVA for the four groups p <0.05 for Per2 and Rev-erbα at ZT12 and Per1 and Per2 at ZT16, p >0.05 for the others; Tukey post hoc tests between groups (when ANOVA shows significant interaction), only different letters between groups p <0.05, share one letter p >0.05; n = 5. Body temperature and cytokine data for this experiment can be found in and .

Article Snippet: For clock gene expression, the following probe sets were used: Per1 (H200242988_m1), Per2 (Hs00256143_m1), Rev-erbα (Hs00253876_m1), Serum amyloid A2 (Hs00605928_g1) and Haptoglobin (Hs01667582_m1).

Techniques: Saline, Expressing

Journal: PLoS ONE

Article Title: Time-Dependent Effects of Localized Inflammation on Peripheral Clock Gene Expression in Rats

doi: 10.1371/journal.pone.0059808

Figure Lengend Snippet: (A, B, C) HepG2 cells were treated with 0, 25, or 100 ng/mL of IL-6 and mRNA was extracted 0.5, 1, 2, and 4 h after the start of the treatment. Relative expression of Per1 (A), Per2 (B) and Rev-erbα (C) was determined. Dose of IL-6 are depicted in the inset of panel A. Two-way ANOVA Time×Condition p >0.05 for all three genes. (D) Expression of SAA2 (serum amyloid A2) and HP (haptoglobin) in selected samples of the same experiment as in (A, B, C). Each data point is the average of 3 culture wells. Student t test between the 1 h-0 ng/mL and 1 h-25 ng/mL groups * p <0.05; n = 5.

Article Snippet: For clock gene expression, the following probe sets were used: Per1 (H200242988_m1), Per2 (Hs00256143_m1), Rev-erbα (Hs00253876_m1), Serum amyloid A2 (Hs00605928_g1) and Haptoglobin (Hs01667582_m1).

Techniques: Expressing

Journal: Oncology reports

Article Title: The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

doi: 10.3892/or.2017.5653

Figure Lengend Snippet: Figure 1. PER2 knockdown by PER2-shRNA in SCC15 cells. (A) Levels of PER2 mRNA were significantly reduced in SCC15 cells transfected with PER2-shRNA-I-III, and the levels of PER2 mRNA were significantly decreased in the PER2-shRNA-I group as compared to the PER2-shRNA-II and PER2-shRNA-III groups. (B) Gel images of PER2 protein level analyzed by western blotting in the SCC15, Control-shRNA, and PER2-shRNA-I-III groups. (C) Expression of PER2 protein was significantly downregulated in SCC15 cells transfected with PER2-shRNA-I, PER2-shRNA-II, or PER2-shRNA-III. Means ± SD from three independent experiments are shown. Significant differences among multiple groups were evaluated using one-way ANOVA; differ- ences between two groups were evaluated using the LSD test. Statistical significance is indicated by asterisks. ***P<0.001.

Article Snippet: The membranes were blocked with 5% skim milk and subsequently incubated overnight at 4 ̊C with mouse monoclonal anti-PER2 antibody (1:500; 19-J6:sc-101105; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) and mouse monoclonal anti-β-actin antibody (1:1000; 60008-1-1g; Santa Cruz Biotechnology, Inc.), respectively, washed three times in PBS, followed by secondary goat monoclonal anti-mouse IgG (1:1000; SA00001-1; Protein Tech, Chicago, IL, uSA) for 1 h at room temperature.

Techniques: Knockdown, shRNA, Transfection, Western Blot, Control, Expressing

Journal: Oncology reports

Article Title: The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

doi: 10.3892/or.2017.5653

Figure Lengend Snippet: Figure 2. Regulation of mRNA expression of tumor-related genes by silenced PER2 in SCC15 cells. The levels of Ki-67, c-Myc, Bcl-2, MDM2, VEGF, MMP2, TIMP-2, p53, and BaxmRNA were determined by real-time PCR in PER2-shRNA-I, Control-shRNA, and SCC15 groups. Means ± SD from three independent experiments are shown. Significant differences among multiple groups were evaluated using one-way ANOVA; differences between two groups were evaluated using the LSD test. Statistical significance is indicated by asterisks. **P<0.01, ***P<0.001.

