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).

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).
<ol>
	<li>Culture HEK293T cells in Dulbecco&#39;s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 &#176;C in a humidified incubator, with an atmosphere of 5% CO2 until they are 80%-90% confluent before seeding for transfection.</li>
	<li>The day before transfection, seed the cells into a 6-well plate at a density of 1 &#215; 106 cells/well in 2 mL/well of complete growth medium. Incubate the plate overnight in a humidified incubator with an atmosphere of 5% CO2. 
	NOTE: Follow the manufacturer&#39;s instructions if using a liposomal-based transfection reagent. This protocol describes the use of a non-liposomal-based reagent. Before starting the transfection, allow the DNA transfection reagent, DNA, and media to equilibrate to room temperature (~15 min).</li>
	<li>For each transfection, add 200 &#181;L of serum-free medium to 1.5 mL microcentrifuge tubes. In each tube, add the following amounts of plasmids: 1,000 ng of pMOWS-ActinLC3dNGLUC; 900 ng of GagPol; 100 ng of VSV-G. 
	NOTE: The amount of plasmids and volume of media listed here are for a 12-well plate. If using a different plate type, adjust the media volume and plasmid amounts to reach the final concentration of 5 ng/&#181;L of transfer plasmid, 4.5 ng/&#181;L of packing plasmid, and 0.5 ng/&#181;L of envelope plasmid. Use any packing plasmid that contains gag- and pol-expressing genes, and any envelope plasmid that contains the VSV-G expressing gene.</li>
	<li>Vortex the DNA transfection reagent vial for 30 s. Pipette 4 &#181;L of the transfection reagent directly into the medium containing the diluted DNA. Mix gently by gently tipping the tube. 
	NOTE: Do not touch the walls of the plastic tubes with the tips. Do not pipette up and down or vortex.</li>
	<li>Incubate the reaction for 15 min at room temperature.</li>
	<li>While incubating the reaction, remove the old medium from the 6-well plate and replace it with 2 mL/well of fresh serum-free media.</li>
	<li>Add each transfection complex to each well in a dropwise manner.</li>
	<li>Gently shake or swirl the plate to ensure even distribution over the entire well surface.</li>
	<li>Incubate the plate overnight at 37 &#176;C in a humidified incubator with an atmosphere of 5% CO2.</li>
	<li>After 24 h, replace the old medium with fresh complete medium (2 mL/well) and return the cells back to the humidified incubator with an atmosphere of 5% CO2 for 72 h.</li>
	<li>After 72 h, harvest the supernatant in a 50 mL conical tube and centrifuge for 10 min at 4,000 &#215; g at 4 &#176;C to remove dead cells and debris. For further purification, use a large 60 mL syringe to pass the supernatant through a 0.20 &#181;m filter. Immediately prepare single-use aliquots of 300 &#181;L and store at -80 &#176;C. 
	&#8203;NOTE: Avoid a freeze-thaw cycle to maintain maximum product activity.</li>
</ol>
2. Retroviral transduction
<ol>
	<li>The day before transducing the cells, seed the target cells at a medium density (PANC1 at 1 &#215; 105 cells/well) in a 12-well plate in 1 mL/well of medium supplemented with 10% FBS and 1% P/S. Incubate the plate overnight at 37 &#176;C in a humidified incubator with an atmosphere of 5% CO2. 
	NOTE: The best cell density has to be established in advance, as different cell types have different attachment abilities. Ideally, use a cell density to obtain 40%-50% confluency.</li>
	<li>Remove the frozen retrovirus aliquots from the -80 &#176;C freezer and thaw on ice before each use. 
	NOTE: Do not refreeze unused aliquots.</li>
	<li>In a 50 mL conical tube, prepare a mixture of viral supernatant and polybrene at a final concentration of 8 &#181;g/mL. 
	NOTE: The subsequent volumes apply to the transduction of a 12-well plate containing a final volume of 500 &#181;L/well. The final viral supernatant volume can be estimated by testing a range of viral dilutions in the presence of polybrene. Higher or lower dilutions can be used depending on the desired expression levels of the transgene and the size of the vessel used.</li>
