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For the quantification of insulin activity, United States Pharmacopeia (USP) general chapter <121> continues to require the rabbit blood sugar test. For new insulin or insulin analogue compounds, those quantitative data are expected for stability or comparability studies. At Sanofi, many rabbits were used to fulfil the authority's requirements to obtain quantitative insulin bioactivity data until the in vivo test was replaced. In order to demonstrate comparability between the in vivo and in vitro test systems, this study was designed to demonstrate equivalency. The measurement of insulin lispro and insulin glargine drug substance and drug product batches, including stress samples (diluted or after temperature stress of 30 min at 80 °C), revealed a clear correlation between the in vitro and in vivo test results. The recovery of quantitative in vitro in-cell Western (ICW) results compared to the in vivo test results was within the predefined acceptance limits of 80% to 125%. Thus, the in vitro ICW cell-based bioassay leads to results that are equivalent to the rabbit blood sugar test per USP <121>, and it is highly suitable for insulin activity quantification. For future development compounds, the in vitro in-cell Western cell-based assay can replace the rabbit blood sugar test required by USP <121>.
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Ferroptosis is a regulated form of cell death caused by the excessive accumulation of iron-dependent lipid peroxidation. It has been implicated in various pathological processes and diseases, and its modulation involves multiple proteins associated with iron and lipid metabolism. To better understand these mechanisms and monitor the ferroptosis process, there is a need for reliable and high-throughput methods to evaluate variations in protein expression levels. In-Cell Western assays provide a simple and rapid assay method for detecting biomarkers and signaling proteins in whole cells using antibodies. This assay involves seeding cells in microtiter plates, followed by fixation/permeabilization and subsequent labeling with primary antibodies and infrared-conjugated secondary antibodies. In this chapter, we introduce the protocol for the In-Cell Western assay for detecting intracellular proteins during ferroptosis.
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Ferroptosis , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular , Hierro/metabolismo , Peroxidación de LípidoRESUMEN
Insulin is a hormone produced by ß-cells of the pancreas and controls the amount of sugar in the blood. Since its discovery over 100 years ago, insulin has been used as a life-saving treatment for people with diabetes. Historically, the biological activity or bioidentity of insulin products has been assessed using an in vivo model. However, reduction in animal experiments is a goal for many worldwide, and there is a need to develop in vitro bioassays to reliably test the biological activity of insulin products. This article describes an in vitro cell-based method to assess the biological activity of insulin glargine, insulin aspart, and insulin lispro in a step-by-step manner.
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The In-Cell Western plate-based immunofluorescence assay is a useful methodology for monitoring protein levels and provides a facile moderate through-put method for PROTAC and degrader optimization. The method is compared to other reported assays used for PROTAC development. The advantages of this method are the greater through-put compared to Western blots due to its plate-based method and the ease to transfer between cells lines. Adherent cell lines are preferred, although suspension cells can be used following recommended modifications and precautions to the protocol. This method requires a high-quality antibody that recognizes the protein epitope in its cellular context, and in general provides data similar to Western blots with higher assay through-put.
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Proteínas , Proteolisis , Línea Celular TumoralRESUMEN
The in-cell western (ICW) is an immunocytochemical technique that has been used to screen for effects of siRNAs, drugs, and small molecule inhibitors. The reduced time and number of cells required to follow this protocol illustrates its semi-high-throughput nature. Performing a successful ICW protocol requires fixing and permeabilizing adherent cells directly in the plate that specifically exposes the epitope of interest. After blocking of non-specific proteins, the cells are incubated overnight with a primary antibody of interest, which is detected via a host-specific near-infrared fluorescently labeled LI-COR secondary antibody. In the final step, the plate is scanned using an Odyssey LI-COR Imaging System or similar, and each of the wells is quantified. For the first time, this technique has been demonstrated to be reproducibly utilized for semi-high-throughput selection of knockout or overexpression clones. Graphical abstract.