Article Snippet: The membranes were blocked with 5% skim milk and subsequently incubated overnight at 4 ̊C with mouse monoclonal anti-PER2 antibody (1:500; 19-J6:sc-101105; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) and mouse monoclonal anti-β-actin antibody (1:1000; 60008-1-1g; Santa Cruz Biotechnology, Inc.), respectively, washed three times in PBS, followed by secondary goat monoclonal anti-mouse IgG (1:1000; SA00001-1; Protein Tech, Chicago, IL, uSA) for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Control

Journal: Oncology reports

Article Title: The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

doi: 10.3892/or.2017.5653

Figure Lengend Snippet: Figure 3. PER2 downregulation promotes cell growth and proliferation. (A) Cell Counting Kit-8 (CCK-8) assay. (B) Histograms show the number of colonies. (C) Colony formation assay. Means ± SD from three independent experiments are shown. Significant differences among multiple groups were evaluated using one-way ANOVA; differences between two groups were evaluated using the LSD test. Statistical significance is indicated by asterisks. *P<0.05, ***P<0.001.

Article Snippet: The membranes were blocked with 5% skim milk and subsequently incubated overnight at 4 ̊C with mouse monoclonal anti-PER2 antibody (1:500; 19-J6:sc-101105; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) and mouse monoclonal anti-β-actin antibody (1:1000; 60008-1-1g; Santa Cruz Biotechnology, Inc.), respectively, washed three times in PBS, followed by secondary goat monoclonal anti-mouse IgG (1:1000; SA00001-1; Protein Tech, Chicago, IL, uSA) for 1 h at room temperature.

Techniques: Cell Counting, CCK-8 Assay, Colony Assay

Journal: Oncology reports

Article Title: The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

doi: 10.3892/or.2017.5653

Figure Lengend Snippet: Figure 4. Effects of PER2 downregulation on cell cycle distribution, cell proliferation, and cell apoptosis in the SCC15 cells. (A) Flow cytometric analysis of the cell cycle. (B) Flow cytometric analysis of cell apoptosis. (C) The proportion of cells in G0/G1 phase, S phase, and G2/M phase to the total number of cells; proliferation index (PI) and apoptosis index (AI) in the three groups. Means ± SD from three independent experiments are shown. Significant differences among multiple groups were evaluated using one-way ANOVA; differences between two groups were evaluated using the LSD test. Statistical significance is indicated by asterisks. *P<0.05, **P<0.01.

Article Snippet: The membranes were blocked with 5% skim milk and subsequently incubated overnight at 4 ̊C with mouse monoclonal anti-PER2 antibody (1:500; 19-J6:sc-101105; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) and mouse monoclonal anti-β-actin antibody (1:1000; 60008-1-1g; Santa Cruz Biotechnology, Inc.), respectively, washed three times in PBS, followed by secondary goat monoclonal anti-mouse IgG (1:1000; SA00001-1; Protein Tech, Chicago, IL, uSA) for 1 h at room temperature.

Techniques:

Journal: Oncology reports

Article Title: The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

doi: 10.3892/or.2017.5653

Figure Lengend Snippet: Figure 5. PER2 downregulation promotes cell migration and invasive ability. (A) Transwell migration assays were performed in PER2-shRNA-I, Control- shRNA, and SCC15 groups. Histograms show the number of SCC15 cells migrating through the inserts. (B) Transwell invasion assays were performed in PER2-shRNA-I, Control-shRNA, and SCC15 groups. Histograms show the number of SCC15 cells invading through the inserts coated with Matrigel. Means ± SD from three independent experiments are shown. Significant differences among multiple groups were evaluated using one-way ANOVA; differ- ences between two groups were evaluated using the LSD test. Statistical significance is indicated by asterisks. ***P<0.001.

Article Snippet: The membranes were blocked with 5% skim milk and subsequently incubated overnight at 4 ̊C with mouse monoclonal anti-PER2 antibody (1:500; 19-J6:sc-101105; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) and mouse monoclonal anti-β-actin antibody (1:1000; 60008-1-1g; Santa Cruz Biotechnology, Inc.), respectively, washed three times in PBS, followed by secondary goat monoclonal anti-mouse IgG (1:1000; SA00001-1; Protein Tech, Chicago, IL, uSA) for 1 h at room temperature.

Techniques: Migration, shRNA, Control

Journal: Oncology reports

Article Title: The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

doi: 10.3892/or.2017.5653

Figure Lengend Snippet: Figure 6. PER2 downregulation increased SCC15 cell growth in vivo. (A) Xenograft that originated from PER2-shRNA-I cells grew larger than SCC15‑derived tumors. (B) Hematoxylin and eosin (H&E) staining of tissues (magnification, x200). Mean ± SD from five independent experiments are shown. Scale bar, 50 µm. Statistical significance is indicated by asterisks. ***P<0.001 (t-test).

