RESUMEN
As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.
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Inmunidad Innata , Proteómica , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Western Blotting/métodos , Espectrometría de Masas/métodos , Inmunoprecipitación/métodos , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Cromatografía Líquida con Espectrometría de MasasRESUMEN
The pathogenesis of acute lung injury is complex. Studies have demonstrated the role of neutrophil extracellular traps (NETs) in the process of lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the underlying mechanism remains unclear. In this study, the regulation of Nrf2 in the formation of NETs, which was pathogenic in LPS-induced ALI, was identified by analyzing the levels of Cit-H3, lung function, lung tissue pathology, lung wet/dry ratio, the inflammatory cells, cytokines and proteins in the bronchoalveolar lavage fluid (BALF) and in addition, the activity of lung myeloperoxidase (MPO) was also measured. Results showed that the levels of Cit-H3 measured by western blot in Nrf2-knockout (KO) mice were higher compared with the WT mice after LPS stimulation. To further investigate the NETs formation was pathogenic during LPS-induced ALI, the Nrf2-KO mice were treated with DNase I. Results showed that DNase I improved lung function and lung tissue pathology and significantly reduced lung wet/dry ratio and proteins in the BALF. Besides, DNase I also attenuated the infiltration of inflammatory cells and the cytokines (TNF-α, IL-1ß) production in the BALF and the activity of lung MPO. Therefore, these results together indicate that Nrf2 may intervene in the release of NETs during LPS-induced ALI in mice.
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Lesión Pulmonar Aguda , Líquido del Lavado Bronquioalveolar , Trampas Extracelulares , Lipopolisacáridos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2 , Animales , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Factor 2 Relacionado con NF-E2/metabolismo , Ratones , Trampas Extracelulares/metabolismo , Líquido del Lavado Bronquioalveolar/química , Masculino , Peroxidasa/metabolismo , Neutrófilos/metabolismo , Pulmón/metabolismo , Pulmón/patología , Interleucina-1beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Desoxirribonucleasa I/metabolismo , Citocinas/metabolismo , Western BlottingRESUMEN
Purpose: This study investigates alterations in intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells (DACs) in lid suture myopia (LSM) rats. Methods: LSM was induced in rats by suturing the right eyes for 4 weeks. Double immunofluorescence staining of ipRGCs and DACs in whole-mount retinas was performed to analyze changes in the density and morphology of control, LSM, and fellow eyes. Real-time quantitative PCR and Western blotting were used to detect related genes and protein expression levels. Results: Significant myopia was induced in the lid-sutured eye, but the fellow eye was not different to control. Decreased ipRGC density with paradoxically increased overall melanopsin expression and enlarged dendritic beads was observed in both the LSM and fellow eyes of the LSM rat retinas. In contrast, DAC changes occurred only in the LSM eyes, with reduced DAC density and tyrosine hydroxylase (TH) expression, sparser dendritic processes, and fewer varicosities. Interestingly, contacts between ipRGCs and DACs in the inner plexiform layer (IPL) and the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and vesicular monoamine transporter protein 2 (VMAT2) mRNA were decreased in the LSM eyes. Conclusions: The ipRGCs and DACs in LSM rat retinas undergo multiple alterations in density, morphology, and related molecule expressions. However, the ipRGC changes alone appear not to be required for the development of myopia, given that myopia is only induced in the lid-sutured eye, and they are unlikely alone to drive the DAC changes. Reduced contacts between ipRGCs and DACs in the LSM eyes may be the structural foundation for the impaired signaling between them. PACAP and VMAT2, strongly associated with ipRGCs and DACs, may play important roles in LSM through complex mechanisms.