	<li>Remove the old medium from the 12-well plate and add 500 &#181;L of the mixture to each well. Incubate the cells with the viral supernatant overnight at 37 &#176;C in a humidified incubator with an atmosphere of 5% CO2. 
	NOTE: Keep two wells with complete medium (no viral supernatant) to use as a control for selection.</li>
	<li>Replace the virus-containing medium with fresh complete growth medium (1 mL/well). Place the cells back in the humidified incubator with an atmosphere of 5% CO2 for 48 h.</li>
</ol>
3. Pooled population selection and maintenance
<ol>
	<li>Replace the growth medium with selection medium (complete growth medium with a final concentration of 1 &#181;g/mL puromycin). Monitor the growth of the cells and change the selection media every 2-3 days. At confluence, expand into a 6-well dish, and then into a 10 cm diameter tissue culture dish.</li>
	<li>Keep the cells in selection medium for at least as long as it takes for the control (untransduced) cells to completely die. 
	NOTE: For successful results, it is recommended that optimal concentrations of puromycin be determined prior to initiating the experimental project. For this, generate a puromycin kill curve to determine the minimum concentration required to kill untransduced cells between 3 and 10 days.</li>
	<li>Once the cells are growing in the selection medium, expand the cells on complete growth media and freeze stock aliquots for the experimental project. 
	NOTE: Record the passage number and avoid working with pooled populations from frozen stock with passage numbers higher than five. Check regularly for mycoplasma contamination prior to performing the assay. Ideally, use a fresh cell batch before each assay to obtain the maximum signal of luciferase.</li>
	<li>Maintain the cells in selection medium and seed enough cells to reach the desired density the day before the assay.</li>
</ol>
4. Compound addition
NOTE: The Selleckchem small molecule library consists of approximately 4,000 compounds arranged in eight rows and 10 columns in fifty 96-well plates at a stock concentration of 10 mM in dimethyl sulfoxide (DMSO).
<ol>
	<li>Aliquot 30 &#181;L of compound into the appropriate well of a source plate that is compatible with a nanoscale acoustic liquid dispenser. Use a 384-well polypropylene plate (384PP plate). Keep these plates sealed and stored at -20 &#176;C. 
	NOTE: The screening protocol described here is for a total of 10 assay plates per day; a fixed concentration of 10 &#181;M was used as the final assay concentration along with a 24 h incubation period.</li>
	<li>Thaw the compound library plates at room temperature. 
	NOTE: Make sure the plate is completely thawed and equilibrated to room temperature. A temperature gradient across the plate may affect liquid handling.</li>
	<li>Dispense 50 nL/well into a 384-well assay plate using a nanoscale acoustic liquid dispenser. 
	NOTE: Ensure the selected source and destination plates in the program matches the ones used for this step.</li>
	<li>Create a dispensing program on a spreadsheet.</li>
	<li>Open the software.</li>
	<li>Open a new protocol. From the Protocol tab, select the following options: Sample plate format, 384PP; Sample plate type, 384PP_DMSO2; and Destination plate type, CellCarrier-384 Ultra PN (Figure 2A).</li>
	<li>Under Pick List, select the import option (Figure 2B) to import the spreadsheet containing the dispensing program (Figure 2C).</li>
	<li>Select the Run protocol option (Figure 2D), verify if the displayed information is correct, and click Run.</li>
	<li>On the new window called Run status (Figure 2E), click on Start and follow the steps displayed on the prompt windows (Figure 2F,G).</li>
</ol>
5. Cell seeding
<ol>
	<li>Trypsinize the cells from a cell culture flask or cell culture Petri dishes and neutralize the trypsin by adding FBS-containing media.</li>
	<li>Transfer the cell suspension to a 50 mL conical tube and centrifuge at 390 &#215; g for 5 min at room temperature. Then, gently remove the supernatant and resuspend the cell pellet in 10 mL of complete growth media.</li>
