Your browser doesn't support javascript.
loading
Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue.
Joseph-Chowdhury, Jazmine-Saskya N; Stransky, Stephanie; Graff, Sarah; Cutler, Ronald; Young, Dejauwne; Kim, Julie S; Madrid-Aliste, Carlos; Aguilan, Jennifer T; Nieves, Edward; Sun, Yan; Yoo, Edwin J; Sidoli, Simone.
Afiliación
  • Joseph-Chowdhury JN; Department of Biochemistry, Albert Einstein College of Medicine.
  • Stransky S; Department of Biochemistry, Albert Einstein College of Medicine.
  • Graff S; Department of Biochemistry, Albert Einstein College of Medicine.
  • Cutler R; Department of Biochemistry, Albert Einstein College of Medicine.
  • Young D; Department of Biochemistry, Albert Einstein College of Medicine.
  • Kim JS; Department of Biochemistry, Albert Einstein College of Medicine.
  • Madrid-Aliste C; Department of Biochemistry, Albert Einstein College of Medicine.
  • Aguilan JT; Department of Biochemistry, Albert Einstein College of Medicine.
  • Nieves E; Department of Biochemistry, Albert Einstein College of Medicine.
  • Sun Y; Department of Biochemistry, Albert Einstein College of Medicine.
  • Yoo EJ; Department of Biochemistry, Albert Einstein College of Medicine.
  • Sidoli S; Department of Biochemistry, Albert Einstein College of Medicine; simone.sidoli@einsteinmed.edu.
J Vis Exp ; (183)2022 05 05.
Article en En | MEDLINE | ID: mdl-35604167
ABSTRACT
Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Histonas / Procesamiento Proteico-Postraduccional / Técnicas de Cultivo Tridimensional de Células / Hígado Tipo de estudio: Guideline Límite: Animals Idioma: En Revista: J Vis Exp Año: 2022 Tipo del documento: Article

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Histonas / Procesamiento Proteico-Postraduccional / Técnicas de Cultivo Tridimensional de Células / Hígado Tipo de estudio: Guideline Límite: Animals Idioma: En Revista: J Vis Exp Año: 2022 Tipo del documento: Article