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Células Amacrinas , Western Blotting , Modelos Animales de Enfermedad , Miopía , Células Ganglionares de la Retina , Opsinas de Bastones , Animales , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/metabolismo , Ratas , Miopía/metabolismo , Células Amacrinas/metabolismo , Células Amacrinas/patología , Opsinas de Bastones/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genética , Masculino , Ratas Sprague-Dawley , Párpados/patología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Recuento de Células , Proteína 2 de Transporte Vesicular de GlutamatoRESUMEN
PURPOSE: Epithelial-mesenchymal transition (EMT) is a crucial pathological process that contributes to proliferative vitreoretinopathy (PVR), and research indicates that factors present in the vitreous that target cells play pivotal roles in regulating EMT. Experimental studies have confirmed that rabbit vitreous (RV) promotes EMT in human retinal pigment epithelial (RPE) cells. The long noncoding RNA (lncRNA) MALAT1 has been implicated in EMT in various diseases. Thus, this study aimed to investigate the involvement of lncRNA MALAT1 in vitreous-induced EMT in RPE cells. METHODS: MALAT1 was knocked down in ARPE-19 cells by short hairpin RNA (shRNA) transfection. Reverse transcription PCR (RTâPCR) was used to evaluate MALAT1 expression, and Western blotting analysis was used to measure the expression of EMT-related proteins. Wound-healing, Transwell, and cell contraction assays were conducted to assess cell migration, invasion, and contraction, respectively. Additionally, cell proliferation was assessed using the CCK-8 assay, and cytoskeletal changes were examined by immunofluorescence. RESULTS: MALAT1 expression was significantly increased in ARPE-19 cells cultured with RV. Silencing MALAT1 effectively suppressed EMT and downregulated the associated factors snail1 and E-cadherin. Furthermore, silencing MALAT1 inhibited the RV-induced migration, invasion, proliferation, and contraction of ARPE-19 cells. Silencing MALAT1 also decreased RV-induced AKT and P53 phosphorylation. CONCLUSIONS: In conclusion, lncRNA MALAT1 participates in regulating vitreous-induced EMT in human RPE cells; these results provide new insight into the pathogenesis of PVR and offer a potential direction for the development of antiproliferative drugs.
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Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Proteínas Proto-Oncogénicas c-akt , ARN Largo no Codificante , Epitelio Pigmentado de la Retina , ARN Largo no Codificante/genética , Transición Epitelial-Mesenquimal/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/patología , Conejos , Animales , Células Cultivadas , Vitreorretinopatía Proliferativa/genética , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patología , Transducción de Señal , Regulación de la Expresión Génica , Western BlottingRESUMEN
Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
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Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Humanos , Transglutaminasas/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Inmunoprecipitación/métodos , Anticuerpos/inmunología , Proteínas de Unión al GTP/inmunologíaRESUMEN
Western blot is a popular biomolecular analysis method for measuring the relative quantities of independent proteins in complex biological samples. However, variability in quantitative western blot data analysis poses a challenge in designing reproducible experiments. The lack of rigorous quantitative approaches in current western blot statistical methodology may result in irreproducible inferences. Here we describe best practices for the design and analysis of western blot experiments, with examples and demonstrations of how different analytical approaches can lead to widely varying outcomes. To facilitate best practices, we have developed the blotRig tool for designing and analyzing western blot experiments to improve their rigor and reproducibility. The blotRig application includes functions for counterbalancing experimental design by lane position, batch management across gels, and analytics with covariates and random effects.
