Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 1. Overexpression of PER2 changes morphology of DDP-resistant cervical cancer cells. (A, B) The mRNA expression of PER2 gene in (A) Hela/DDP as well as (B) SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment by RT-qPCR. (C) Morphology changes in Hela/DDP as well as SiHa/DDP cells transfected by pcDNA3.1-PER2 and/or hEGF treatment under a light microscope. Bar = 100 μm. Magnifying power: 200× and 400×. ***P < 0.001; ****p < 0.0001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Light Microscopy

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 2. Overexpression of PER2 reduces proliferation as well as facilitates apoptosis via PI3K/AKT pathway in DDP-resistant cervical cancer cells. (A, B) The cell viability of (A) Hela/DDP cells and (B) SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment according to CCK-8 assay. (C-F) The apoptotic levels of (C, D) Hela/DDP cells and (E, F) SiHa/DDP cells treated with pcDNA3.1-PER2 and/or hEGF through Annexin V-FITC/PI staining. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Over Expression, Transfection, CCK-8 Assay, Staining

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 3. Overexpression of PER2 decreases migration of DDP-resistant cervical cancer cells by PI3K/AKT pathway. (A-D) The relative migration was detected in (A, B) Hela/DDP cells and (C, D) SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment through transwell assay. **p < 0.01; ****p < 0.0001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Over Expression, Migration, Transfection, Transwell Assay

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 4. Forced PER2 expression weakens nuclear translocation of NF-κB p65 by PI3K/AKT pathway in DDP-resistant cervical cancer cells. (A) Immunofluorescence for detecting the nuclear translocation of NF-κB p65 in Hela/DDP cells as well as SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment. Bar = 25 μm. (B-D) Western blot for the expression of NF-κB p65 in the nucleus and cytoplasm of Hela/DDP cells and SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment. ****P < 0.0001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Expressing, Translocation Assay, Immunofluorescence, Transfection, Western Blot

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 5. Overexpression of PER2 regulates clock circadian and ameliorates drug resistance through PI3K/AKT pathway in DDP-resistant cervical cancer cells. (A) Representative images of western blot. (B-M) Quantitative results of (B, C) PER2, (D, E) CLOCK, (F, G) BMAL1, (H, I) CRY1, (J, K) MRP1 and (L, M) MDR1 in Hela/ DDP as well as SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment. **P < 0.01; ****p < 0.0001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Over Expression, Western Blot, Transfection

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 6. Overexpression of PER2 suppresses EMT phenotype in DDP-resistant cervical cancer cells. (A) Representative images of western blot. (B-M) Quantitative results of (B, C) PI3K, (D, E) p-PI3K, (F, G) AKT, (H, I) p-AKT, (J, K) Snail, (L, M) Twist, (N, O) Vimentin and (P, Q) E-cadherin in Hela/DDP as well as SiHa/DDP cells with pcDNA3.1-PER2 transfection and/or hEGF treatment. **P < 0.01; ***P < 0.001; ****p < 0.0001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Over Expression, Western Blot, Transfection

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 8. Immunohistochemistry for the expression of Ki-67 and PCNA in tumor tissues from Hela/DDP cells-induced nude mice injected with cisplatin when PER2 expression peaked and trough. (A) Ki-67. (B) PCNA. Magnification: 200× and 400×.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Immunohistochemistry, Expressing, Injection

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 7. Rhythmic expression of PER2 affects the therapeutic effects of cisplatin in Hela/DDP cells-induced nude mouse model. (A) The qPCR for detecting the rhythmic expression of PER2 at different time points in Hela/DDP cells-induced nude mice. (B) Tumor growth curves in Hela/DDP cells-induced nude mice treated with cisplatin at the time point when PER2 expression was highest or lowest. (C, D) Images of nude mice and removed tumors. **P < 0.01.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Expressing

