Acquisition of Temporal HIF Transcriptional Activity Using a Secreted Luciferase Assay

2018 ◽  
pp. 37-44
Author(s):  
Miguel A. S. Cavadas ◽  
Cormac T. Taylor ◽  
Alex Cheong
Keyword(s):  
Transcriptional Activity ◽  
Luciferase Assay

P1608Inhibition of GATA4 dimerization suppress hypertrophic responses

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Shimizu ◽  
Y Sunagawa ◽  
K Hara ◽  
A Hishiki ◽  
Y Katanasaka ◽  
...  
Keyword(s):  
Heart Failure ◽  
Dna Binding ◽  
Cardiac Hypertrophy ◽  
Zinc Finger ◽  
Therapeutic Target ◽  
Transcriptional Activity ◽  
Luciferase Assay ◽  
Zinc Finger Motif ◽  
Zinc Finger Domain ◽  
Acetylation Site

Abstract Introduction Hypertrophic signals eventually reach the nuclei of cardiomyocytes, change patterns of gene expression, and cause the development of heart failure. During the development of heart failure, intrinsic histone acetyltransferase called p300 induce GATA4 acetylation. Acetylated GATA4 increases its DNA binding, up-regulates cardiac hypertrophic response genes, and lead to heart failure. A zinc finger protein, GATA4 is the transcription factor that expression level is high in heart. It has been reported that GATA1, the same GATA family, regulates transcriptional activity through its homo-dimerization. However, GATA4 homo-dimerization and its relationship to hypertrophic responses are still unknown. Purpose To clarify the relationship between GATA4 homo-dimerization and transcriptional activity and investigate whether inhibition of this homo-dimerization become therapeutic target for cardiac hypertrophy. Methods GST pull-down and DNA pull-down assay were performed using GST fusion full length and deletion mutants of GATA4 and biotin-conjugated ET-1 promoter probe including a GATA element. Recombinant C-zinc finger domain (256–326), including C-zinc finger motif (256–295) and acetylation site (308–326) was cross-linked using glutaraldehyde and subjected to silver staining. An expression plasmid with three GATA4-acetylation site mutant-conjugated with nuclear localization sequence (3xG4D) was constructed. Immunoprecipitation and western blotting were performed using nuclear extract from HEK293T cells expressing p300, GATA4, and 3xG4D. Luciferase assay was using ANF and ET-1 promoter sequences. Neonatal rat cultured cardiomyocyte expressed 3xG4D and then stimulated with phenylephrine (PE) for 48 hours. Next cardiomyocytes stained with α-actinin antibody and measured the cell surface area. Results The acetylation site of GATA4 was required for the dimerization of GATA4. But, C-zinc finger motif (256–295) and the acetylation site were required for the DNA binding. Recombinant C-zinc finger domain formed not only a homo-dimer but also a multimer. Co-expression of p300 increased the formation of homo-dimer as well as the acetylation of GATA4 in HEK293T cells. The GATA4 homo-dimer was disrupted by acetyl-deficient GATA4 or HAT-deficient p300 mutant. Overexpression of 3xG4D prevented the dimerization of GATA4, but not acetylation of GATA4. The result of luciferase assay showed that overexpression of 3xG4D prevented p300/GATA-induced ANF and ET-1 promoter activities. Furthermore, overexpression of 3xG4D inhibited phenylephrine-induced cardiomyocyte hypertrophy. Conclusions These results suggest that GATA4 dimerization may play an important role in hypertrophy-response gene activation. Thus, it is likely that inhabitation of GATA4 dimerization become therapeutic target for cardiac hypertrophy.

scholarly journals Cinobufagin Suppresses Melanoma Cell Growth by Inhibiting LEF1