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Western Blotting , Reproducibilidad de los Resultados , Western Blotting/métodos , Western Blotting/normas , Proyectos de Investigación , Programas Informáticos , HumanosRESUMEN
Purpose: This study aimed to explore the impact of HSPA13 on epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells and proliferative vitreoretinopathy (PVR) development, along with its associated molecular mechanisms. Methods: HSPA13 expression was evaluated in epiretinal membranes (ERMs) from patients with PVR using immunohistochemistry. The effects of HSPA13 knockdown on TGFß1-induced EMT in hESC-RPE cells were studied through quantitative PCR (qPCR), Western blot, and wound healing assays. Intracellular Ca2+ levels were measured using Fluo-8/AM incubation. A rat PVR model was induced by the intravitreal injection of RPE cells combined with platelet-rich plasma (PRP). RNA-seq was applied to study the molecular mechanism of HSPA13 knockdown-mediated EMT inhibition. Results: HSPA13 was found in human ERMs and its expression increased with TGFß1 treatment in hESC-RPE cells. Knockdown of HSPA13 inhibited TGFß1-induced EMT and migration. In the PVR rat model, HSPA13 was expressed in the ERMs and its knockdown in RPE cells reduced the development of PVR. Consistent with these observations, RNA-seq showed a global suppression of TGFß1-induced EMT and migration by shHSPA13 in RPE cells. Mechanistically, TGFß1 treatment increased intracellular Ca2+ levels, leading to an upregulation of HSPA13 expression. Downregulation of HSPA13 hindered the phosphorylation of PI3K/Akt in TGFß1-induced RPE cells. Conclusions: Our study revealed the involvement of HSPA13 in PVR development, as well as in TGFß1-induced EMT of RPE through the PI3K/Akt signaling pathway. Targeting HSPA13-related pathways involved in regulating EMT in RPE cells could serve as a novel therapeutic approach for patients with PVR.
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Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Proteínas HSP70 de Choque Térmico , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Humanos , Ratas , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Vitreorretinopatía Proliferativa/genética , Vitreorretinopatía Proliferativa/patología , Vitreorretinopatía Proliferativa/metabolismo , Masculino , Western Blotting , Células Cultivadas , Ratas Sprague-Dawley , Movimiento Celular , InmunohistoquímicaRESUMEN
Histones and their posttranslational modifications (PTMs) are critical regulators of gene expression. Differentiation, environmental stressors, xenobiotics, and major human diseases cause significant changes in histone variants and PTMs. Western blotting is the mainstay methodology for detection of histones and their PTMs in the majority of studies. Surprisingly, despite their high abundance in cells, immunoblotting of histones typically involves loading of large protein amounts that are normally used for detection of sparse cellular proteins. We systematically examined technical factors in the Western-blotting-based detection of human histones with >30 antibodies. We found that under multiple protein transfer conditions, many histone epitopes on polyvinylidene fluoride (PVDF) membranes had a very low antibody accessibility, which was dramatically increased by the addition of a simple denaturation step. Denaturation of membrane-bound proteins also enhanced the specificity of some histone antibodies. In comparison to standard PVDF membranes, the sensitivity of histone detection on standard nitrocellulose membranes was typically much higher, which was further increased by the inclusion of the same denaturation step. Optimized protocols increased by >100-times detection sensitivity for the genotoxic marker γ-H2AX with two monoclonal antibodies. The impact of denaturation and nitrocellulose use varied for different histones, but for each histone, it was generally similar for antibodies targeting N-terminal and C-terminal regions. In summary, denaturation of membrane-bound histones strongly improves their detection by Westerns, resulting in more accurate measurements and permitting analyses with small biological samples.
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Histonas , Histonas/química , Histonas/metabolismo , Histonas/análisis , Humanos , Western Blotting , Polivinilos/química , Polímeros de FluorocarbonoRESUMEN
Retinal neurodegenerative diseases, including hypertensive retinopathy, involve progressive damage to retinal neurons, leading to visual impairment. In this study, we investigated the pathological mechanisms underlying retinal neurodegeneration in spontaneously hypertensive rats (SHR), using Wistar Kyoto (WKY) rats as normotensive controls. We observed that SHR exhibited significantly higher blood pressure and decreased retinal thickness, indicating retinal neurodegeneration. Molecular tests including quantitative real-time polymerase chain reaction, immunoblot, and immunofluorescent staining showed elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α, apoptotic markers (Fas, FasL, caspase-8, active caspase-3, and cleaved poly (ADP-ribose) polymerase), and necroptotic markers (receptor-interacting protein kinase-1 and -3) in SHR retinas. Additionally, we found elevated transforming growth factor-ß (TGF-ß) levels in the retinal pigment epithelium (RPE) of SHR, with a decrease in lecithin retinol acyltransferase (LRAT), which regulates retinoid metabolism and photoreceptor health. In human RPE cells (ARPE-19), TGF-ß administration suppressed mRNA and protein levels of LRAT; and vactosertib, a selective inhibitor of TGF-ß receptor kinase type 1, reversed the effect of TGF-ß. These findings suggest that hypertension-induced retinal neurodegeneration involves inflammation, apoptosis, necroptosis, and disrupted retinoid metabolism, providing potential therapeutic targets for hypertensive retinopathy.
