Autophagy Detection During Oncogene-Induced Senescence Using Fluorescence Microscopy.

Abstract:: 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.

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