Journal: Gene

Article Title: Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

doi: 10.1016/j.gene.2021.146003

Figure Lengend Snippet: Fig. 9. Cisplatin therapy at the peak of PER2 expression ameliorates chemotherapy resistance and EMT in cervical cancer. (A) Representative images of western blot. Assessment of the expression of (B) PER2, (C) CLOCK, (D) BMAL1, (E) CRY1, (F) MRY1, (G) MRP1, (H) PI3K, (I) p-PI3K, (J) p-PI3K/PI3K, (K) AKT, (L) p-AKT, (M) p- AKT/AKT, (N) Snail, (O) Twist, (P) Vimentin and (Q) E-cadherin in tumor tissues from Hela/DDP cells-induced nude mice treated with Cisplatin at the peak or trough of PER2 expression. *P < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The sample was transferred onto the membrane at a gel volume of 1.5 mA/cm2 for 1.5 h. The membrane was added with 5% skimmed milk powder + TBST, and shaken in a shaker at room temperature lasting 1 h. The membranes were added with primary antibodies against PER2 (67513-1-Ig; Proteintech), CLOCK (18094-1-AP; Proteintech), BMAL1 (14268-1-AP; Proteintech), CRY1 (13474-1-AP; Proteintech), MRP1 (67228–1-Ig; Proteintech), MDR1 (22336-1-AP; Proteintech), PI3K (20584-1-AP; Proteintech), p-PI3K (17366S; CST), AKT (10176-2-AP; Proteintech), p-AKT (66444-1-Ig; Proteintech), Snail1 (13099-1-AP; Proteintech), Twist (25465–1-AP; Proteintech), E-cadherin (20874-1- AP; Proteintech), Vimentin (10366-1-AP; Proteintech) and GAPDH (60004-1-Ig; Proteintech) which was diluted at 1:1000 with 5% BSA + TBS, and shaken overnight at 4 ◦C.

Techniques: Expressing, Western Blot

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: 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: Chronobiology international

Article Title: Down-regulation of circadian clock gene period 2 in uterine endometrial stromal cells of pregnant rats during decidualization.

doi: 10.3109/07420528.2010.522289

Figure Lengend Snippet: FIGURE 1. Profiles of circadian Per2-dLuc oscillations in UESCs of pregnant rats at day 4.50 and day 6.50 of gestation. UESCs were placed in fresh medium supplemented with DXM (each 0.1 µM: panels B and D) or vehicle (as a control: panels A and C) and cultured for 2 h. At time 0 h, UESCs were placed in fresh medium supplemented with 0.1 mM luciferin. (a) Real-time bioluminescent activity was monitored for 4 days. P1–3: first to third peaks, T1–3: first to third troughs. Panels A and B: day 4.50 of gestation; panels C and D: day 6.50 of gestation. (b) Differences between peak and trough were normalized to the values of the control cells treated with vehicle, and data are means ±SEM of three independent determinations. Asterisk indicates statistically significant difference ( p < .05).

Article Snippet: Membranes were blocked in 10 mM TrisHCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, and 0.05% Tween 20 with 10% nonfat dry milk and incubated at 4°C with primary antibodies, polyclonal anti-human PER2 antibody (H-90) (1:1000; Santa Cruz Biotechnology), and monoclonal anti-α-tubulin antibody DM1A (1:3000; Sigma) and then with the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:1000; Santa Cruz Biotechnology) and goat anti-mouse IgG antibody (1:1500) to Chronobiology International C hr on ob io l I nt D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y T ec hn is ch e U ni ve rs ite it E in dh ov en o n 06 /2 5/ 14 Fo r pe rs on al u se o nl y. detect each protein, respectively.

Techniques: Control, Cell Culture, Activity Assay

Journal: Chronobiology international

Article Title: Down-regulation of circadian clock gene period 2 in uterine endometrial stromal cells of pregnant rats during decidualization.

doi: 10.3109/07420528.2010.522289

Figure Lengend Snippet: FIGURE 2. Circadian rhythms of PER2 protein in uteri of pregnant rats at days 4.33–5.16 of gestation (implantation stage) estimated by Western blotting and fluorescent immunohistochemistry. Pregnant rats were killed at 4-h intervals over a daily cycle (ZT 0, 08:00 h). (a) Pro- teins (30 µg) prepared from a part of uterine horns were separated on SDS-PAGE and analyzed by immunodetection using anti-mPER2 antibody. Data are means ±SEM from three independent experiments normalized to the values given by α-tubulin. Values with different letters are significantly different ( p < .05). Striped areas, during the night. (b) Parts of uterine horns at ZT 4 and ZT 12 were subjected to fluorescent immunohistochemistry. The square regions shown in HE (A, E) were magnified from a serial section subjected to immunofluor- escent studies (B, F) and Hoechst staining (blue). PER2 and Hoechst staining merged image (C, G). The square regions shown in merged image (C, G) were magnified (D, H). Bars: 150 µm (A, E), 50 µm (B, C, F, G), and 25 µm (D, H).