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Background: Extracellular signal-regulated kinases (ERKs) are important signaling mediators in mammalian cells and, as a result, one of the major areas of research focus. The detection and quantification of ERK phosphorylation as an index of activation is normally conducted using immunoblotting, which does not allow high-throughput drug screening. Plate-based immunocytochemical assays provide a cheaper and relatively high-throughput alternative method for quantifying ERK phosphorylation. Here, we present optimization steps aimed to increase assay sensitivity and reduce variance and cost using the LI-COR In-Cell Western (I-CW) system in a recombinant CHO-K1 cell line, over-expressing the human delta-opioid receptor (hDOPr) as a model. Methods: Cells cultured in 96-well microassay plates were stimulated with three standard/selective DOPr agonists (SNC80, ADL5859, and DADLE) and a novel selective DOPr agonist (PN6047) to elicit a phospho-ERK response as an index of activation. A number of experimental conditions were investigated during the assay development. Key results: Preliminary experiments revealed a clearly visible edge-effect which significantly increased assay variance across the plate and which was reduced by pre-incubation for 30 min at room temperature. ERK phosphorylation was detectable as early as 1 min after agonist addition, with a distinct peak at 3-5 min. Optimization of the cell seeding densities showed that 25,000 cells per well have the lowest basal phospho-ERK response and an optimal agonist ERK1/2 signal. Pre-incubation with apyrase (an ATPase) did not reduce the basal or agonist responses. All agonists produced concentration-dependent increases in phospho-ERK activation, and pertussis toxin was able to attenuate these ERK responses. Naltrindole, which is a selective DOPr antagonist, was able to antagonize the DOPr-mediated ERK activation of the ligands. Conclusion: We have developed an optimization protocol and highlighted a number of considerations when performing this high-throughput fluorescence immunocytochemical (ICC) assay measuring ERK phosphorylation in the human DOPr. The optimized protocol was found to be a more conducive option for the screening of delta agonists. This provides a basis for additional assay development to investigate opioid pharmacology. This protocol should be widely applicable for measuring ERK phosphorylation in any cell line and investigating other protein targets in GPCR drug discovery.
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Long interspersed nuclear element 1 (LINE-1) is a dominant autonomous retrotransposon in human genomes which plays a role in affecting the structure and function of somatic genomes, resulting in human disorders including genetic disease and cancer. LINE-1 encoded ORF1p protein which possesses RNA-binding and nucleic acid chaperone activity, and interacts with LINE-1 RNA to form a ribonucleoprotein particle (RNP). ORF1p can be detected in many kinds of tumors and its overexpression has been regarded as a hallmark of histologically aggressive cancers. In this study, we developed an In-Cell Western (ICW) assay in T47D cells to screen the compounds which can decrease the expression of ORF1p. Using this assay, we screened 1,947 compounds from the natural products library of Target Mol and Selleckchem, among which three compounds, Hydroxyprogesterone, 2,2':5',2â³-Terthiophene and Ethynyl estradiol displayed potency in diminishing LINE-1 ORF1p expression level. Further mechanistic studies indicated the compounds act by affecting LINE-1 RNA transcription. Notably, we demonstrated that the compounds have an inhibitory effect on the proliferation of several lung and breast cancer cell lines. Taken together, we established a high throughput screening system for ORF1p expression inhibitors and the identified compounds provide some clues to the development of a novel anti-tumor therapeutic strategy by targeting ORF1p.
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Evaluation of in vivo potencies plays an important role in drug discovery. Traditionally, the cellular activity and percent of plasma protein binding of a test agent are evaluated separately, with the plasma protein binding-adjusted cellular potency computation used to estimate in vivo potency. This process is costly, takes weeks to complete, and is increasingly unreliable for compounds that bind extensively to plasma proteins. Described in this article is a simple, high-throughput human plasma in-cell Western (ICW) assay that directly incorporates plasma protein binding into a cellular pharmacodynamic assay to provide a rapid and accurate estimate of in vivo potencies. The assay is versatile and can be readily employed for various targets that require short treatment periods for displaying maximal biological responses. © 2021 Wiley Periodicals LLC. Basic Protocol: Concentration-dependent human plasma ICW assay to determine test compound IC50 against the target of interest.