	<li>Perform cell counting.</li>
	<li>Prepare the cell suspension with 4.6 &#215; 107 cells in 230 mL of complete culture media. 
	NOTE: This volume and cell density is for 10x 384-well assay plates plus a dead volume of two 384-well plates.</li>
	<li>Dispense 50 &#181;L of cell suspension into each well of a 384-well assay plate, prepared in section 4. 
	NOTE: Cell seeding can be done either manually or by using a bulk dispenser.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified incubator with an atmosphere of 5% CO2 for 24 h.</li>
</ol>
6. Harvesting the cellular supernatant
NOTE: The liquid handling robotic platform used here performs liquid handling with a multichannel arm for 96-tips. If no liquid-handling automation is available, the protocol can be adapted to low-throughput format by using multichannel pipettes.
<ol>
	<li>Configure the deck of the lab automation workstation as shown in Figure 3.</li>
	<li>Place the disposable 96-tip stack on position P1 (Figure 3A). 
	NOTE: Each 96-tip stack is formed of eight disposable racks. When using a multichannel arm for 96-tips, the 384-well plate is divided in four quadrants. Thus, each tip stack is enough to transfer the supernatant from two assay plates into two empty, solid-black, 384-well plates. The entire tip stack needs to be replaced after each run of two plates.</li>
	<li>Place the assay plates on positions P2 and P4 (Figure 3A).</li>
	<li>Place the empty, solid-black, 384-well plates on positions P3 and P5 (Figure 3A). 
	NOTE: This flexible and capable robotic platform has been adapted for this assay and a special program was written (Figure 3B).</li>
	<li>Get the tips from position P1.</li>
	<li>Aspirate 10 &#181;L of supernatant from the plate on position P2 and transfer into the empty, solid-black plate on position P3. 
	NOTE: The tips should be positioned at an appropriate depth inside the wells to aspirate the supernatant without disturbing the cell monolayer at the bottom of the well.</li>
	<li>Drop the tips in waste on position P6 (Figure 3A).</li>
	<li>Repeat steps 6.5-6.7 for the remaining wells of the plate on position P2, and then repeat the same steps to transfer the supernatant from position P4 to position P5. 
	NOTE: Be sure to collect the supernatant and dispense on the corresponding wells into the empty, solid-black plate (Figure 3C,D). As secreted dNGLUC is very stable in cell culture medium, the plates can be sealed and kept for up to 7 days at 4 &#176;C in the dark.</li>
</ol>
7. Luciferase assay
NOTE: The dNGLUC used in the reporter exhibits flash kinetics with rapid signal decay. Due to the rapid decay of luminescence after adding substrate (coelenterazine), the plate reader should be set to measure the luminescence signal in the supernatants; inject the substrate to a well and read that well after a few seconds. For this reason, use a plate reader that is capable of monitoring luminescence and equipped with a substrate injector to ensure the time between the injection and read steps will be uniform for all samples. The settings used on the plate reader can be found in Figure 4.
<ol>
	<li>Prepare native coelenterazine as a 1 mg/mL stock solution in acidified methanol (10 &#181;L of 3 M HCl to 1 mL of methanol). 
	NOTE: Follow local health and safety guidance regarding the handling of methanol in the lab and avoid contact with skin. Prepare the fresh working substrate solution before starting the assay.</li>
	<li>Initialize the injector pump (Figure 5A).</li>
	<li>Rinse the tubing with deionized water (Figure 5B-D).</li>
	<li>Rinse the tubing with methanol.</li>
	<li>While rinsing the tubing, prepare the working substrate solution by diluting the substrate 1:100 (for one 384-well plate, add 220 &#181;L from the substrate stock solution into 21.8 mL of 1x phosphate-buffered saline [PBS]).</li>
	<li>Rinse the tubing with the substrate working solution (Figure 5B-D).</li>
	<li>Load the plate into the reader and start the measurement using the settings described in Figure 4.</li>
	<li>Repeat steps 7.6-7.7 for all assay plates.</li>