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Apoptosis , Células Fotorreceptoras de Vertebrados , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Epitelio Pigmentado de la Retina , Animales , Ratas , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Masculino , Modelos Animales de Enfermedad , Presión Sanguínea/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/etiología , Retinopatía Hipertensiva/metabolismo , Western Blotting , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/genética , HumanosRESUMEN
Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
Asunto(s)
Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Proteína Huntingtina , Inmunoprecipitación , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/inmunología , Inmunoprecipitación/métodos , Técnica del Anticuerpo Fluorescente/métodos , Anticuerpos/inmunología , Animales , Enfermedad de Huntington/inmunología , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/genética , Células HEK293RESUMEN
Antemortem serodiagnosis of aspergillosis remains challenging in Sphenisciformes. Protein electrophoresis, serology (antibody, antigen) by ELISA, and gliotoxin detection provide variable diagnostic value. In the present study, a commercially available Western blot (WB) validated for use in humans and dolphins was adapted for use with penguin samples. Using the same method and reagents, samples were analyzed from multiple institutions in the United States and one facility in France. This was inclusive of normal juvenile African penguins (Spheniscus demersus, n = 10) and various species of penguins in the United States with confirmed infection (n = 9) as well as 52 samples from Humboldt penguins (Spheniscus humboldti) in France. Cumulative WB scores (based on reactivity to different antigens) were found to be significantly higher in the group of penguins with confirmed infection (p < 0.0001). Significant differences were also observed between the clinically normal penguins in the two populations, with higher scores in the United States (median score 1.0, 95%CI [0-5], min 0, max 11) compared to France (median score 0,95%CI [0-0], min 0, max 5). The utilization of the WB as a diagnostic tool is inconclusive due to the use of samples from varying institutions, environmental background, age, and stages of infection. However, this tool may provide an overview of antigen reactivity in penguins infected with Aspergillus to help design a more robust serology assay and further understand the humoral immune response during infection.
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Anticuerpos Antifúngicos , Aspergilosis , Aspergillus , Enfermedades de las Aves , Western Blotting , Spheniscidae , Animales , Aspergilosis/veterinaria , Aspergilosis/diagnóstico , Estados Unidos , Francia , Western Blotting/veterinaria , Aspergillus/inmunología , Anticuerpos Antifúngicos/sangre , Enfermedades de las Aves/diagnóstico , Enfermedades de las Aves/microbiología , Enfermedades de las Aves/inmunologíaRESUMEN
Purpose: This study aims to investigate the relationship among STRA6, circadian rhythm, and choroidal neovascularization (CNV) formation, as well as the regulatory mechanism of STRA6 in CNV under circadian rhythm disturbances. Methods: C57BL/6J male mice (aged 6 weeks) were randomly divided into control and jet lag groups (using a time shift method every 4 days to disrupt the molecular clock's capacity to synchronize with a stable rhythm). A laser-induced CNV model was established in both the control and the jet lag group after 2 weeks of jet lag. The size of CNV lesions and vascular leakage were detected by morphological and imaging examination on the seventh day post laser. STRA6 was screened by full transcriptome sequencing. Bioinformatics analysis was conducted to assess the variation and association of STRA6 in the GSE29801 dataset. The effects of STRA6 were evaluated both in vivo and in vitro. The pathway mechanism was further elucidated and confirmed through immunofluorescence of paraffin sections and Western blotting. Results: The disturbance of circadian rhythm promotes the formation of CNV. Patients with age-related macular degeneration (AMD) exhibited higher levels of STRA6 expression compared to the control group, and STRA6 was enriched in pathways related to angiogenesis. In addition, CLOCK and BMAL1, which are initiators that drive the circadian cycle, had regulatory effects on STRA6. Knocking down STRA6 reversed the promotion of CNV formation caused by circadian rhythm disturbance in vivo, and it also affected the proliferation, migration, and VEGF secretion of RPE cells without circadian rhythm in vitro, as well as impacting endothelial cells. Through activation of the JAK2/STAT3/VEGFA signaling pathway in unsynchronized RPE cells, STRA6 promotes CNV formation. Conclusions: This study suggests that STRA6 reduces CNV production by inhibiting JAK2/STAT3 phosphorylation after circadian rhythm disturbance. The results suggest that STRA6 may be a new direction for the treatment of AMD.