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Bioensayo , Descubrimiento de Drogas , Proteínas Sanguíneas/metabolismo , Humanos , Unión ProteicaRESUMEN
Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers, including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that through the activities of the viral protein kinase conserved across herpesviruses and two cellular proteins, ATM and KAP1, a lytic cycle amplification loop is established, and disruption of this loop disables the EBV lytic cascade. We therefore devised a high-throughput screening assay, screened a small-molecule-compound library, and identified 17 candidates that impair the release of lytically replicated EBV. The identified compounds will (i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses, and (ii) be developed into anticancer agents, as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for antiherpesviral and anticancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small-molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even antiherpesviral potential, as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both antiherpesviral and anticancer drugs.
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Antivirales/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Herpesvirus Humano 4/efectos de los fármacos , Proteínas Quinasas/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Transactivadores/genética , Proteína 28 que Contiene Motivos Tripartito/genética , Antivirales/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/virología , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Regulación de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crecimiento & desarrollo , Herpesvirus Humano 4/metabolismo , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Lisogenia/efectos de los fármacos , Fosforilación , Proteínas Quinasas/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Transactivadores/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación ViralRESUMEN
Sickle-cell disease (SCD) is a debilitating hematological disorder with very few approved treatment options. Therapeutic reactivation of fetal hemoglobin (HbF) is one of the most pursued methods for ameliorating the systemic manifestations of SCD. Despite this, very few pharmacological agents have advanced to clinical trials or marketing for use. In this study, we report the development of an HbF in situ intracellular immunoblot assay coupled to a high-throughput drug screen to identify Food and Drug Administration (FDA) approved drugs that can be repurposed clinically for treatment of SCD. Using this assay we evaluated the National Institute of Health (NIH) Clinical Collection (NCC), a publicly available library of 725 small molecules, and found nine candidates that can significantly re-express HbF in erythroid cell lines as well as primary erythroblasts derived from SCD patients. Furthermore, we show the strong effects on HbF expression of these candidates to occur with minimal cytotoxicity in 7 of the 9 drugs. Given these data and their proven history of use for other indications, we hypothesize that several of these candidate drugs warrant further investigation for use in SCD.
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BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.
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Infecciones Bacterianas/diagnóstico , Técnicas Bacteriológicas/métodos , Chlamydia trachomatis/crecimiento & desarrollo , Escherichia coli/crecimiento & desarrollo , Shigella flexneri/crecimiento & desarrollo , Línea Celular , Chlamydia trachomatis/aislamiento & purificación , Recuento de Colonia Microbiana , Escherichia coli/aislamiento & purificación , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Biológicos , Reproducibilidad de los Resultados , Shigella flexneri/aislamiento & purificaciónRESUMEN
Synaptic adhesion molecules, including presynaptic neurexins (NRXNs) and post-synaptic leucine-rich repeat transmembrane (LRRTM) proteins are important for development and maintenance of brain neuronal networks. NRXNs are probably the best characterized synaptic adhesion molecules, and one of the major presynaptic organizer proteins. The LRRTMs were found as ligands for NRXNs. Many of the synaptic adhesion proteins have been linked to neurological cognitive disorders, such as schizophrenia and autism spectrum disorders, making them targets of interest for both biological studies, and towards drug development. Therefore, we decided to develop a screening method to target the adhesion proteins, here the LRRTM-NRXN interaction, to find small molecule probes for further studies in cellular settings. To our knowledge, no potent small molecule compounds against the neuronal synaptic adhesion proteins are available. We utilized the AlphaScreen technology, and developed an assay targeting the NRXN-LRRTM2 interaction. We carried out screening of 2000 compounds and identified hits with moderate IC50-values. We also established an orthogonal in-cell Western blot assay to validate hits. This paves way for future development of specific high affinity compounds by further high throughput screening of larger compound libraries using the methods established here. The method could also be applied to screening other NRXN-ligand interactions.
