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 autophagy-modulating drugs.
Keywords: 3-MA, 3-methyladenine; Cyto-ID; FBS, fetal bovine serum; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 beta; MAP3K6, mitogen-activated protein kinase kinase kinase 6; MDC, monodansylcadaverine; MTOR, mechanistic target of rapamycin; NS, nonsilencing; RAB5A, member RAS oncogene family; RNA interference screen; RNAi, RNA interference; SQSTM1, sequestosome 1; WNK2, WNK lysine deficient protein kinase 2; autophagy; autophagy flux; autophagy response; mRFP, monomeric red fluorescent protein; shRNA, short-hairpin RNA; small molecule screen; spectrophotometric assay.