	<li>Once finished with all the assay plates, rinse the tubing with methanol.</li>
	<li>Rinse the tubing with deionized water. 
	&#8203;NOTE: At the end of this step, raw luciferase values are obtained that are correlative to the cellular ATG4B activity. For normalization to cell numbers, the next steps are necessary by counting the cell number in each well by fluorescence microscopy.</li>
</ol>
8. Cell fixation and staining
NOTE: This step can be performed manually with the aid of a multichannel pipette or by using a bulk dispenser.
<ol>
	<li>Fix the cells with 4% paraformaldehyde (in 1x PBS) for 15 min. 
	NOTE: Follow local health and safety guidance regarding the handling of paraformaldehyde in the lab and avoid contact with skin. Perform this step in a safety hood if possible.</li>
	<li>Wash three times with 1x PBS.</li>
	<li>Stain the nuclei with Hoechst 33342 diluted 1:5,000 in 1x PBS for 15 min.</li>
	<li>Wash three times with 1x PBS.</li>
</ol>
9. Image acquisition
NOTE: Perform image acquisition using an automated microscope. As an alternative to image acquisition to determine number of cells, the intracellular luciferase activity can also be determined. There are advantages and disadvantages with regards to whether one normalizes to cell numbers or to intracellular luciferase activity, which is discussed below. We find that determining cell numbers is less invasive and results in lower variability than determining intracellular luciferase values.
<ol>
	<li>Launch the microscope operating software (Figure 6).</li>
	<li>In the Setup tab, choose the correct predefined plate type. If the plate type is not preset, enter the plate dimensions manually.</li>
	<li>Load the plate into the microscope by clicking on Eject and Load option.</li>
	<li>Then, choose the 20x Air (numerical aperture [NA]: 0.4) objective. 
	NOTE: Ensure the objective collar is set to the correct value, allowing proper focus with different plate types.</li>
	<li>Under Channel Selection, choose Hoechst 33342. 
	NOTE: The channel settings for time, power, and height have to be optimized according to the plate type used.</li>
	<li>On Define Layout, select all wells from the plate and four fields from the well.</li>
	<li>On Online Jobs, select the corresponding folder to transfer the data to the analysis software.</li>
</ol>
10. Image analysis
NOTE: Any image analysis software can be used to segment and count cell nuclei from the acquired images. Here, we describe the steps to use a specific online software that is compatible to multiple automated microscopes files.
<ol>
	<li>Launch the image analysis software.</li>
	<li>Go to the Image Analysis tab to start the image segmentation (Figure 7A).</li>
	<li>In the Input Image tab, click on the + sign to add a new building block (Figure 7A).</li>
	<li>From the list, select the Find Nuclei option (Figure 7A) and select Hoechst 33342 as the channel option (Figure 7B).</li>
	<li>Visually inspect the segmented objects on the image and select the most accurate segmentation method. 
	NOTE: For this experiment, we used method C (Figure 7C). Each method option has subcategories that can be adjusted to obtain the best segmentation possible.</li>
	<li>Then, click on the Define Results tab (Figure 7C) and select Standard Output as the Method option.</li>
	<li>From the subcategory, select Nuclei-number of objects and Object count (Figure 7C).</li>
	<li>Save the analysis pipeline using the Save analysis to Disk or Save analysis to Database option.</li>
	<li>Under the Batch Analysis tab, select the data to be analyzed from the tree.</li>
	<li>Under Method, select the analysis pipeline saved in step 10.8. 
	NOTE: It is also possible to upload a script file from outside the referenced software or upload an existing analysis saved to the database.</li>
	<li>Click on Run Analysis to start the analysis (Figure 7D). At the end of this workflow, two datasets are generated: raw luciferase values from the supernatants and the cell number in each well. Use both to normalize the luciferase value per cell.</li>
</ol>