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Proteínas de Unión al Calcio/antagonistas & inhibidores , Enfermedades del Sistema Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/antagonistas & inhibidores , Proteínas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Drosophila , Proteínas Repetidas Ricas en Leucina , Ratones , Modelos Moleculares , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMEN
BACKGROUND: The two isoforms of the eukaryotic Elongation Factor 1A (eEF1A1 and eEF1A2), sustain the progression/aggressiveness of cancer cells. Thus, they are considered promising therapeutic targets and prognostic markers. It follows that their precise quantification is of utmost relevance in research and development. The simultaneous quantification of A1 and A2 proteins in the cells helps the comprehension of cancer biology mechanisms and response to drug treatments. However, the high homology at the amino-acidic level (92%) can cause antibodies cross-reaction. Moreover, the commonly employed western blotting just gives semi-quantitative data and does not allow the detection of both protein targets within the same cell. Thus, we developed an in cell western (ICW) technique to bypass the above limitations. METHODS: Firstly, relevant antibodies cross-reaction was excluded by immunohistochemistry on normal pancreatic tissue; then eEF1A1-A2 protein levels were quantitated by ICW in prostate and colorectal cancer cell lines in 96 well plates under different conditions, which include: 1) drug treatment, 2) siRNA silencing, 3) cell seeding density. RESULTS: We show that: 1) eEF1A1-A2 levels vary depending on the cell type following drug treatment, 2) ICW can accurately detect eEF1A1-A2 protein levels following siRNA silencing, 3) cell seeding density influences eEF1A1-A2 levels, depending on cell type. CONCLUSIONS: ICW is a valuable tool to specifically determine the intracellular level of eEF1A1-A2 proteins thus contributing to better define their role as potential therapeutic targets and prognostic markers in human tumors as well as for drug effects screening.
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Western Blotting/métodos , Factor 1 de Elongación Peptídica/aislamiento & purificación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Humanos , Espacio Intracelular/química , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factor 1 de Elongación Peptídica/antagonistas & inhibidores , Factor 1 de Elongación Peptídica/metabolismoRESUMEN
INTRODUCTION: Elevation in the level of intracellular cAMP is known to induce astrocytic differentiation of C6 glioma cells by unknown mechanisms. METHODS: Therefore, cytoskeletal protein genes (phalloidin) fluorescents to investigate morphological changes, cell proliferation assay, MTT assay, flow cytometry, western blotting, in-cell western, immune-cytochemical (protein expression and localization), and oxygen electrodes (oxygen consumption rate) after a treatment with 0.25 mM dbcAMP were conducted. RESULTS: Undifferentiated cells (media without dbcAMP) showed a flat polygonal appearance, whereas those cultured in the presence of 0.25 mM dbcAMP exhibited a more differentiated astrocytic morphology. They had more numerous neurite-like thin processes. The cell proliferation of differentiated c6 glioma reduced at day 2 and then started to increase at day 3 till day 5 compared to undifferentiated c6 glioma cells. In terms of flow-cytometry data, dbcAMP had no apoptotic effect on the C6 glioma cells. There was an increase in the protein expression GFAP (specific marker for astrocytes). There was no significant effect between undifferentiated and 5-day differentiation regarding their response to glucose 10 mM. In addition, there were no significant effects of glucose on the basal of 5-day differentiation of C6 glioma cells. However, there was a significant correlation between the concentration of glucose and inhibition of the basal oxygen consumption. Finally, glucose 10 mM did not stimulate NAD (P)H levels of C6 glioma cells. CONCLUSION: The above results showed that cAMP induce C6 glioma cells differentiation without affecting its bioenergetics. Therefore cAMP is considered to be the best differentiating agent.
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Astrocitos/fisiología , Diferenciación Celular/fisiología , AMP Cíclico/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/metabolismo , Glucosa/metabolismo , Consumo de Oxígeno/fisiología , Animales , Astrocitos/metabolismo , Línea Celular Tumoral , RatasRESUMEN
G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB1) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB1 levels and/or signaling may be beneficial for HD patients while decreasing CB1 levels and/or signaling may be detrimental. Recent studies have found that Gαi/o-biased CB1 agonists activate extracellular signal-regulated kinase (ERK), increase CB1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB1 agonists that are ß-arrestin1-biased were found to reduce CB1 protein levels and cell viability. Measuring agonist bias of known and novel CB1 agonists will provide important data that predict CB1-specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.
