Abstract
The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagymodulating drugs.
Recommended Citation
S. Guo et al., "A Rapid and High Content Assay that Measures Cyto-ID-stained Autophagic Compartments and Estimates Autophagy Flux with Potential Clinical Applications," Autophagy, vol. 11, no. 3, pp. 560 - 572, Taylor and Francis Group; Taylor and Francis, Jan 2015.
The definitive version is available at https://doi.org/10.1080/15548627.2015.1017181
Department(s)
Chemistry
Publication Status
Open Access
Keywords and Phrases
Autophagy flux; Autophagy response; Cyto-ID; RNA interference screen; Small molecule screen; Spectrophotometric assay; Utophagy
International Standard Serial Number (ISSN)
1554-8635; 1554-8627
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2024 Taylor and Francis Group; Taylor and Francis, All rights reserved.
Publication Date
01 Jan 2015
PubMed ID
25714620
Comments
Industrial Technology Research Institute, Grant None