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: Scientific Reports

Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

doi: 10.1038/s41598-018-36736-y

Figure Lengend Snippet: Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.

Article Snippet: For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA). hP2-GP49 was used in the current study.

Techniques: CRISPR, Plasmid Preparation, Software, Sequencing, Expressing, Transfection, Clone Assay

Journal: Scientific Reports

Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

doi: 10.1038/s41598-018-36736-y

Figure Lengend Snippet: Efficient generation of Bmal1 and Per2 knockout clones in U2OS cells by the all-in-one vector. ( A ) Function of Bmal1 is essential for the clock in U2OS cells. Bmal1 promoter driven Luciferase rhythms are completely disrupted by overexpressing a mutant BMAL1 lacking the DNA-binding domain. Transduction efficiency by the adenovirus was ~100% (see Supplementary Fig. 5) as shown in the merged image between bright field and GFP. Expression levels of the transgene were ~10-fold higher than those of endogenous Bmal1 (right panel). *Indicates a nonspecific band. Note that the mutant BMAL1 is smaller than the endogenous one. The scale bar represents 50 μm. ( B ) Circadian rhythms are still observed when Bmal1 exon 6 and 7 are targeted by transfection of the all-in-one CRISPR-Cas9 vector, and positive cells were selected by FACS and subjected to bioluminescence assay. Amplitude was reduced in both cases. ( C ) Arrhythmic clones are easily selected by single cell isolation and expansion from the FACS sorted U2OS cells. Four arrhythmic clones from Bmal1 exon 6 targeted cells are shown along with two control traces. Raw bioluminescence rhythms are shown. Efficiency of knockouts (number of arrhythmic clones/total number) for E6 = 23/37 (62%); for E8 = 9/17 (53%); for E9 = 5/16 (31%). ( D ) Arrhythmicity is not due to off-target effect of the all-in-one CRISPR-Cas9. All arrhythmic clones tested were rescued by expressing wt BMAL1 protein using the adenoviral vector. Two representative cases are shown. Blue: arrhythmic clone; Green: the arrhythmic clone plus AV- wtBmal1 ; Black: control cell. ( E ) Knockouts were confirmed by immunoblots and sequencing (Supplementary Fig. 2). Representative arrhythmic clones from three different experiments ( Bmal1 exon 6, 8 and 9) are shown. Note that the BMAL1-rescued sample was under-loaded to show the difference in size clearly between endogenous and transgenic BMAL1 due to 3XFlag tag. ( F,G ) Knockout clones for Per2 were efficiently generated by the all-in-one vector targeting exon 5. Three knockout clones are shown, which was confirmed by immunoblots ( G ) and sequencing (Supplementary Fig. 4). Note that all knockout clones (red) show damped rhythms compared to control cells (black). The same control traces and the same scale for Y-axis were used in all three graphs. The graphs were de-trended to compare amplitudes between control and knockout samples. ( G ) PER2 exhibits robust rhythms in U2OS (left panel). The samples were harvested at the indicated times after 2-hr serum shock. The majority of clones with damped rhythms show absence of PER2 (middle panel). Note that serially diluted control samples (1, 1/2 and 1/3) were loaded next to the candidate samples to show the detection of PER2 at those levels and the corresponding absence in knockout clones. Note that clones 5–13 and 5–62 retain PER2 expression. *Indicates a nonspecific band. ( H ) Knockout was independently confirmed by a novel monoclonal antibody. Probing of time-course samples with the antibody showed similarly robust oscillations (Supplementary Fig. 7).

Article Snippet: For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA). hP2-GP49 was used in the current study.

Techniques: Knock-Out, Clone Assay, Plasmid Preparation, Luciferase, Mutagenesis, Binding Assay, Transduction, Expressing, Transfection, CRISPR, ATP Bioluminescent Assay, Single-cell Isolation, Western Blot, Sequencing, Transgenic Assay, Generated

Journal: Scientific Reports

Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

doi: 10.1038/s41598-018-36736-y

Figure Lengend Snippet: PCR primers for T7E1 assay.

Article Snippet: For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA). hP2-GP49 was used in the current study.

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: 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