2020 ◽  
Vol 21 (18) ◽  
pp. 6706
Author(s):  
Geon-Hee Kim ◽  
Xue-Quan Fang ◽  
Woo-Jin Lim ◽  
Jooho Park ◽  
Tae-Bong Kang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Growth ◽  
Tumor Growth ◽  
Cancer Cells ◽  
Melanoma Cell ◽  
Transcriptional Activity ◽  
Melanoma Cells ◽  
Lung Cancer Cells ◽  
Luciferase Assay ◽  
Dependent Manner

Constitutive activation of the β-catenin dependent canonical Wnt signaling pathway, which enhances tumor growth and progression in multiple types of cancer, is commonly observed in melanoma. LEF1 activates β-catenin/TCF4 transcriptional activity, promoting tumor growth and progression. Although several reports have shown that LEF1 is highly expressed in melanoma, the functional role of LEF1 in melanoma growth is not fully understood. While A375, A2058, and G361 melanoma cells exhibit abnormally high LEF1 expression, lung cancer cells express lower LEF1 levels. A luciferase assay-based high throughput screening (HTS) with a natural compound library showed that cinobufagin suppressed β-catenin/TCF4 transcriptional activity by inhibiting LEF1 expression. Cinobufagin decreases LEF1 expression in a dose-dependent manner and Wnt/β-catenin target genes such as Axin-2, cyclin D1, and c-Myc in melanoma cell lines. Cinobufagin sensitively attenuates cell viability and induces apoptosis in LEF1 expressing melanoma cells compared to LEF1-low expressing lung cancer cells. In addition, ectopic LEF1 expression is sufficient to attenuate cinobufagin-induced apoptosis and cell growth retardation in melanoma cells. Thus, we suggest that cinobufagin is a potential anti-melanoma drug that suppresses tumor-promoting Wnt/β-catenin signaling via LEF1 inhibition.

Triptolide Synergistically Enhances Temozolomide-Induced Apoptosis and Potentiates Inhibition of NF-κB Signaling in Glioma Initiating Cells

2014 ◽  
Vol 42 (02) ◽  
pp. 485-503 ◽  
Author(s):  
Ke Sai ◽  
Wen-Yu Li ◽  
Yin-Sheng Chen ◽  
Jian Wang ◽  
Su Guan ◽  
...  
Keyword(s):  
Western Blot ◽  
Transcriptional Activity ◽  
Combined Treatment ◽  
Acquired Resistance ◽  
Luciferase Assay ◽  
Solid Cancer ◽  
Tumor Sphere ◽  
Glioma Initiating Cells ◽  
Induced Apoptosis ◽  
Sphere Formation

Glioblastoma multiforme (GBM) is a lethal solid cancer in adults. Temozolomide (TMZ) is a first-line chemotherapeutic agent but the efficacy is limited by intrinsic and acquired resistance in GBM. Triptolide (TPL), a derivative from traditional Chinese medicine, demonstrated anti-tumor activity. In this study, we explored the interaction of TPL and TMZ in glioma-initiating cells (GICs) and the potential mechanism. A GIC line (GIC-1) was successfully established. Cell viability of GIC-1 after treatment was measured using a CCK-8 assay. The interaction between TPL and TMZ was calculated from Chou–Talalay equations and isobologram. Self-renewal was evaluated with tumor sphere formation assay. Apoptosis was assessed with flow cytometry and western blot. Luciferase assay was employed to measure NF-κB transcriptional activity. The expression of NF-κB downstream genes, NF-κB nuclear translocalization and phoshorylation of IκBα and p65 were evaluated using western blot. We found that GIC-1 cells were resistant to TMZ, with the expected IC50 of 705.7 μmol/L. Co-treatment with TPL yielded a more than three-fold dose reduction of TMZ. TPL significantly increased the percentage of apoptotic cells and suppressed the tumor sphere formation when combined with TMZ. Phosphorylation of IκBα and p65 coupled with NF-κB nuclear translocalization were notably inhibited after a combined treatment. Co-incubation synergistically repressed NF-κB transcriptional activity and downstream gene expression. TPL sensitizes GICs to TMZ by synergistically enhancing apoptosis, which is likely resulting from the augmented repression of NF-κB signaling. TPL is therefore a potential chemosensitizer in the treatment of GBM.