High-Throughput Screening Analysis for 4B Cysteine Peptidase Autophagy Inhibitor

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The authors have no conflicts of interest to disclose.

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 &#946;-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. ...

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 identified as a cellular stress response related to several diseases, such as neurodegenerative disorders, pathogen infection, and various types of cancer. The function of autophagy in cancer is complex and dependent on the type, stage, and status of the tumor. It can suppress tumorigenesis through autophagic degradation of damaged cells, but can also promote the survival of advanced tumors by improving cell survival during stressful conditions, such as hypoxia, nutrient deprivation, and cytotoxic damage2,4,5,6.
Several studies have shown that autophagy inhibition provides a benefit as an anticancer strategy. Thus, the inhibition of critical steps, such as autophagosome formation or its fusion with the lysosome, could be an effective method for cancer control2,4,5,6. Growing evidence has shown that ATG4B is involved in certain pathological conditions, and it has gained attention as a potential anticancer target2,3,4. For instance, it was observed that colorectal cancer cells and human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells had significantly higher ATG4B expression levels than adjacent normal cells2,4. In prostate cancer cells, inhibition of ATG4B resulted in a cell line-specific susceptibility to chemotherapy and radiotherapy7. Recently, strong evidence has emerged that pancreatic ductal adenocarcinoma (PDAC) is particularly vulnerable to ATG4B inhibition. For instance, in a genetically engineered mouse model, it was shown that intermittent loss of ATG4B function reduces PDAC tumor growth and increases survival3,4. Overall, ATG4B is highly overexpressed in some cancer types, is related to the progression of tumor, and is linked to cancer therapy resistance2,4,8.
The ATG4 cysteine proteases in mammals have four family members, ATG4A-ATG4D. These proteins exhibit some target selectivity toward the LC3/GABARAP (ATG8) family of proteins9,10,11 and may have additional functions not linked to their protease activity12,13. Furthermore, ATG4 functions in regulating a novel type of post-translational modification, the ATG8-ylation of proteins11,12. While ATG4B and its main substrate LC3B are the most widely studied, a picture is emerging that suggests a complex role for each subfamily member in the regulation of autophagic and non-autophagic processes. This is further corroborated by a complex network of post-translational modifications that regulate ATG4B activity via phosphorylation, acetylation, glycosylation, and nitrosylation9,10,11,12,13.
Several known ATG4B inhibitors have been published2,4,14,15. While these are suitable as research tools, their pharmacodynamic profile, selectivity, or potency have yet precluded them from development as preclinical candidates4,16. Overall, there is an urgent need to identify more potent and selective compounds. Often, the compounds are good biochemical inhibitors of protein function, yet their efficacy in cell-based assays is poor. There are multiple assays to monitor ATG4B activity, including biochemical methods and cell-based assays4. We have previously developed a simple, luminescence-based, high-throughput assay for monitoring ATG4B activity in cells8,17. This assay utilizes a luciferase protein from Gaussia princeps (GLUC) that is stable and active in the extracellular milieu and can be inducibly released from cells in response to ATG4B proteolytic activity18,19.
In this reporter construct, dNGLUC is linked to the actin cytoskeleton of cells. A protease-specific linker can be introduced between the &#946;-actin anchor and dNGLUC, turning the secretion dependent on cleavage of the linker. We used the full-length open reading frame of LC3B between &#946;-actin and dNGLUC, to be able to monitor LC3B cleavage17,18,19. Although the secretion mechanism of dNGLUC is poorly understood, it is specific for monitoring ATG4B activity, does not depend on overall autophagy as it occurs in ATG5 knockout cells, and is mediated by non-conventional mechanisms that do not require a classical signal peptide4,18,19. We have successfully used this reporter to screen small molecules and siRNA libraries, and have identified novel regulators of ATG4B activity, such as the Akt protein kinases8. This paper describes a detailed protocol for the use of this luciferase reporter in a semi-automated, high-throughput screening format.

