Measurement of autophagy via LC3 western blotting following DNA-damage-induced senescence

Hitomi Yamamoto-Imoto; Eiji Hara; Shuhei Nakamura; Tamotsu Yoshimori

doi:10.1016/j.xpro.2022.101539

Measurement of autophagy via LC3 western blotting following DNA-damage-induced senescence

STAR Protoc. 2022 Sep 16;3(3):101539. doi: 10.1016/j.xpro.2022.101539. Epub 2022 Jul 11.

Authors

Hitomi Yamamoto-Imoto¹, Eiji Hara², Shuhei Nakamura³, Tamotsu Yoshimori⁴

Affiliations

¹ Department of Genetics, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
² Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Osaka 565-0871, Japan; Immunology Frontier Research Center (IFReC), Osaka University, Suita, Osaka 565-0871, Japan.
³ Department of Genetics, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; Institute for Advanced Co-Creation Studies, Osaka University, Suita, Osaka 565-0871, Japan; Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: [email protected].
⁴ Department of Genetics, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: [email protected].

Abstract

Senescent cells accumulation is associated with aging and age-related diseases, and recent findings suggest that autophagy, the activity of the intracellular degradation system, decreases during senescence. In this protocol, we detail steps to induce cellular senescence in response to DNA damage, evaluate the senescent state using SA-β-gal staining and western blot for p21, LAMP1, and Lamin B1, and detect autophagy via LC3 western blotting. This protocol can be used in most cell lines and for various types of senescent cells. For complete details on the use and execution of this protocol, please refer to Yamamoto-Imoto et al. (2022).

Keywords: Cell Biology; Cell culture; Cell-based Assays.

Publication types

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

MeSH terms

Autophagy*
Blotting, Western
Cellular Senescence* / genetics
DNA
DNA Damage

Substances

DNA