scholarly journals Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

2019 ◽  
Author(s):  
Alejandro Sarrion-Perdigones ◽  
Lyra Chang ◽  
Yezabel Gonzalez ◽  
Tatiana Gallego-Flores ◽  
Damian W. Young ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Emission Spectra ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Research Fields ◽  
Experimental Errors ◽  
Cellular Pathways ◽  
Multiple Signaling ◽  
Simultaneous Assessment ◽  
Spectra Deconvolution

AbstractSensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases have arisen as possible genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. Here, we expanded luciferase multiplexing in post-lysis endpoint luciferase assays from two towards six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. Using synthetic assembly cloning, all six luciferase reporter units are stitched together into one plasmid; facilitating delivery of all reporter units through a process we named solotransfection, minimizing experimental errors. We engineered a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We were able to monitor the effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, as well as its broad application across different research fields.

scholarly journals Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Alejandro Sarrion-Perdigones ◽  
Lyra Chang ◽  
Yezabel Gonzalez ◽  
Tatiana Gallego-Flores ◽  
Damian W. Young ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Emission Spectra ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Research Fields ◽  
Experimental Errors ◽  
Cellular Pathways ◽  
Multiple Signaling ◽  
Simultaneous Assessment ◽  
Spectra Deconvolution

AbstractSensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.

scholarly journals CHOP and c-JUN up-regulate the mutant Z α1-antitrypsin, exacerbating its aggregation and liver proteotoxicity

2020 ◽  
Vol 295 (38) ◽  
pp. 13213-13223
Author(s):  
Sergio Attanasio ◽  
Rosa Ferriero ◽  
Gwladys Gernoux ◽  
Rossella De Cegli ◽  
Annamaria Carissimo ◽  
...  
Keyword(s):  
Liver Disease ◽  
Er Stress ◽  
Proteinase Inhibitors ◽  
Pediatric Population ◽  
Transcriptional Response ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Primary Role ◽  
Loss Of Function ◽  
Human Livers

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop−/− mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop−/− mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter–based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.

scholarly journals Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

2012 ◽  
Vol 18 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Ellen Siebring-van Olst ◽  
Christie Vermeulen ◽  
Renee X. de Menezes ◽  
Michael Howell ◽  
Egbert F. Smit ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Chemical Components ◽  
Simple Liquid ◽  
Luciferase Reporter Construct ◽  
Reagent Cost ◽  
Firefly Luciferase Gene

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

scholarly journals MicroRNA-122 Inhibits Lipid Droplet Formation and Hepatic Triglyceride Accumulation via Yin Yang 1

2017 ◽  
Vol 44 (4) ◽  
pp. 1651-1664 ◽  
Author(s):  
Guo-yi Wu ◽  
Chen Rui ◽  
Ji-qiao Chen ◽  
Eiketsu Sho ◽  
Shan-shan Zhan ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Lipid Droplet ◽  
Droplet Formation ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Hepatic Triglyceride ◽  
Yin Yang 1 ◽  
Lipid Droplet Formation ◽  
Huh7 Cells ◽  
Yin Yang

Background/Aims: An increase in intracellular lipid droplet formation and hepatic triglyceride (TG) content usually results in nonalcoholic fatty liver disease. However, the mechanisms underlying the regulation of hepatic TG homeostasis remain unclear. Methods: Oil red O staining and TG measurement were performed to determine the lipid content. miRNA expression was evaluated by quantitative PCR. A luciferase assay was performed to validate the regulation of Yin Yang 1 (YY1) by microRNA (miR)-122. The effects of miR-122 expression on YY1 and its mechanisms involving the farnesoid X receptor and small heterodimer partner (FXR-SHP) pathway were evaluated by quantitative PCR and Western blot analyses. Results: miR-122 was downregulated in free fatty acid (FFA)-induced steatotic hepatocytes, and streptozotocin and high-fat diet (STZ-HFD) induced nonalcoholic steatohepatitis (NASH) in mice. Transfection of hepatocytes with miR-122 mimics before FFA induction inhibited lipid droplet formation and TG accumulation in vitro. These results were verified by overexpressing miR-122 in the livers of STZ-HFD-induced NASH mice. The 3’-untranslated region (3’UTR) of YY1 mRNA is predicted to contain an evolutionarily conserved miR-122 binding site. In silico searches, a luciferase reporter assay and quantitative PCR analysis confirmed that miR-122 directly bound to the YY1 3’UTR to negatively regulate YY1 mRNA in HepG2 and Huh7 cells. The (FXR-SHP) signaling axis, which is downstream of YY1, may play a key role in the mechanism of miR-122-regulated lipid homeostasis. YY1-FXR-SHP signaling, which is negatively regulated by FFA, was enhanced by miR-122 overexpression. This finding was also confirmed by overexpression of miR-122 in the livers of NASH mice. Conclusions: The present results indicate that miR-122 plays an important role in lipid (particularly TG) accumulation in the liver by reducing YY1 mRNA stability to upregulate FXR-SHP signaling.

