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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 &#181;L Disposable Tips - Non-filtered, Pure, Nested 8 Stack (Passive Stack)</td>
			<td>Tecan</td>
			<td>30038609</td>
			<td>Disposable 96-tip rack</td>
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		<tr>
			<td>BioTek MultiFlo</td>
			<td>BioTek</td>
			<td></td>
			<td>bulk dispenser</td>
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		<tr>
			<td>Coelenterazine</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-205904</td>
			<td>substrate</td>
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		<tr>
			<td>Columbus Image analysis software</td>
			<td>Perkin Elmer</td>
			<td>Version 2.9.1</td>
			<td>image analysis software</td>
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			<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&#181;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>
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		<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>
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		<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

689 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 

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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, ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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 ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

POC Method and Integrated Decision Support Tool for Estimating Anemia

A Point-of-Care Method with Integrated Decision Support Tool to Estimate Anemia at Population Level

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point of care. To address this caveat, a novel method based on pooled capillary blood and a portable autoanalyzer was developed for the estimation of Hb. Additionally, custom software was developed for near-real-time integration of the Hb values from the auto analyzer to the server. Moreover, a decision support tool that can immediately categorize the participants into different stages of anemia was developed. The decision support tool was designed based on the World Health Organization (WHO) cut-off for anemia at the population level and was available for all age and gender groups. This simple and user-friendly tool could&#160;easily be used by front-line health workers who have limited technical skills. Overall, the method developed could&#160;be used at the point of care and is accurate. This high-throughput method could&#160;be used for screening anemia at the population level for all age and gender groups.

Author Spotlight: Developing a Point-of-Care Hemoglobin Estimation Method for Anemia Management

An accurate hemoglobin estimation method is lacking at the point of care and may hinder population-based programs for treating anemia. Therefore, we developed a point-of-care method based on pooled capillary blood and an auto-analyzer integrated into a custom software application to categorize the hemoglobin values into different grades of anemia.

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point ...