Article Snippet: The membranes were blocked with 5% skim milk and subsequently incubated overnight at 4 ̊C with mouse monoclonal anti-PER2 antibody (1:500; 19-J6:sc-101105; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) and mouse monoclonal anti-β-actin antibody (1:1000; 60008-1-1g; Santa Cruz Biotechnology, Inc.), respectively, washed three times in PBS, followed by secondary goat monoclonal anti-mouse IgG (1:1000; SA00001-1; Protein Tech, Chicago, IL, uSA) for 1 h at room temperature.

Techniques: In Vivo, shRNA, Staining

Journal: International Journal of Oral Science

Article Title: PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis

doi: 10.1038/s41368-021-00123-7

Figure Lengend Snippet: Dysregulated signalling molecules in the ameloblasts of neonatal offspring of circadian disruption pregnant mice. An environmental circadian disruption model was constructed in 10- to 12-week-old pregnant mice. a Total protein of mandibular first molar germs of the offspring (PN3) was extracted. Compared to the control group, the protein levels of PER2, PPARγ and AMELX decreased in the disturbance group. b The mandibles of the offspring (PN5) were dissected. Compared to the control group, the expression levels of PER2, PPARγ, AKT1-Ser473 and β-catenin-Ser552 were reduced in ameloblasts in the disturbance group. ** P < 0.01; *** P < 0.001. Bar = 20 μm

Article Snippet: Primary antibodies included PER2 (NBP2-24616, Novus Biologicals), BMAL1 (NB100-2288, Novus Biologicals), PPARγ (A0270, ABclonal Technology, China), AKT1-Ser473 (AP0140, ABclonal Technology, China), β-catenin-Ser552 (AP0579, ABclonal Technology, China), AKT1 (A11016, ABclonal Technology, China), AMELX (ab153915, Abcam), β-catenin (A11343, ABclonal Technology, China), LAMIN B1 (A1910, ABclonal Technology, China), β-tubulin (PMK081M, BioPM, China), GAPDH (PMK042M, BioPM, China), CK14 (MA5-11599, ThermoFisher Scientific), F-actin (ab205, Abcam), and E-cadherin (sc-8426, Santa Cruz).

Techniques: Disruption, Construct, Control, Expressing

Journal: International Journal of Oral Science

Article Title: PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis

doi: 10.1038/s41368-021-00123-7

Figure Lengend Snippet: Altered signalling molecules expression and differentiation inhibition in Per2 -knockdown ALC cells. a Construction of Per2 knockdown ALC cell line. The knockdown efficiency of ALC- Per2 -sh was examined by qRT-PCR and western blot. b , c Expression of PPARγ, AMELX, AKT1-Ser473 and β-catenin-Ser552 was reduced in ALC- Per2 -sh. d , e ALC-Con cells and ALC- Per2 -sh cells were cultured in differentiation-inducing medium. On days 3, 7, 14 and 21 of differentiation induction, in ALC- Per2 -sh cells, ALP staining weakened ( e ), and the transcription levels of Alp and Ocn decreased ( d ). * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Primary antibodies included PER2 (NBP2-24616, Novus Biologicals), BMAL1 (NB100-2288, Novus Biologicals), PPARγ (A0270, ABclonal Technology, China), AKT1-Ser473 (AP0140, ABclonal Technology, China), β-catenin-Ser552 (AP0579, ABclonal Technology, China), AKT1 (A11016, ABclonal Technology, China), AMELX (ab153915, Abcam), β-catenin (A11343, ABclonal Technology, China), LAMIN B1 (A1910, ABclonal Technology, China), β-tubulin (PMK081M, BioPM, China), GAPDH (PMK042M, BioPM, China), CK14 (MA5-11599, ThermoFisher Scientific), F-actin (ab205, Abcam), and E-cadherin (sc-8426, Santa Cruz).