scholarly journals Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts

2006 ◽  
Vol 12 (1) ◽  
pp. 140-144 ◽  
Author(s):  
Michael K. Hancock ◽  
Myleen N. Medina ◽  
Brendan M. Smith ◽  
Anthony P. Orth
Keyword(s):  
High Throughput ◽  
Dynamic Range ◽  
Cell Lysis ◽  
Single Step ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Activity Measurements ◽  
Simple Detection ◽  
Reporter Assays ◽  
Orbital Mixing

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

Effect of Down-Regulation of miR-23b-3p on the Differentiation of Acute Myeloid Leukemia via Wilms Cancer Gene 1

2021 ◽  
Vol 11 (7) ◽  
pp. 1377-1382
Author(s):  
Lixia Cao ◽  
Jing Zhang ◽  
Huijuan Ren ◽  
Yanqiu Han
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Hot Spot ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Blank Group ◽  
Acute Myeloid

miRNA has always been a hot spot research. We assessed the effect of down-regulation of miR-23b-3p on the differentiation of acute myeloid leukemia (AML). Human AML cell line U937 was divided into blank group, NC group and miR-23b-3p low expression group (transfected with miR-23b-3p inhibitor) and miR-23b-3p followed by analysis of WT1 level and relationship between miR-23b-3p and WT1 by dual luciferase reporter assay. All-trans retinoic acid is used to induce differentiation, and then the morphological changes of cells and CD11b level were detected. When miR-23b-3p level was reduced, WT1 mRNA and protein level was also decreased. Dual luciferase assay showed that miR-23b-3p bound to WT1 3’-UTR. Inhibition of miR-23b-3p significantly decreased cell proliferation. Swiss Giemsa staining showed that most of cells were in the differentiation stage with low miR-23b-3p expression. The differentiation marker CD11b was significantly higher than other groups, indicating that low miR-23b-3p expression can promote cell differentiation and reduce cell proliferation to a certain extent. Under low miR-23b-3p expression, the positive rate of CD11b was significantly increased. Down-regulating miR-23b-3p can inhibit WT1 to a certain extent and promote the differentiation of AML, which provides a guidance for the gene-level treatment of AML.

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

2021 ◽  
Author(s):  
Jie Lan ◽  
Chunhui Sun ◽  
Xinping Liang ◽  
Ruixin Ma ◽  
Yuhua Ji ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Transcriptional Activation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Thyroid Dysgenesis ◽  
Type Protein ◽  
Wild Type Protein

Abstract Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.

scholarly journals Luteolin Inhibits Respiratory Syncytial Virus Replication by Regulating the MiR-155/SOCS1/STAT1 Signaling Pathway

2020 ◽  
Author(s):  
Saisai Wang ◽  
Yiting Ling ◽  
Yuanyuan Yao ◽  
Gang Zheng ◽  
Wenbin Chen
Keyword(s):  
Respiratory Syncytial Virus ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Virus Replication ◽  
Janus Kinase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Cytokine Signaling ◽  
Stat1 Signaling ◽  
Syncytial Virus

Abstract Background: Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in infants, children, immunocompromised adults, and elderly individuals. Currently, there are few therapeutic options available to prevent RSV infection. The present study aimed to investigate the effects of luteolin on RSV replication and the related mechanisms. Material and methods: We pretreated cells and mice with luteolin before infection with RSV, the virus titer, expressions of RSV-F, interferon (IFN)-stimulated genes (ISGs), and production of IFN-α and IFN-β were determined by plaque assay, RT-qPCR, and ELISA, respectively. The activation of Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) signaling pathway was detected by Western blotting and luciferase assay. Proteins which negatively regulates STAT1 was determined by Western blotting. Then cells were transfected with suppressor of cytokine signaling 1 (SOCS1) plasmid and virus replication and ISGs expression was determined. Luciferase reporter assay and Western blotting was performed to detect the relationship between SOCS1 and miR-155. Results: Luteolin inhibited RSV replication, as shown by the decreased viral titer and RSV-F mRNA expression both in vitro and in vivo. The antiviral activity of luteolin was attributed to the enhanced phosphorylation of STAT1, resulting in the increased production of ISGs. Further study showed that SOCS1 was downregulated by luteolin and SOCS1 is a direct target of microRNA-155 (miR-155). Inhibition of miR-155 rescued luteolin-mediated SOCS1 downregulation, whereas upregulation of miR-155 enhanced the inhibitory effect of luteolin. Conclusion: Luteolin inhibits RSV replication by regulating the miR-155/SOCS1/STAT1 signaling pathway.

scholarly journals Dihydromyricetin promotes autophagy and attenuates renal interstitial fibrosis by regulating miR-155-5p/PTEN signaling in diabetic nephropathy

2019 ◽  
Author(s):  
Liming Guo ◽  
Kuibi Tan ◽  
Qun Luo ◽  
Xu Bai
Keyword(s):  
Diabetic Nephropathy ◽  
Signaling Pathway ◽  
Rat Model ◽  
Interstitial Fibrosis ◽  
Mtor Signaling ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Mtor Signaling Pathway ◽  
Renal Interstitial Fibrosis ◽  
Gene Assay

Diabetic nephropathy (DN) is the most common complication of diabetes and is prone to kidney failure. Dihydromyricetin (DHM) has been reported to have a variety of pharmacological activities. This study aims to explore the effect of DHM on DN and the underlying molecular mechanism. An in vivo DN rat model was established. The degree of renal interstitial fibrosis (RIF) was detected by hematoxylin-eosin (HE) staining, Masson's trichrome staining, and immunohistochemistry (IHC). In vitro, NRK-52E cells were divided into four groups: normal glucose (NG), high glucose (HG), HG+DHM, and HG+rapamycin (autophagy inhibitor). The levels of autophagy- and fibrosis-related proteins were analyzed by western blotting. The expression of miR-155-5p and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and their relationship were assessed by quantitative reverse transcription (qRT)-PCR and dual luciferase reporter gene assay. Our results showed that RIF was increased in DN rat model and in HG-induced NRK-52E cells. DHM treatment attenuated the increased RIF and also increased autophagy. MiR-155-5p expression was increased, while PTEN expression was decreased in DN rat and cell model, and DHM reversed both effects. Dual luciferase assay showed that PTEN was the target gene of miR-155-5p. DHM inhibited HG-induced fibrosis and promoted autophagy by inhibiting miR-155-5p expression in NRK-52E cells. In addition, DHM promoted autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway. In conclusion, DHM promotes autophagy and attenuates RIF by regulating the miR-155-5p/PTEN signaling and PI3K/AKT/mTOR signaling pathway in DN.

scholarly journals US28: HCMV’s Swiss Army Knife

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 445 ◽  
Author(s):  
Benjamin Krishna ◽  
William Miller ◽  
Christine O’Connor
Keyword(s):  
Latent Infection ◽  
Viral Latency ◽  
Lytic Replication ◽  
G Protein Coupled Receptors ◽  
Disease States ◽  
Cellular Pathways ◽  
The Impact ◽  
Multiple Signaling ◽  
G Protein Coupled ◽  
Multifunctional Properties

US28 is one of four G protein coupled receptors (GPCRs) encoded by human cytomegalovirus (HCMV). The US28 protein (pUS28) is a potent signaling molecule that alters a variety of cellular pathways that ultimately alter the host cell environment. This viral GPCR is expressed not only in the context of lytic replication but also during viral latency, highlighting its multifunctional properties. pUS28 is a functional GPCR, and its manipulation of multiple signaling pathways likely impacts HCMV pathogenesis. Herein, we will discuss the impact of pUS28 on both lytic and latent infection, pUS28-mediated signaling and its downstream consequences, and the influence this viral GPCR may have on disease states, including cardiovascular disease and cancer. We will also discuss the potential for and progress towards exploiting pUS28 as a novel therapeutic to combat HCMV.

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