Functional characterization of chicken glucocorticoid and mineralocorticoid receptors

2010 ◽  
Vol 298 (5) ◽  
pp. R1257-R1268 ◽  
Author(s):  
Monika Proszkowiec-Weglarz ◽  
Tom E. Porter
Keyword(s):  
Cellular Localization ◽  
Transcriptional Activity ◽  
Functional Characterization ◽  
Nuclear Hormone Receptor ◽  
Avian Species ◽  
Full Length ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Dependent Manner ◽  
Specific Expression

Glucocorticoid (GR) and mineralocorticoid (MR) receptors are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Little is known about the function of GR and MR in avian species. Recently, the chicken homologue of the GR (cGR) gene was cloned, and its tissue-specific expression was characterized, whereas the full-length sequence of the chicken MR (cMR) gene remains unknown. Therefore, the aims of this project were to clone the full-length cMR and to functionally characterize both chicken receptors. Cos-7 cells were transiently transfected with cGR or cMR expression vectors along with a glucocorticoid response element-luciferase (GRE-Luc) reporter construct. Transfected cells were then treated with increasing doses of corticosterone (CORT) or aldosterone (ALDO) alone and with GR or MR antagonists (ZK98299 and spironolactone, respectively). Transactivation of cGR or cMR was evaluated by luciferase assay. CORT and ALDO induced cGR- and cMR-driven transcriptional activity in a dose-dependent manner. Each receptor responded to both steroids, but cMR transcriptional activity was induced by lower levels of CORT and ALDO than cGR. Coexpression of both chicken corticosteroid receptors in Cos-7 cells had no synergistic or additive effect on CORT- or ALDO-induced transcriptional activity. Corticosteroid-dependent transactivation of cGR and cMR was partially blocked by antagonists. ZK98299 showed high specificity to cGR, while spironolactone had agonist properties toward both receptors. Immunocytochemistry was used to assess the cellular localization of both receptors. Corticosteroids induced translocation of both receptors into the nucleus. The functional properties of cGR and cMR determined in this study will be helpful in defining the physiological roles of GR and MR in avian species.

The New -474(C→T) Substitution Discovered in the HBG2 Promoter of a Sardinian δβ-Thalassemia Carrier

2016 ◽  
Vol 136 (3) ◽  
pp. 178-185 ◽  
Author(s):  
Sandro Trova ◽  
Paolo Mereu ◽  
Elena Cocco ◽  
Bruno Masala ◽  
Laura Manca ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Regulatory Mechanism ◽  
Luciferase Assay ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Per Se ◽  
In Cis

During a screening for hemoglobinopathies, we found a carrier of the Sardinian δβ-thalassemia condition. The proband's hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α2Aγ2 and α2Gγ2 instead of nearly 100% α2Aγ2. In order to explain the unusual γ-chain ratio, sequencing of the Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the Gγ promoter. Results indicated that the observed in vivo Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

scholarly journals A Novel Tumor Suppressor Gene, ZNF24, Inhibits the Development of NSCLC by Inhibiting the WNT Signaling Pathway to Induce Cell Senescence

2021 ◽  
Vol 11 ◽  
Author(s):  
Bo Pang ◽  
Yong Wang ◽  
Xiaoyan Chang
Keyword(s):  
Tumor Suppressor ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Ectopic Expression ◽  
Wnt Signaling Pathway ◽  
Cell Senescence ◽  
Nsclc Cell ◽  
Luciferase Assay