<table><tbody><tr><td>50 &amp;micro;L Disposable Tips - Non-filtered, Pure, Nested 8 Stack (Passive Stack)</td><td>Tecan</td><td>30038609</td><td>Disposable 96-tip rack</td></tr><tr><td>BioTek MultiFlo</td><td>BioTek</td><td></td><td>bulk dispenser</td></tr><tr><td>Coelenterazine</td><td>Santa Cruz Biotechnology</td><td>sc-205904</td><td>substrate</td></tr><tr><td>Columbus Image analysis software</td><td>Perkin Elmer</td><td>Version 2.9.1</td><td>image analysis software</td></tr><tr><td>DPBS (1x)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Echo Qualified 384-Well Polypropylene Microplate, Clear, Non-sterile</td><td>Beckman Coulter</td><td>001-14555</td><td>384PP plate</td></tr><tr><td>EnVision II</td><td>Perkin Elmer</td><td></td><td>luminescence plate reader</td></tr><tr><td>Express pick Library (96-well)-L3600-Z369949-100&amp;micro;L</td><td>Selleckchem</td><td>L3600</td><td>Selleckchem</td></tr><tr><td>FMK9A</td><td>MedChemExpress</td><td>HY-100522</td><td></td></tr><tr><td>Greiner FLUOTRAC 200 384 well plates</td><td>Greiner Bio-One</td><td>781076</td><td>solid-black 384-well plates</td></tr><tr><td>Harmony Imaging software</td><td>Perkin Elmer</td><td>Version 5.1</td><td>imaging software</td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water</td><td>ThermoFisher</td><td>H3570</td><td>Hoechst 33342</td></tr><tr><td>Labcyte Echo 550 series with Echo Cherry Pick software</td><td>Labcyte/Beckman Coulter</td><td></td><td>nanoscale acoustic liquid dispenser</td></tr><tr><td>Milli-Q water</td><td></td><td></td><td>deionized water</td></tr><tr><td>Opera Phenix High-Content Screening System</td><td>Perkin Elmer</td><td></td><td>automated microscope</td></tr><tr><td>Paraformaldehyde solution 4% in PBS</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>PhenoPlate 384-well, black, optically clear flat-bottom, tissue-culture treated, lids</td><td>Perkin Elmer</td><td>6057300</td><td>CellCarrier-384 Ultra PN</td></tr><tr><td>pMOWS-ActinLC3dNGLUC</td><td></td><td></td><td>Obtained from Dr. Robin Ketteler (Department of Human Medicine, Medical School Berlin)</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>Merck</td><td>TR-1003-G</td><td>polybrene</td></tr><tr><td>Puromycin dihydrochloride, 98%, Thermo Scientific Chemicals</td><td>ThermoFisher</td><td>J61278.ME</td><td>Puromycin</td></tr><tr><td>Tecan Freedom EVO 200 robot</td><td>Tecan</td><td></td><td>liquid handling robotic platform</td></tr><tr><td>X-tremeGENE HP DNA Transfection Reagent Roche</td><td>Merck</td><td>6366244001</td><td>DNA transfection reagent</td></tr></tbody></table>

cell-based drug screening for inhibitors of autophagy related 4b cysteine peptidase

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.

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</...

Watch this Scientific Journal Video about A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells at JoVE.com

Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells 

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins ...

cell-based-drug-screening-for-inhibitors-autophagy-related-4b

Research

JoVE Journal

Biology

778 Views.  University College London. Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

Video: Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells 

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Biochemistry

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Immunology and Infection

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicine

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

This paper details monitoring autophagy flux to identify new molecules by cell-based imaging screening.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify ...

Virtual Screening and Validation of UCHL3 Inhibitors Using Molecular Docking

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately involved in a myriad of physiological and pathological processes. Therefore, developing small molecule inhibitors targeting Ubiquitin C-Terminal Hydrolase L3 (UCHL3) is of great significance. This protocol aims to establish a process for virtual screening and in vitro validation of small molecule inhibitors of cysteine DUB represented by UCHL3. Firstly, potential inhibitors of UCHL3 are virtually screened using molecular docking technology, and the interaction between drugs and protein targets is visualized. Subsequently, the effectiveness of the screened drug, Danshensu, is verified through in vitro activity inhibition assays. Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and hemagglutinin-ubiquitin-vinyl sulfone (HA-Ub-VS) are used as probes for in vitro activity testing, as they can competitively bind to DUB with small molecule inhibitors to assess the activity of UCHL3. The results indicate that Danshensu has a good binding affinity with UCHL3 in molecular docking, and it can competitively inhibit the activity of UCHL3 with HA-Ub-VS. These findings provide important references for further research and development of therapeutic drugs targeting UCHL3.

The experiment used here shows a method of molecular docking combined with probe technologies to predict and validate the interaction between small molecules of traditional Chinese medicine and protein targets.

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately ...

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Summary

Explore More Videos

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Study of Protein-protein Interactions in Autophagy Research

The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology