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Mishra P, Rai S, Manjithaya R. A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast . Biochem Biophys Rep. 2017;11:138-146doi: 10.1016/j.bbrep.2017.07.008.
Mishra, P., Rai, S., & Manjithaya, R. (2017). A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast . Biochemistry and biophysics reports, 11138-146. https://doi.org/10.1016/j.bbrep.2017.07.008
Mishra, Piyush, et al. "A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast ." Biochemistry and biophysics reports vol. 11 (2017): 138-146. doi: https://doi.org/10.1016/j.bbrep.2017.07.008
Mishra P, Rai S, Manjithaya R. A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast . Biochem Biophys Rep. 2017 Jul 20;11:138-146. doi: 10.1016/j.bbrep.2017.07.008. eCollection 2017 Sep. PMID: 28955778; PMCID: PMC5614714.
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Biochem Biophys Rep. 2017 Jul 20;11:138-146. doi: 10.1016/j.bbrep.2017.07.008. eCollection 2017 Sep.

A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast .

Biochemistry and biophysics reports

Piyush Mishra, Shashank Rai, Ravi Manjithaya

Affiliations

  1. Autophagy Lab, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.

PMID: 28955778 PMCID: PMC5614714 DOI: 10.1016/j.bbrep.2017.07.008

Abstract

BACKGROUND: Macroautophagy is a cellular response to starvation wherein superfluous and damaged cytoplasmic constituents are degraded to provide energy for survival and to maintain cellular homeostasis. Dysfunctional autophagy is attributed to disease progression in several pathological conditions and therefore, autophagy has appeared as a potential pharmacological target for such conditions.

OBJECTIVE: In search of potential drugs that modulate autophagy, identifying small molecule effectors of autophagy is the primary step. The conventional autophagy assays have a limitation that they cannot be scaled down to a high throughput format, therefore, novel sensitive assays are needed to discover new candidate molecules. Keeping this rationale in mind, a dual luciferase based assay was developed in the yeast

METHODS: Firefly and

RESULTS AND CONCLUSION: The assay was more sensitive than conventional assays and could be scaled down to a 384 well format using an automated system. A good Z-factor score indicated that the assay is highly suitable for High Throughput Screening (HTS) of small molecule libraries. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy.

Keywords: Autophagy; High throughput screening; Luciferase; Peroxisome; Pexophagy; Small molecules

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