Article Snippet: Membranes were blocked in 10 mM TrisHCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, and 0.05% Tween 20 with 10% nonfat dry milk and incubated at 4°C with primary antibodies, polyclonal anti-human PER2 antibody (H-90) (1:1000; Santa Cruz Biotechnology), and monoclonal anti-α-tubulin antibody DM1A (1:3000; Sigma) and then with the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:1000; Santa Cruz Biotechnology) and goat anti-mouse IgG antibody (1:1500) to Chronobiology International C hr on ob io l I nt D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y T ec hn is ch e U ni ve rs ite it E in dh ov en o n 06 /2 5/ 14 Fo r pe rs on al u se o nl y. detect each protein, respectively.

Techniques: Western Blot, Immunohistochemistry, SDS Page, Immunodetection, Staining

Journal: Chronobiology international

Article Title: Down-regulation of circadian clock gene period 2 in uterine endometrial stromal cells of pregnant rats during decidualization.

doi: 10.3109/07420528.2010.522289

Figure Lengend Snippet: FIGURE 3. Circadian rhythms of PER2 protein in uteri of pregnant rats at days 6.33–7.16 of gestation (decidualization stage) estimated by Western blotting and fluorescent immunohistochemistry. Pregnant rats were killed at 4-h intervals over daily cycle (ZT 0, 08:00 h). (a) Pro- teins (30 µg) prepared from a part of uterine horns were separated on SDS-PAGE and analyzed by immunodetection using anti-mPER2 antibody. Data are means ± SEM from three independent experiments normalized to the values given by α-tubulin. Values with different letters are significantly different ( p < .05). Striped areas, during the night. (b) Parts of uterine horns at ZT 4 and ZT 12 were subjected to fluorescent immunohistochemistry. The square regions shown in HE (A, E) were magnified from a serial section subjected to immunofluor- escent studies (B, F) and Hoechst staining (blue). PER2 and Hoechst staining merged image (C, G). The square regions shown in merged image (C, G) were magnified (D, H). Bars: 150 µm (A, E), 50 µm (B, C, F, G), and 25 µm (D, H).

Article Snippet: Membranes were blocked in 10 mM TrisHCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, and 0.05% Tween 20 with 10% nonfat dry milk and incubated at 4°C with primary antibodies, polyclonal anti-human PER2 antibody (H-90) (1:1000; Santa Cruz Biotechnology), and monoclonal anti-α-tubulin antibody DM1A (1:3000; Sigma) and then with the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:1000; Santa Cruz Biotechnology) and goat anti-mouse IgG antibody (1:1500) to Chronobiology International C hr on ob io l I nt D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y T ec hn is ch e U ni ve rs ite it E in dh ov en o n 06 /2 5/ 14 Fo r pe rs on al u se o nl y. detect each protein, respectively.

Techniques: Western Blot, Immunohistochemistry, SDS Page, Immunodetection, Staining

Journal: Chronobiology international

Article Title: Down-regulation of circadian clock gene period 2 in uterine endometrial stromal cells of pregnant rats during decidualization.

doi: 10.3109/07420528.2010.522289

Figure Lengend Snippet: FIGURE 4. Circadian rhythms of Per2 and Vegf transcripts in UESCs of pregnant rats during implantation and decidualization stages. RNA was extracted from a part of uterine horns and reverse transcribed. The resulting cDNA was used for real-time qPCR using Per2- and Vegf- specific primers. (a) Left panel, Per2 mRNA expression was significantly changed in UESCs of pregnant rats during implantation (days 4.33– 5.16 of gestation). Right panel, Per2 mRNA expression was not changed in UESCs of pregnant rats during decidualization (days 6.33–7.16 of gestation). (b) Left panel, Vegf mRNA expression was significantly changed in UESCs of pregnant rats during implantation (days 4.33–5.16 of gestation). Right panel, Vegf mRNA expression was not changed in UESCs of pregnant rats during decidualization (days 6.33–7.16 of gestation). Values with different letters are significantly different ( p < .05). Striped areas, during the night.