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Neovascularización Coroidal , Ritmo Circadiano , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/genética , Neovascularización Coroidal/fisiopatología , Animales , Ritmo Circadiano/fisiología , Ratones , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Regulación de la Expresión Génica/fisiología , Western Blotting , Humanos , Proliferación Celular/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Connexins are essential gap junction proteins that play pivotal roles in intercellular communication in various organs of mammals. Connexin-43 (Cx43) is expressed in various components of the immune system, and there is extensive evidence of its participation in inflammation responses. The involvement of Cx43 in macrophage functionality involves the purinergic signaling pathway. Macrophages contribute to defenses against inflammatory reactions such as bacterial sepsis and peritonitis. Several assays can identify the presence and activity of Cx43 in macrophages. Real-time polymerase chain reaction (PCR) can measure the relative mRNA expression of Cx43, whereas western blotting can detect protein expression levels. Using immunofluorescence assays, it is possible to analyze the expression and observe the localization of Cx43 in cells or tissues. Moreover, connexin-mediated gap junction intercellular communication can be evaluated using functional assays such as microinjection of fluorescent dyes or scrape loading-dye transfer. The use of selective inhibitors contributes to this understanding and reinforces the role of connexins in various processes. Here, we discuss these methods to evaluate Cx43 and macrophage gap junctions.
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Conexina 43 , Uniones Comunicantes , Macrófagos , Conexina 43/metabolismo , Conexina 43/genética , Macrófagos/metabolismo , Macrófagos/inmunología , Animales , Uniones Comunicantes/metabolismo , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Comunicación Celular , Western Blotting , Técnica del Anticuerpo Fluorescente , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Feline morbillivirus (FeMV) has been associated with feline health, although its exact role in pathogenesis is still debated. In this study, an indirect enzyme-linked immunosorbent assay (i-ELISA) targeting a recombinant matrix protein of FeMV (rFeMV-M) was developed and assessed in comparison to a Western blotting (WB) assay. The i-ELISA was evaluated using blood samples from 136 cats that were additionally tested with real-time reverse-transcription PCR (RT-qPCR). The i-ELISA exhibited a sensitivity of 90.1%, specificity of 75.6%, positive predictive value of 88.2%, and negative predictive value of 79.1%. The agreement between i-ELISA and WB analyses was substantial (a κ coefficient of 0.664 with a 95% confidence interval of 0.529 to 0.799). Within the study group, 68.4% (93/136) of the cats were serologically positive in the i-ELISA and 66.9% (91/136) in the WB assay, with 11.8% (11/93) of false positivity with the i-ELISA. However, only 8.1% (11/136) of the cats tested positive for FeMV using RT-qPCR (p < 0.001). The developed i-ELISA proved effective in identifying FeMV-infected cats and indicated the prevalence of FeMV exposure. Combining FeMV antibody detection through i-ELISA with FeMV RT-qPCR could offer a comprehensive method to determine and monitor FeMV infection status. Nevertheless, this assay still requires refinement due to a significant number of false positive results, which can lead to the misdiagnosis of cats without antibodies as having antibodies. This study also provided the first evidence of seroprevalence against FeMV among cat populations in Thailand, contributing valuable insights into the geographic distribution and prevalence of this virus.
