Protocol to stimulate and delineate alternative lengthening of telomeres in human U2OS cells

Jia-Min Zhang; Lee Zou

doi:10.1016/j.xpro.2022.101594

Protocol to stimulate and delineate alternative lengthening of telomeres in human U2OS cells

STAR Protoc. 2022 Jul 31;3(3):101594. doi: 10.1016/j.xpro.2022.101594. eCollection 2022 Sep 16.

Authors

Jia-Min Zhang¹, Lee Zou²

Affiliations

¹ Department of Orthopedics, Tongji Hospital, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration, Tongji University, Ministry of Education, Shanghai 200065, China. Electronic address: zhangjiamin@tongji.edu.cn.
² Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address: zou.lee@mgh.harvard.edu.

Abstract

Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe a protocol to stimulate the formation of ALT-associated PML bodies (APBs) and ALT activity by tethering PML-IV to telomeres in human U2OS cells. Through immunofluorescence, in situ hybridization, and microscopy, we analyze dynamics of telomere clustering, visualize recruitment of DNA repair proteins to APBs, and measure telomere DNA synthesis during ALT. This protocol provides a unique approach to delineate the ALT pathway. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Keywords: Cancer; Cell Biology; In Situ Hybridization; Microscopy; Molecular Biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Replication
Humans
Telomerase* / genetics
Telomere* / genetics

Substances

Telomerase