Article Snippet: Membranes were blocked in 10 mM TrisHCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, and 0.05% Tween 20 with 10% nonfat dry milk and incubated at 4°C with primary antibodies, polyclonal anti-human PER2 antibody (H-90) (1:1000; Santa Cruz Biotechnology), and monoclonal anti-α-tubulin antibody DM1A (1:3000; Sigma) and then with the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:1000; Santa Cruz Biotechnology) and goat anti-mouse IgG antibody (1:1500) to Chronobiology International C hr on ob io l I nt D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y T ec hn is ch e U ni ve rs ite it E in dh ov en o n 06 /2 5/ 14 Fo r pe rs on al u se o nl y. detect each protein, respectively.

Techniques: Reverse Transcription, Expressing

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-020-00496-5

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

Article Snippet: For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation.

Techniques: Binding Assay, Construct, Plasmid Preparation

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-020-00496-5

Figure Lengend Snippet: Wild-type mouse embryonic fibroblasts (WT MEFs) were transfected with miR-24-3p or miR-25-3p mimics in a dose-dependent manner (25, 50, and 100 nM) and then assayed for the expression levels of a miR-24-3p and miR-25-3p using real-time qPCR ( n = 4) and b quantified PER2 protein expression by Western blotting ( n = 3). To determine the effects of miR-24-3p and miR-25-3p on Per2 transcription, Period2::Luc knock-in mouse embryonic fibroblasts (Per2::Luc KI MEFs) were transfected with 0.5 µg of miR-24-3p- and miR-25-3p-overexpressing and control vectors, and then, actinomycin D (a potent transcription inhibitor) was added for 30 h after dexamethasone synchronization. c The results of real-time bioluminescence recordings are represented in raw data format after actinomycin D (solid line) and DMSO (dashed line) treatment. d Per2 mRNA was quantified after an additional 0 and 6 h of actinomycin D treatment (Per2::Luc KI MEFS were harvested as indicated by the arrowheads), and the data were normalized to that of the control vector ( n = 4). Data are presented as the means ± SE, and the significance was assessed by one-way ANOVA, * p < 0.05 compared to the control groups.

Article Snippet: For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation.

Techniques: Transfection, Expressing, Western Blot, Knock-In, Plasmid Preparation

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-020-00496-5

Figure Lengend Snippet: The results of real-time bioluminescence recordings are represented in raw and detrended data formats. a , b Per2::Luc KI MEFs were transfected with miR-24-3p or miR-25-3p mimics for gain-of-function studies. c , d For loss-of-function studies, miR-24-3p or miR-25-3p inhibitors were transfected into the Per2::Luc KI MEFs. Bioluminescence patterns were recorded for 3 days and normalized by the control group. e Relative periods and area under curve were calculated from the raw data, which is presented in bar graphs. f , g Per2 promoter-driven luciferase (Per2pro-Luc) mutants were transfected into NIH3T3 fibroblasts with an miR-24-3p inhibitor, miR-25-3p inhibitor, or miR-control oligomer. Data are presented as the means ± SE ( n = 4), and significance was assessed by one-way ANOVA, * p < 0.01 compared to the control group.

Article Snippet: For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation.

Techniques: Transfection, Luciferase

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-020-00496-5

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

Article Snippet: For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation.

Techniques: Transfection, Binding Assay, Plasmid Preparation, Luciferase

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-020-00496-5

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

Article Snippet: For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation.

Techniques: Expressing, Transduction

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-020-00496-5

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

Article Snippet: For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation.

Techniques: Expressing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Daily feeding rhythm of KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 h of darkness; SL, short-light treatment of 8 h of light and 16 h of darkness. Recorded the times of feeding of each mouse from 0:00–2:00, 6:00–8:00,12:00:14:00, and 18:00–20:00 during the trial period using surveillance cameras. Statistical analyses were conducted using two-way ANOVA. * p < 0.05, significant difference; ** or *** p < 0.01 or p < 0.001, extremely significant difference. Results are presented as means ± SEM (n = 4 per group).