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Anticuerpos Antivirales , Enfermedades de los Gatos , Ensayo de Inmunoadsorción Enzimática , Infecciones por Morbillivirus , Morbillivirus , Sensibilidad y Especificidad , Animales , Gatos , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Morbillivirus/inmunología , Enfermedades de los Gatos/virología , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/inmunología , Infecciones por Morbillivirus/veterinaria , Infecciones por Morbillivirus/diagnóstico , Infecciones por Morbillivirus/inmunología , Infecciones por Morbillivirus/virología , Proteínas Recombinantes/inmunología , Femenino , Western Blotting/veterinaria , Masculino , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/genéticaRESUMEN
Purpose: The purpose of this study was to examine possible involvement of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1/Flt-1 in pigment epithelium-derived factor (PEDF)-promoted survival of retinal neurons. Methods: Survival of growth factor-deprived retinal ganglion cells (RGCs) and R28 cells and activation of ERK-1/-2 MAP kinases were assessed in the presence of PEDF, placental growth factor (PlGF), and VEGF using cell cultures, viability assays and quantitation of ERK-1/-2 phosphorylation. VEGFR-1/Flt-1 expression was determined using quantitative PCR (qPCR) and Western blotting. VEGFR-1/Flt-1 was knocked down in R28 cells by small interfering RNA (siRNA). Binding of a PEDF-IgG Fc fusion protein (PEDF-Fc) to retinal neurons, immobilized VEGFR-1/Flt-1 and VEGFR-1/Flt-1-derived peptides was studied using binding assays and peptide scanning. Results: PEDF in combination with PlGF stimulated increased cell survival and ERK-1/-2 MAP kinase activation compared to effects of either factor alone. VEGFR-1/Flt-1 expression in RGCs and R28 cells was significantly upregulated by hypoxia, VEGF, and PEDF. VEGFR-1/Flt-1 ligands (VEGF and PlGF) or soluble VEGFR-1 (sflt-1) competed with PEDF-Fc for binding to R28 cells. Depleting R28 cells of VEGFR-1/Flt-1 resulted in reduced PEDF-Fc binding when comparing VEGFR-1/Flt-1 siRNA- and control siRNA-treated cells. PEDF-Fc interacted with immobilized sflt-1, which was specifically blocked by VEGF and PlGF. PEDF-Fc binding sites were mapped to VEGFR-1/Flt-1 extracellular domains D3 and D4. Peptides corresponding to D3 and D4 specifically inhibited PEDF-Fc binding to R28 cells. These peptides and sflt-1 significantly inhibited PEDF-promoted survival of R28 cells. Conclusions: These results suggest that PEDF can target VEGFR-1/Flt-1 and this interaction plays a significant role in PEDF-mediated neuroprotection in the retina.
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Western Blotting , Supervivencia Celular , Proteínas del Ojo , Factores de Crecimiento Nervioso , Serpinas , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Animales , Ratas , Células Cultivadas , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/genética , Fosforilación , Células Ganglionares de la Retina/metabolismo , Serpinas/metabolismo , Serpinas/farmacología , Serpinas/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVES: The aim of this study was to investigate the role and molecular mechanism of proline/arginine-rich end leucine-rich repeat protein (PRELP), a secreted protein in extracellular matrix, in oral squamous cell carcinoma (OSCC) progression. DESIGN: PRELP expression in OSCC was analyzed in the Gene Set Enrichment (GSE) 138206, GSE37991, and GSE23558 datasets as well as cell lines. Also, PRELP expression and its relationship with prognosis and immune infiltration in head and neck squamous cell carcinoma (HNSCC) were confirmed by bioinformatics analysis. The proliferation, apoptosis, invasion, epithelial-to-mesenchymal transition (EMT) and NF-κB activation were detected after alteration of PRELP expression in OSCC cells using CCK-8, EdU, flow cytometry, Transwell, real-time PCR, immunofluorescence and Western blot. Additionally, an NF-κB inhibitor PDTC was used to confirm the regulation mechanism of PRELP. RESULTS: The expression of PRELP in OSCC tissues, cells and in HNSCC samples was low. HNSCC patients with higher PRELP expression was associated with longer overall survival. A positive correlation between PRELP expression and immune cell infiltration was found in HNSCC. Upregulation of PRELP inhibited, whereas PRELP silencing promoted, the proliferation, invasion and EMT of OSCC cells. Also, overexpression of PRELP promoted cell apoptosis. Mechanistically, PRELP suppressed p65 phosphorylation and nuclear translocation. And PDTC treatment partially reversed the influences of PRELP knockdown on the malignant behaviors in OSCC cells. CONCLUSION: PRELP suppressed OSCC progression via inactivation of the NF-κB pathway. Targeting PRELP may be a potential approach for OSCC treatment.
