RESUMEN
Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.
Asunto(s)
Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente Directa , Histonas/metabolismo , Microscopía Confocal , Análisis de la Célula Individual/métodos , Factores de Transcripción/metabolismo , Animales , Apoptosis , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Núcleo Celular/patología , Proliferación Celular , Fibroblastos/metabolismo , Humanos , Cinética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fosforilación , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Vitiligo is an acquired disorder characterized by circumscribed depigmented macules devoid of identifiable melanocytes. Complex genetic, immunological, neural and self destructive mechanisms interplay in its pathogenesis. According to autocytotoxic hypothesis, oxidative stress has been suggested to be the initial pathogenic event in melanocyte degeneration. AIMS: The aim of our investigation was to evaluate the role of oxidative stress by measuring levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in lesional and normal skin of patients with vitiligo and in the skin of normal controls. METHODS: We determined the activity of SOD in lesional and non-lesional skin and CAT in lesional skin only of 25 vitiligo patients and 25 controls by using the spectrophotometric assay and Aebi's method, respectively. RESULTS: There was statistically significant increase in the levels of SOD in vitiliginous and non vitiliginous skin of patient group compared to the control group (P < 0.001). No significant difference was found between the levels of SOD in lesional skin and non-lesional skin of vitiligo patients. The levels of CAT in the skin of patients were found to be significantly lower than those of controls (P < 0.001). CONCLUSIONS: There is increased oxidative stress in vitiligo as is indicated by high levels of SOD and low levels of CAT in the skin of vitiligo patients.