human per2 promoter sequence  (Addgene inc)

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2025.1525844

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

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

Techniques:

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2025.1525844

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

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

Techniques: Cell Culture, Immunocytochemistry, Isolation

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2025.1525844

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

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

Techniques: Expressing

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2025.1525844

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

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

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

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2025.1525844

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

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

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

Journal: Theranostics

Article Title: Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model

doi: 10.7150/thno.70893

Figure Lengend Snippet: Expression of clock proteins in 3D chondrogenic pellets. Representative immunohistochemistry of chondrogenic pellets at day 11 (2D) + day 10 (3D) stained with primary antibodies to clock factors CLOCK, PER2 and BMAL1. N = 3. Negative control sections were incubated without the primary antibody. Panels on the right show higher magnifications of images on the left panels. Scale bars = 100 µm.

Article Snippet: Promoter sequences of human BMAL1 and PER2 genes were synthesised (Thermo Fisher) and subcloned into this vector upstream of the sequence encoding the luciferase enzyme (vector maps in ) using the NEBuilder® HiFi DNA Assembly (NEB).

Techniques: Expressing, Immunohistochemistry, Staining, Negative Control, Incubation

Journal: Theranostics

Article Title: Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model

doi: 10.7150/thno.70893

Figure Lengend Snippet: Expression of clock proteins are readily detectable in hESCs. (A). Representative IF of clock factors in hESCs. MAN13 cells were fixed and incubated with antibodies against CLOCK, BMAL1, PER2 and CRY1, followed by Alexa Fluor plus 488 antibody. Nuclei were stained with DAPI. Scale bars represent 100 μm. Representative images from N = 3 except CRY1 (N = 2). (B). Co-expression of CLOCK (in green) and OCT4 (in red) proteins in MAN13 hESCs by IF. Nuclei were stained with DAPI. Scale bar = 100 μm. Representative images of N = 2.

Article Snippet: Promoter sequences of human BMAL1 and PER2 genes were synthesised (Thermo Fisher) and subcloned into this vector upstream of the sequence encoding the luciferase enzyme (vector maps in ) using the NEBuilder® HiFi DNA Assembly (NEB).

Techniques: Expressing, Incubation, Staining