ObjectiveUnderstanding the characteristics of tumor suppressor genes (TSGs) is of great significance for the development of new targeted treatment strategies for non-small cell lung cancer (NSCLC). Therefore, this present article is to explore the underlying molecular mechanism of ZFN24 inhibiting the development of NSCLC.MethodsWe performed RT-PCR and Western blotting for evaluating associated RNA and protein expression. CCK8, colony forming and sphere-forming assays were used to evaluate the proliferation and stemness of NSCLC cells. NSCLC cell senescence was examined by β-galactosidase staining assay. Luciferase assay was performed to evaluate β-catenin transcriptional activity. The effect of ZNF24 on NSCLC cells in vivo was evaluated by the xenograft tumor experiment.ResultsEctopic expression of ZNF24 significantly inhibited cell viability, colony forming ability, and stemness of NSCLC cells. WNT signaling pathway was inhibited by ZNF24 resulting in NSCLC cell senescence. β-catenin transcriptional activity was significantly inhibited by ZNF24 (P < 0.05). Ectopic expression of ZNF24 significantly inhibited xenotransplant tumors growth in vivo (P < 0.05).ConclusionZNF24 could notably inhibit the development of NSCLC by inhibiting the WNT signaling pathway.

scholarly journals Phorbol Esters with Wnt Signal-Augmenting Effects Isolated from Excoecaria Indica

2012 ◽  
Vol 7 (4) ◽  
pp. 1934578X1200700 ◽  
Author(s):  
Tatsuhiro Yamaguchi ◽  
Kazufumi Toume ◽  
Midori A. Arai ◽  
Firoj Ahmed ◽  
Samir K. Sadhu ◽  
...  
Keyword(s):  
Natural Products ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Phorbol Esters ◽  
Wnt Signaling Pathway ◽  
Fold Increase ◽  
Luciferase Assay ◽  
Bioassay Guided Fractionation ◽  
Wnt Signal

A screening of natural products using a luciferase assay targeting the Wnt signaling pathway was carried out, and the bioassay-guided fractionation of Excoecaria indica (Euphorbiaceae) collected from Bangladesh afforded three phorbol esters (1 – 3). These compounds exhibited Wnt signal-augmenting effects with 1 causing a 25-fold increase in TCF/β-catenin (TOP) transcriptional activity at 95 nM.

scholarly journals Dexamethasone suppresses Smad3 pathway in osteoblastic cells

2005 ◽  
Vol 185 (1) ◽  
pp. 131-138 ◽  
Author(s):  
Mei-Fway Iu ◽  
Hiroshi Kaji ◽  
Hideaki Sowa ◽  
Junko Naito ◽  
Toshitsugu Sugimoto ◽  
...  
Keyword(s):  
Bone Formation ◽  
Transcriptional Activity ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Luciferase Assay ◽  
Osteoblastic Cells ◽  
Specific Response ◽  
Type I ◽  
Alp Activity ◽  
Umr 106

Central in the pathogenesis of glucocorticoid (GC)-induced osteoporosis is the effects of GC on bone formation. However, the mechanism of GC-inhibited bone formation is not well known. Transforming growth factor (TGF)-β is most abundant in bone matrix compared with other tissues, and we have recently proposed that Smad3, a TGF-β signaling molecule, is important for promoting bone formation. However, no reports have been available about the effects of GC on Smad3 in osteoblasts. In the present study, we investigated whether dexamethasone (Dex), an active GC analog, would affect the expression and activity of Smad3 in mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Dex significantly suppressed Smad3-stimulated alkaline phosphatase (ALP) activity, although it did not affect TGF-β-inhibited ALP activity in MC3T3-E1 cells. Moreover, pretreatment with Dex suppressed TGF-β-enhanced expression of type I collagen in MC3T3-E1 and UMR-106 cells. In the luciferase assay using p3TP-Lux with a Smad3-specific response element, Dex significantly suppressed the transcriptional activity induced by TGF-β as well as Smad3. However, Dex did not affect the expression of Smad3 in these cells at both mRNA and protein levels. In conclusion, the present study indicates that Dex inhibits ALP activity and type I collagen expression, presumably by suppressing Smad3-induced transcriptional activity but not by modulating Smad3 expression in osteoblastic cells.

