Autophagy Detection During Oncogene-Induced Senescence Using Fluorescence Microscopy.
Oncogene-induced senescence (OIS) is a highly dynamic process, involving several different effector mechanisms, the multitude and combination of which likely determines the quality of the phenotype (Pérez-Mancera et al., Nat Rev Cancer 14:547-558, 2014). Autophagy, a cellular degradation process, has been proposed to be one of these senescence effectors, although its functional relevance seems highly context dependent (Hoare et al., Semin Cancer Biol 21:397-404, 2011). A number of methods for monitoring autophagy are available, and several excellent protocols have been published in this journal (Klionsky et al., Autophagy 8:445-544, 2012; Tooze et al., Methods Mol Biol 1270:155-165, 2015; Tabata et al., Methods Mol Biol 931:449-466, 2013; Young and Tooze, Methods Mol Biol 445:147-157, 2008). The same principles apply to models of OIS in culture. Thus, in this chapter, we describe how to generate OIS cells using human diploid fibroblasts (HDFs), the best-characterized cell model of OIS, and how to detect autophagy, particularly focusing on immunofluorescence methods.
Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge, CB2 0RE, UK.
Masako
M
Narita
Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge, CB2 0RE, UK. masashi.narita@cruk.cam.ac.uk.
Masashi
M
Narita
D001343:Autophagy / Q000235:genetics*; D015415:Biomarkers; D016922:Cellular Senescence / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D009857:Oncogenes / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D015415:Biomarkers
Autophagy; Immunofluorescence; Lysosome; Senescence; mTOR