Identification and Validation of Novel Autophagy Regulators Using an Endogenous Readout siGENOME Screen

Methods Mol Biol. 2019:1880:359-374. doi: 10.1007/978-1-4939-8873-0_24.

Abstract

Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.

Keywords: Autophagy; Autophagy regulation; Endogenous readout; Hit identification; LC3 puncta; Large data set analysis; Screen analysis; siGENOME screen.

MeSH terms

  • Animals
  • Autophagy / genetics*
  • Cell Culture Techniques / instrumentation
  • Cell Culture Techniques / methods
  • Cell Line
  • Gene Knockdown Techniques / instrumentation
  • Gene Knockdown Techniques / methods*
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods
  • Humans
  • Microtubule-Associated Proteins / genetics
  • RNA Interference
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • Transfection / instrumentation
  • Transfection / methods

Substances

  • Microtubule-Associated Proteins
  • RNA, Small Interfering