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Agonistas de Receptores de Cannabinoides/farmacología , Técnica del Anticuerpo Fluorescente/métodos , Enfermedad de Huntington/tratamiento farmacológico , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Agonistas de Receptores de Cannabinoides/uso terapéutico , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular/efectos de los fármacos , Cuerpo Estriado/patología , Endocannabinoides/metabolismo , Técnica del Anticuerpo Fluorescente/instrumentación , Células HEK293 , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Ligandos , Ratones , Receptor Cannabinoide CB1/agonistasRESUMEN
Rho-associated kinase (ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system. Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase (ERK) signaling pathway, but its effect on microglial migration was unknown. Therefore, in this study, we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord, and we examined the underlying mechanisms. The microglia were treated with Y27632, fasudil and/or the ERK inhibitor U0126. Cellular morphology was observed by immunofluorescence. Transwell chambers were used to assess cell migration. ERK levels were measured by in-cell western blot assay. Y27632 and fasudil increased microglial migration, and the microglia were irregularly shaped and had many small processes. These inhibitors also upregulated the levels of phosphorylated ERK protein. The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil. These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.
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To perform cell-based assays using fluorescence as the readout there is a fundamental need to identify individual cellular objects. In the majority of cases this requires the addition of a DNA dye or so-called nuclear counterstain and these have become integral to assay design. End-point assays can use live or fixed cells and thus it is beneficial if such reagents are cell membrane-permeant.Further, membrane-permeant DNA dyes can open new opportunities in dynamic real time assays with caveats according to the impact of their interaction with the chromatin in live cells. As cell-based assays offer information on the in vitro toxicity of treatments, cell viability has become a basic readout and cell membrane-impermeant fluorescent DNA-specific dyes can provide this information.In the case of both nuclear counterstaining and viability reporting, it is beneficial if the DNA dyes employed are suitably spectrally separated to permit multi-color experimental design. Methods will be described for these two important assay readouts.
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Bioensayo/métodos , Sondas de ADN , Colorantes Fluorescentes , Antraquinonas/farmacología , Biomarcadores , Supervivencia Celular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Imagen MolecularRESUMEN
AIMS: New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates. METHODS: An 'in-cell western' assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay ('myoblot') to be applied to the study of differentiated myoblast cultures. RESULTS: After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin and other muscle-associated proteins in muscle cell cultures. We are able to distinguish accurately between the different sets of patients based on their dystrophin expression and detect dystrophin restoration after treatment. CONCLUSIONS: We expect that this new tool to quantify muscle proteins in DMD and other muscle disorders will aid in their diagnosis and in the development of new therapies.
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Western Blotting/métodos , Distrofina/análisis , Técnica del Anticuerpo Fluorescente , Distrofia Muscular de Duchenne , Mioblastos , Técnicas de Cultivo de Célula/métodos , HumanosRESUMEN
Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.
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Anticuerpos Neutralizantes/inmunología , Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Virus Hantaan/inmunología , Replicación Viral/inmunología , Células A549 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales , Antivirales/aislamiento & purificación , Línea Celular , Chlorocebus aethiops , ARN Helicasas DEAD-box/farmacología , Células HEK293 , Virus Hantaan/efectos de los fármacos , Virus Hantaan/genética , Fiebre Hemorrágica con Síndrome Renal/tratamiento farmacológico , Fiebre Hemorrágica con Síndrome Renal/prevención & control , Humanos , Inmunidad Humoral , Interferones/farmacología , Células Vero , Proteínas Virales/metabolismo , Vacunas ViralesRESUMEN
BACKGROUND: Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction. METHODS: Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. RESULTS: Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all apelin isoforms for the shared C-terminal region responsible for receptor binding and activation. CONCLUSIONS: AR activation by all apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with apelin-55 providing an alternative bioactive isoform despite the addition of 19N-terminal residues relative to apelin-36. GENERAL SIGNIFICANCE: Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system.