Protocol for Establishing a Multiplex Image-based Autophagy RNAi Screen in Cell Cultures.

Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy is regulated provides an opportunity to explore druggable targets and subsequently develop treatment strategies for these diseases. To identify novel autophagy regulators, we developed an image-based phenotypic RNAi screening approach using autophagic marker proteins at endogenous levels (Jung et al., 2017). In contrast to previously performed autophagy screens, this approach does not use overexpressed, tagged autophagy marker proteins but rather detects autophagic structures at endogenous levels. Furthermore, we monitored early and late phases of autophagy in parallel while other screens employed only a single autophagosome marker mostly GFP-LC3B. Here, we describe this multiplex screening protocol in detail and discuss general considerations about how to establish image-based siRNA screens.