Published: June 30th, 2023
Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.
Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.
Autophagy is a conserved metabolic process that allows cells to keep intracellular homeostasis and respond to stress by degrading aged, defective, or unnecessary cellular contents via the lysosomes1,2,3. Under some pathophysiologic conditions, this process acts as a crucial cellular response to nutrient and oxygen deprivation, resulting in recycled nutrients and lipids, allowing the cells to adapt to their metabolic needs2,3,4. Autophagy has also been i....
NOTE: The assay process is outlined in Figure 1. See the Table of Materials for details related to all materials, reagents, and equipment used in this protocol.
1. Retrovirus production
NOTE: The plasmid encoding the ActinLC3dNGLUC is pMOWS-ActinLC3dNGLUC20. Use a low-passage number of cells for high-titer virus production (ideally less than P20).
In a previous publication8, we successfully used this assay to screen small molecule and siRNA libraries and identified novel regulators of ATG4B. Here, we describe the protocol and representative results of this luciferase reporter in a semi-automated, high-throughput screening format. Figure 8 shows an example of the raw data analysis for both cell nuclei and luminescence. A typical result of a luminescence measurement is depicted in Figure 8A
This protocol describes a cell-based reporter-gene assay for the identification of ATG4B inhibitors. The identification of primary hits is based on luciferase activity upon the treatment of cells expressing the full-length open reading frame of LC3B between β-actin and dNGLUC. Some advantages of this assay are that it is sensitive, highly quantitative, and noninvasive, as it can detect dNGLUC without lysing the cells. This paper presents a detailed protocol for generating a stable cell line and a primary screening. .......
This work was supported by UK Medical Research Council core funding to the MRC-UCL University Unit Grant Ref MC_U12266B, MRC Dementia Platform Grant UK MR/M02492X/1, Pancreatic Cancer UK (grant reference 2018RIF_15), and the UCL Therapeutic Acceleration Support scheme, supported by funding from MRC Confidence in Concept 2020 UCL MC/PC/19054. The plasmid encoding the ActinLC3dNGLUC (pMOWS-ActinLC3dNGLUC) was obtained from Dr. Robin Ketteler (Department of Human Medicine, Medical School Berlin).....
|50 µL Disposable Tips - Non-filtered, Pure, Nested 8 Stack (Passive Stack)
|Disposable 96-tip rack
|Santa Cruz Biotechnology
|Columbus Image analysis software
|image analysis software
|Echo Qualified 384-Well Polypropylene Microplate, Clear, Non-sterile
|luminescence plate reader
|Express pick Library (96-well)-L3600-Z369949-100µL
|Greiner FLUOTRAC 200 384 well plates
|solid-black 384-well plates
|Harmony Imaging software
|Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water
|Labcyte Echo 550 series with Echo Cherry Pick software
|nanoscale acoustic liquid dispenser
|Opera Phenix High-Content Screening System
|Paraformaldehyde solution 4% in PBS
|Santa Cruz Biotechnology
|PhenoPlate 384-well, black, optically clear flat-bottom, tissue-culture treated, lids
|CellCarrier-384 Ultra PN
|Obtained from Dr. Robin Ketteler (Department of Human Medicine, Medical School Berlin)
|Polybrene Infection / Transfection Reagent
|Puromycin dihydrochloride, 98%, Thermo Scientific Chemicals
|Tecan Freedom EVO 200 robot
|liquid handling robotic platform
|X-tremeGENE HP DNA Transfection Reagent Roche
|DNA transfection reagent
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