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Gene Knockout

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Relative expression of colonic and cecal epithelial circadian rhythm genes and Per2 protein in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 -/- ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 h of darkness; SL, short-light treatment of 8 h of light and 16 h of darkness. Representative charts of the expression levels of Per2 gene and protein of colonic epithelium (A) and cecal epithelium (B) , Per1 gene, Per3 gene, Cry1 gene, Cry2 gene, Clock gene, Bmal1 gene, and Rev-erbα gene of colonic epithelium (C) and cecal epithelium (D) . Expressions of circadian genes and Per2 protein were determined in Per2 knockout and wild-type mice under NLD and SL by PCR and Western blot method. Statistical analyses were conducted using two-way ANOVA. * p < 0.05, significant difference; ** or *** p < 0.01 or p < 0.001, extremely significant difference. Results are presented as means ± SEM (n = 6 per group).

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Expressing, Gene Knockout, Knock-Out, Western Blot

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Relative expression of colonic and cecal epithelial SCFAs transporter genes in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 h of darkness; SL, short-light treatment of 8 h of light and 16 h of darkness. Representative charts of the expression levels of Nhe1 gene, Nhe2 gene, Nhe3 gene, Mct1 gene, Mct4 gene, Ae2 gene, and Na + /K + ATPase gene of colonic epithelium (A) and cecal epithelium (B) . Expressions of SCFAs transporter genes were determined in Per2 knockout and wild-type mice under NLD and SL by PCR method. Statistical analyses were conducted using two-way ANOVA. * p < 0.05, significant difference; ** or *** p < 0.01 or p < 0.001, extremely significant difference. Results are presented as means ± SEM (n = 6 per group).

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Expressing, Gene Knockout, Knock-Out

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Relative expression of colonic and cecal epithelial inflammatory and barrier genes in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 h of darkness; SL, short-light treatment of 8 h light and 16 h darkness. Representative charts of the expression levels of Il-1β gene, Il-6 gene, Tnf-α gene, Zo-1 gene, Claudin-1 gene, and Occludin gene of colonic epithelium (A) and cecal epithelium (B) . Expressions of inflammatory and barrier genes were determined in Per2 knockout and wild-type mice under NLD and SL by PCR method. Statistical analyses were conducted using two-way ANOVA. * p < 0.05, significant difference; ** or *** p < 0.01, or p < 0.001, extremely significant difference. Results are presented as means ± SEM (n = 6 per group).

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Expressing, Gene Knockout, Knock-Out

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Relative expression of colonic and cecal epithelial inflammatory pathways-related genes in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 of h darkness; SL, short-light treatment of 8 h light and 16 h darkness. Representative charts of the expression levels of Tlr2 gene, Tlr4 gene, Myd88 gene, and Nf-κb p65 gene of colonic epithelium (A) and cecal epithelium (B) . Expressions of inflammatory pathways-related were determined in Per2 knockout and wild-type mice under NLD and SL by PCR method. Statistical analyses were conducted using two-way ANOVA. * p < 0.05, significant difference; ** or *** p < 0.01 or p < 0.001, extremely significant difference. Results are presented as means ± SEM (n = 6 per group).

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Expressing, Gene Knockout, Knock-Out

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Bacterial diversities and compositions in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 h of darkness; SL, short-light treatment of 8 h of light and 16 h of darkness. (A) Compositions of colonic and cecal microbiota at the phylum, family, and order levels in KO and CON mice under NLD and SL, the bacterial relative abundance in <0.05% were belonged to others. (B) Venn diagrams based on OTUs in colonic content (B1) and cecal content (B2), the number of unique OTUs were represented by the unoverlapped portion. (C) Principal coordinate analysis plots of unweighted UniFrac distances of microbiota in colonic and cecal contents in KO and CON mice under NLD and SL. n = 6 per group for 16S rRNA sequencing.