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Carcinoma de Células Escamosas , Transición Epitelial-Mesenquimal , Proteínas de la Matriz Extracelular , Glicoproteínas , Neoplasias de la Boca , FN-kappa B , Humanos , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Citometría de Flujo , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/genética , FN-kappa B/metabolismo , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMEN
Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
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Anticuerpos , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Sinaptotagmina I , Sinaptotagmina I/inmunología , Sinaptotagmina I/metabolismo , Humanos , Citometría de Flujo/métodos , Inmunoprecipitación/métodos , Técnica del Anticuerpo Fluorescente/métodos , Anticuerpos/inmunologíaRESUMEN
Purpose: Metastatic uveal melanoma (UM) treatment is difficult, and effective treatments are urgently needed. We aimed to explore the role of heme oxygenase 1 (HO-1) in UM and provide new therapeutic strategies for UM. Methods: Bioinformatics was used to analyze the relationship between HMOX1 and immunity in UM and other tumors. Cell Counting Kit-8, Western blot, immunofluorescence staining, wound healing, and Transwell assays were used. A subcutaneous transplanted UM tumor model was used in mice to verify the therapeutic effect. Results: In UM, the expression level of HMOX1 was strongly correlated with the immune score and the infiltration level of various immune cells. ZnPP can inhibit the growth of UM cells, promote cell apoptosis, and block the cell cycle at G0/G1 phase in vitro. HO-1 knockout can effectively inhibit the proliferation of UM cells. ZnPP effectively inhibited the growth of UM and promoted the infiltration of CD8+ T cells in a subcutaneous tumor transplantation model. Conclusions: These results indicate that targeting HO-1 in UM has the potential for independent targeted immunotherapy or adjuvant immunotherapy.
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Apoptosis , Linfocitos T CD8-positivos , Movimiento Celular , Proliferación Celular , Hemo-Oxigenasa 1 , Linfocitos Infiltrantes de Tumor , Melanoma , Neoplasias de la Úvea , Neoplasias de la Úvea/patología , Neoplasias de la Úvea/inmunología , Neoplasias de la Úvea/metabolismo , Animales , Melanoma/patología , Melanoma/inmunología , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Humanos , Invasividad Neoplásica , Western Blotting , Protoporfirinas/farmacología , Línea Celular Tumoral , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Purpose: Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFß and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFß2-induced TM changes and ocular hypertension (OHT). Methods: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFß signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. Results: We found that the small-molecule Wnt activators (GSK3ß inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFß signaling as well as TGFß2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and ß-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFß and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFß2-induced OHT in perfusion-cultured human eyes. Conclusions: Our results showed that small-molecule Wnt activators have the potential for treating TGFß signaling-induced OHT in patients with POAG.
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Glaucoma de Ángulo Abierto , Glucógeno Sintasa Quinasa 3 beta , Presión Intraocular , Malla Trabecular , Humanos , Malla Trabecular/metabolismo , Malla Trabecular/efectos de los fármacos , Presión Intraocular/fisiología , Presión Intraocular/efectos de los fármacos , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Células Cultivadas , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Western Blotting , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/fisiología , Hipertensión Ocular/metabolismo , Hipertensión Ocular/tratamiento farmacológico , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta2/farmacologíaRESUMEN
Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.