scholarly journals Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2396
Author(s):  
Diana Azenha ◽  
Santiago Hernandez-Perez ◽  
Yuse Martin ◽  
Marta S. Viegas ◽  
Alexandra Martins ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Rna Processing ◽  
Familial Breast Cancer ◽  
Transcriptional Activity ◽  
Cancer Susceptibility ◽  
Exon Skipping ◽  
Luciferase Assay ◽  
Genetic Changes ◽  
Multifunctional Protein ◽  
Physiological Processes

Claspin is a multifunctional protein that participates in physiological processes essential for cell homeostasis that are often defective in cancer, namely due to genetic changes. It is conceivable that Claspin gene (CLSPN) alterations may contribute to cancer development. Therefore, CLSPN germline alterations were characterized in sporadic and familial breast cancer and glioma samples, as well as in six cancer cell lines. Their association to cancer susceptibility and functional impact were investigated. Eight variants were identified (c.-68C>T, c.17G>A, c.1574A>G, c.2230T>C, c.2028+16G>A, c.3595-3597del, and c.3839C>T). CLSPN c.1574A>G (p.Asn525Ser) was significantly associated with breast cancer and was shown to cause partial exon skipping and decreased Claspin expression and Chk1 activation in a minigene splicing assay and in signalling experiments, respectively. CLSPN c.2028+16G>A was significantly associated with familial breast cancer and glioma, whereas c.2230T>C (p.Ser744Pro), was exclusively detected in breast cancer and glioma patients, but not in healthy controls. The remaining variants lacked a significant association with cancer. Nevertheless, the c.-68C>T promoter variant increased transcriptional activity in a luciferase assay. In conclusion, some of the CLSPN variants identified in the present study appear to modulate Claspin’s function by altering CLSPN transcription and RNA processing, as well as Chk1 activation.

scholarly journals Identification of SMAD3 as a Novel Mediator of Inflammation in Human Myometrium In Vitro

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Martha Lappas
Keyword(s):  
Preterm Birth ◽  
Proinflammatory Cytokines ◽  
Transcriptional Activity ◽  
Transforming Growth Factor ◽  
Luciferase Assay ◽  
Human Myometrium ◽  
Loss Of Function ◽  
Myometrial Cells ◽  
Polycytidylic Acid

Preterm birth remains the primary cause of early neonatal death and is a major determinant for long-term health consequences. Aberrant intrauterine inflammation and infection are known to augment the synthesis of proinflammatory cytokines and induce uterine contractions, which can subsequently lead to preterm birth. The transforming growth factor-β (TGF-β) superfamily members regulate numerous cellular processes through the activation of intracellular mediators known as mothers against decapentaplegic homolog (SMADs). Studies in nongestational tissues have shown that SMAD3 plays a role in immune regulation and inflammation; however, its role in human labour remains unknown. Thus, the present study aimed at (i) characterising the expression of SMAD3 in the human myometrium; (ii) determining the effect of bacterial and viral products and proinflammatory cytokines on SMAD3 transcriptional activity in primary human myometrial cells; and (iii) investigating the effect of SMAD3 siRNA knockdown on the production of prolabour mediators in primary human myometrial cells. Phosphorylated (i.e., active) SMAD3 protein expression was lower in the myometrium after spontaneous term labour compared to the myometrium from nonlabouring women. Using a luciferase assay, the proinflammatory cytokines IL-1β and TNF, and viral analogue polyinosinic : polycytidylic acid (poly(I : C)) significantly reduced SMAD3 transcriptional activity in human primary myometrial cells. Loss-of-function studies found that SMAD3 knockdown in myometrial cells significantly increased IL-1β- and poly(I : C)-induced proinflammatory cytokines (IL-1A, IL-6), chemokines (IL-8, MCP-1), the adhesion molecule ICAM-1, COX-2 mRNA expression, and subsequent PGF2α release. In conclusion, SMAD3 deficiency is associated with increased production of proinflammatory and prolabour mediators in the human myometrium.

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