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Gene Knockout, Sequencing

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: LEfSe analysis and cluster heatmap of in intestinal differential microbial genus classifications in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h of light and 12 h of darkness; SL, short-light treatment of 8 h of light and 16 h darkness. (A) Linear discriminant analysis (LDA) plus effect size (LEfSe) at bacterial genus levels in KO and CON mice under NLD and SL, only LDA score in over than 3.5 was marked. (B) Cluster heatmap of differential bacterial genus levels in KO and CON mice under NLD and SL. (C) Classification the differential bacterial genus in family levels. (D) Classification the differential bacterial genus in phylum levels. n = 6 per group for 16S rRNA sequencing.

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Gene Knockout, Sequencing

Journal: Frontiers in Immunology

Article Title: Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice

doi: 10.3389/fimmu.2022.848248

Figure Lengend Snippet: Predicted of bacterial functions of KEGG pathway via PICRUSt2 in KO and CON mice under NLD and SL. KO, Per2 gene knockout ( Period2 −/− ) mice; CON, wild-type ( Period2 +/+ ) mice; NLD, normal light–dark cycle treatment of 12 h light and 12 h darkness; SL, short-light treatment of 8 h light and 16 h darkness. (A) The bacterial functional predictions of KEGG secondary pathway via metabolism. (B) The amino acid metabolism pathways predicted via metabolism. (C) The carbohydrate metabolism pathways predicted via metabolism. n = 6 per group for 16S rRNA sequencing.

Article Snippet: Briefly, primary antibodies used in this work were Per2 (GTX129688, GeneTex, Irvine, CA, USA), and Gapdh (GTX100118, GeneTex, Irvine, CA, USA).

Techniques: Gene Knockout, Functional Assay, Sequencing

Journal: Cell death & disease

Article Title: Loss of thyroid gland circadian PER2 rhythmicity in aged mice and its potential association with thyroid cancer development.

doi: 10.1038/s41419-022-05342-2

Figure Lengend Snippet: Fig. 6 Relationship between PER2 immunoexpression and aging in the human thyroid gland. A In non-tumor thyroid, PER2 immunostaining is more frequently observed in the thyroid follicular epithelium of individuals aged <60 years than in those aged >60 years. B PER2 is more frequently expressed in PTCs of individuals aged <60 years than in those aged >60 years. Scale bar, 20 μm.

Article Snippet: The primary antibody was anti-rabbit PER2 (biorbyt, 1:100) and Ki-67 (Cell Signaling Technology, 1:200).

Techniques: Immunostaining

Journal: Molecular Brain

Article Title: DA-JC1 improves learning and memory by antagonizing Aβ31–35-induced circadian rhythm disorder

doi: 10.1186/s13041-019-0432-9

Figure Lengend Snippet: The effect of DA-JC1 on Aβ31–35-induced abnormal expression of Per2 in HT22 hippocampal cells. ( a ) mRNA levels of Per2 were assayed at indicated circadian times. ( b ) Representative immunoblots show the levels of Per2 at CT16. Quantifications of Western blots were obtained by using β-actin as a loading control. ( c ) mRNA expression of Per2 in the hippocampal tissue at different time points was detected. Data are expressed as means ± SEM (n = 6 per group). * P < 0.05 compared with the control group; # P < 0.05 compared with the Aβ31–35 group

Article Snippet: The membrane was blocked for 2 h at room temperature with 5% skimmed milk, then incubated with anti-SYP primary antibody (Santa Cruz, sc-17,750), anti-GAP43 primary antibody (Santa Cruz, sc-17,790), anti-PER1 primary antibody (Abcam, ab3443), and anti-PER2 primary antibody(Santa Cruz, sc-25,363) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Molecular Brain

Article Title: DA-JC1 improves learning and memory by antagonizing Aβ31–35-induced circadian rhythm disorder

doi: 10.1186/s13041-019-0432-9

Figure Lengend Snippet: The role of Per2 interference using lentivirus on circadian rhythm disorder in mice. ( a ) A laser scanning confocal microscope was used to observe the GFP-positive cells. Scale bar = 100 μm. ( n = 3 per group). ( b ) Representative Western blot and quantification analysis showing the protein expression of Per2. ( n = 5 per group). ( c ) Representative locomotor activity records of each group and the free-running period of the locomotor activity rhythm in each group. (n = 5 per group). ( d ) The point-by-point average activity in DD of all the mice at each one-hour interval in the actogram. (n = 5 per group). Data are expressed as means ± SEM. * P < 0.05 compared with the control group

Article Snippet: The membrane was blocked for 2 h at room temperature with 5% skimmed milk, then incubated with anti-SYP primary antibody (Santa Cruz, sc-17,750), anti-GAP43 primary antibody (Santa Cruz, sc-17,790), anti-PER1 primary antibody (Abcam, ab3443), and anti-PER2 primary antibody(Santa Cruz, sc-25,363) overnight at 4 °C.