Techniques: Expressing, Inhibition, Knockdown, Quantitative RT-PCR, Western Blot, Cell Culture, Staining

Journal: International Journal of Oral Science

Article Title: PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis

doi: 10.1038/s41368-021-00123-7

Figure Lengend Snippet: β-catenin translocated into the nucleus, and the subcellular localization of E-cadherin changed in Per2 -knockdown ALC cells. a , b ALC-Con cells and ALC- Per2 -sh cells were cultured in differentiation-inducing medium. On days 7, 14 and 21 of differentiation induction, in ALC- Per2 -sh cells, β-catenin expression increased in the nucleus ( a ) and decreased in the cytoplasm ( b ) compared to that in control cells; c Per2 knockdown altered the subcellular localization of E-cadherin in ALC cells. Original magnification, ×100. * P < 0.05; ** P < 0.01

Article Snippet: Primary antibodies included PER2 (NBP2-24616, Novus Biologicals), BMAL1 (NB100-2288, Novus Biologicals), PPARγ (A0270, ABclonal Technology, China), AKT1-Ser473 (AP0140, ABclonal Technology, China), β-catenin-Ser552 (AP0579, ABclonal Technology, China), AKT1 (A11016, ABclonal Technology, China), AMELX (ab153915, Abcam), β-catenin (A11343, ABclonal Technology, China), LAMIN B1 (A1910, ABclonal Technology, China), β-tubulin (PMK081M, BioPM, China), GAPDH (PMK042M, BioPM, China), CK14 (MA5-11599, ThermoFisher Scientific), F-actin (ab205, Abcam), and E-cadherin (sc-8426, Santa Cruz).

Techniques: Knockdown, Cell Culture, Expressing, Control

Journal: International Journal of Oral Science

Article Title: PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis

doi: 10.1038/s41368-021-00123-7

Figure Lengend Snippet: Overexpression of PPARγ partially rescued the altered signalling molecule expression and weakened ALP staining and ALP activity in Per2 -knockdown ALC cells. a PPARγ was overexpressed in ALC- Per2 -sh cells transfected with plasmids. With the increase in PPARγ expression, PER2 expression remained unchanged, while AKT1 and β-catenin phosphorylation levels were enhanced in the ALC- Per2 -sh-pEnCMV- Pparγ group. b ALP staining and ALP activity were enhanced in ALC- Per2 -sh-pEnCMV- Pparγ cells compared to those in ALC- Per2 -sh-pEnCMV cells but still weakened compared to those in ALC-Con-pEnCMV cells. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Primary antibodies included PER2 (NBP2-24616, Novus Biologicals), BMAL1 (NB100-2288, Novus Biologicals), PPARγ (A0270, ABclonal Technology, China), AKT1-Ser473 (AP0140, ABclonal Technology, China), β-catenin-Ser552 (AP0579, ABclonal Technology, China), AKT1 (A11016, ABclonal Technology, China), AMELX (ab153915, Abcam), β-catenin (A11343, ABclonal Technology, China), LAMIN B1 (A1910, ABclonal Technology, China), β-tubulin (PMK081M, BioPM, China), GAPDH (PMK042M, BioPM, China), CK14 (MA5-11599, ThermoFisher Scientific), F-actin (ab205, Abcam), and E-cadherin (sc-8426, Santa Cruz).

Techniques: Over Expression, Expressing, Staining, Activity Assay, Knockdown, Transfection, Phospho-proteomics

Journal: International Journal of Oral Science

Article Title: PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis

doi: 10.1038/s41368-021-00123-7

Figure Lengend Snippet: Overexpression of PPARγ reversed β-catenin subcellular localization in Per2 -knockdown ALC cells. ALC-Con and ALC- Per2 -sh were transfected with plasmids. a , b In the differentiation assay, the expression of β-catenin was obviously decreased in the nucleus ( a ) and cytoplasm ( b ) in ALC- Per2 -sh-pEnCMV- Pparγ cells. c Cell immunofluorescence showed that β-catenin translocated into the nucleus in ALC- Per2 -sh-pEnCMV cells compared with ALC-Con-pEnCMV cells but was reversed in ALC- Per2 -sh-pEnCMV- Pparγ cells. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Primary antibodies included PER2 (NBP2-24616, Novus Biologicals), BMAL1 (NB100-2288, Novus Biologicals), PPARγ (A0270, ABclonal Technology, China), AKT1-Ser473 (AP0140, ABclonal Technology, China), β-catenin-Ser552 (AP0579, ABclonal Technology, China), AKT1 (A11016, ABclonal Technology, China), AMELX (ab153915, Abcam), β-catenin (A11343, ABclonal Technology, China), LAMIN B1 (A1910, ABclonal Technology, China), β-tubulin (PMK081M, BioPM, China), GAPDH (PMK042M, BioPM, China), CK14 (MA5-11599, ThermoFisher Scientific), F-actin (ab205, Abcam), and E-cadherin (sc-8426, Santa Cruz).

Techniques: Over Expression, Knockdown, Transfection, Differentiation Assay, Expressing, Immunofluorescence

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