Synchronization of human retinal pigment epithelial-1 cells in mitosis.
J Cell Sci
; 133(18)2020 09 17.
Article
em En
| MEDLINE
| ID: mdl-32878943
ABSTRACT
Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cinetocoros
/
Mitose
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
J Cell Sci
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Reino Unido