Dupont Nicolas, Orhon Idil, Bauvy Chantal, Codogno Patrice
INSERM U1151-CNRS UMR 8253, Institut Necker Enfants-Malades, University Paris Descartes, Paris cedex 14, France.
INSERM U1151-CNRS UMR 8253, Institut Necker Enfants-Malades, University Paris Descartes, Paris cedex 14, France.
Methods Enzymol. 2014;543:73-88. doi: 10.1016/B978-0-12-801329-8.00004-0.
Macroautophagy (hereafter referred to as autophagy), a central mechanism mediating the lysosomal degradation of cytoplasmic components, can be stimulated by a wide panel of adverse stimuli, including a panoply of anticancer agents. The central autophagic organelle is the autophagosome, a double membrane-bound vacuole that sequesters the cytoplasmic material destined to disposal. The ultimate destiny of the autophagosome is to fuse with a lysosome, resulting in the degradation of the autophagic cargo. In this setting, it is important to discriminate whether a particular stimulus actually promotes autophagy or it simply blocks the fusion of autophagosomes with lysosomes. To this aim, the methods that assess autophagy should assess not only the number of autophagosomes but also the so-called autophagic flux, that is, the clearance of the autophagy cargo from the lysosomal compartment. Here, we present a compendium of methods to assess the autophagic flux in cultured malignant cells. This approach should allow for the study of the intimate link between autophagy and oncometabolism in several experimental paradigms.
巨自噬（以下简称自噬）是介导细胞质成分溶酶体降解的核心机制，可被多种不良刺激所激活，包括众多抗癌药物。自噬的核心细胞器是自噬体，它是一种由双层膜包裹的液泡，能够隔离注定要被清除的细胞质物质。自噬体的最终命运是与溶酶体融合，从而导致自噬货物的降解。在这种情况下，区分特定刺激是真正促进自噬还是仅仅阻断自噬体与溶酶体的融合非常重要。为此，评估自噬的方法不仅应评估自噬体的数量，还应评估所谓的自噬通量，即自噬货物从溶酶体区室的清除情况。在此，我们介绍了一系列评估培养的恶性细胞中自噬通量的方法。这种方法应有助于在多种实验范式中研究自噬与肿瘤代谢之间的密切联系。