Techniques: Microscopy, Western Blot, Expressing, Activity Assay

Journal: Molecular Brain

Article Title: DA-JC1 improves learning and memory by antagonizing Aβ31–35-induced circadian rhythm disorder

doi: 10.1186/s13041-019-0432-9

Figure Lengend Snippet: The change in the expression of Per2 after interference with Per2. ( a ) A fluorescence microscope was used to determine the optimal multiplicity of infection (MOI). Scale bar = 100 μm. ( b ) Typical diagram of a fluorescence microscope at an MOI of 60, representative Western blot and quantification analysis of PER2. Scale bar = 50 μm. Data are expressed as means ± SEM (n = 6 per group). * P < 0.05 compared with the control group

Article Snippet: The membrane was blocked for 2 h at room temperature with 5% skimmed milk, then incubated with anti-SYP primary antibody (Santa Cruz, sc-17,750), anti-GAP43 primary antibody (Santa Cruz, sc-17,790), anti-PER1 primary antibody (Abcam, ab3443), and anti-PER2 primary antibody(Santa Cruz, sc-25,363) overnight at 4 °C.

Techniques: Expressing, Fluorescence, Microscopy, Infection, Western Blot

Journal: Molecular Brain

Article Title: DA-JC1 improves learning and memory by antagonizing Aβ31–35-induced circadian rhythm disorder

doi: 10.1186/s13041-019-0432-9

Figure Lengend Snippet: The change in learning and memory abilities after interference in Per2 using lentivirus. ( a ) The escape latency in hidden platform acquisition testing on each day is shown for each group of mice. ( b ) Representative swim paths of mice on the sixth training day after finishing the hidden platform test and the percentage of time spent and distance traveled in the four quadrants. Zone-NE indicates the target quadrant. ( c ) Representative Western blot and quantification analysis of SYP. ( d ) Representative Western blot and quantification analysis of GAP-43. Data are expressed as means ± SEM (n = 6 per group). * P < 0.05 compared with the control group

Article Snippet: The membrane was blocked for 2 h at room temperature with 5% skimmed milk, then incubated with anti-SYP primary antibody (Santa Cruz, sc-17,750), anti-GAP43 primary antibody (Santa Cruz, sc-17,790), anti-PER1 primary antibody (Abcam, ab3443), and anti-PER2 primary antibody(Santa Cruz, sc-25,363) overnight at 4 °C.

Techniques: Western Blot

Journal: Molecular Brain

Article Title: DA-JC1 improves learning and memory by antagonizing Aβ31–35-induced circadian rhythm disorder

doi: 10.1186/s13041-019-0432-9

Figure Lengend Snippet: The effect of interference in Per2 using lentivirus on DA-JC1’s improvement of abnormal learning and memory capacity induced by Aβ31–35. ( a ) The escape latency in hidden platform acquisition testing at each day. ( b ) The percentage of time spent and the percentage of distance traveled in the four quadrants. Zone-NE indicates the target quadrant. ( c ) Representative Western blot and quantification analysis of SYP and GAP-43. Data are expressed as means ± SEM (n = 6 per group). * P < 0.05 compared with the control group

Article Snippet: The membrane was blocked for 2 h at room temperature with 5% skimmed milk, then incubated with anti-SYP primary antibody (Santa Cruz, sc-17,750), anti-GAP43 primary antibody (Santa Cruz, sc-17,790), anti-PER1 primary antibody (Abcam, ab3443), and anti-PER2 primary antibody(Santa Cruz, sc-25,363) overnight at 4 °C.

Techniques: Western Blot