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

<ol>
	<li>Kocaturk, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+as+a+molecular+target+for+cancer+treatment.">Autophagy as a molecular target for cancer treatment.</a> European Journal of Pharmaceutical Sciences. 134, 116-137 (2019).</li><li>Fu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+ATG4+in+cancer+therapy.">Targeting ATG4 in cancer therapy.</a> Cancers. 11 (5), 649 (2019).</li><li>Towers, C. G., Thorburn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+autophagy.">Therapeutic targeting of autophagy.</a> EBioMedicine. 14, 15-23 (2016).</li><li>Agrotis, A., Ketteler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+ATG4B+as+drug+target+for+treatment+of+solid+tumours-the+knowns+and+the+unknowns.">On ATG4B as drug target for treatment of solid tumours-the knowns and the unknowns.</a> Cells. 9 (1), 53 (2019).</li><li>Levy, J. M. M., Towers, C. G., Thorburn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+autophagy+in+cancer.">Targeting autophagy in cancer.</a> Nature Reviews Cancer. 17 (9), 528-542 (2017).</li><li>Kimmelman, A. C., White, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+tumor+metabolism.">Autophagy and tumor metabolism.</a> Cell Metabolism. 25 (5), 1037-1043 (2017).</li><li>Tran, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+role+of+ATG4B+as+target+for+autophagy+inhibition+in+prostate+cancer+therapy.">Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy.</a> Biochemical and Biophysical Research Communications. 441 (4), 726-731 (2013).</li><li>Pengo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+kinases+and+phosphatases+that+regulate+ATG4B+activity+by+siRNA+and+small+molecule+screening+in+cells.">Identification of kinases and phosphatases that regulate ATG4B activity by siRNA and small molecule screening in cells.</a> Frontiers in Cell and Developmental Biology. 6, 148 (2018).</li><li>Kauffman, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delipidation+of+mammalian+Atg8-family+proteins+by+each+of+the+four+ATG4+proteases.">Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.</a> Autophagy. 14 (6), 992-1010 (2018).</li><li>Tanida, I., Sou, Y. -. S., Minematsu-Ikeguchi, N., Ueno, T., Kominami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg8L/Apg8L+is+the+fourth+mammalian+modifier+of+mammalian+Atg8+conjugation+mediated+by+human+Atg4B,+Atg7+and+Atg3.">Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3.</a> The FEBS Journal. 273 (11), 2553-2562 (2006).</li><li>Agrotis, A., Pengo, N., Burden, J. J., Ketteler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundancy+of+human+ATG4+protease+isoforms+in+autophagy+and+LC3/GABARAP+processing+revealed+in+cells.">Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells.</a> Autophagy. 15 (6), 976-997 (2019).</li><li>Nguyen, T. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg8+family+LC3/GABARAP+proteins+are+crucial+for+autophagosome-lysosome+fusion+but+not+autophagosome+formation+during+PINK1/Parkin+mitophagy+and+starvation.">Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.</a> The Journal of Cell Biology. 215 (6), 857-874 (2016).</li><li>Ketteler, R., Tooze, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATG4:+More+than+a+protease.">ATG4: More than a protease.</a> Trends in Cell Biology. 31 (7), 515-516 (2021).</li><li>Zhang, L., Li, J., Ouyang, L., Liu, B., Cheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+roles+of+Atg4+proteases+from+autophagy+modulation+to+targeted+cancer+therapy.">Unraveling the roles of Atg4 proteases from autophagy modulation to targeted cancer therapy.</a> Cancer Letters. 373 (1), 19-26 (2016).</li><li>Fern&#225;ndez, &. #. 1. 9. 3. ;. F., L&#243;pez-Ot&#237;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+and+pathologic+relevance+of+autophagy+proteases.">The functional and pathologic relevance of autophagy proteases.</a> The Journal of Clinical Investigation. 125 (1), 33-41 (2015).</li><li>Maruyama, T., Noda, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy-regulating+protease+Atg4:+structure,+function,+regulation+and+inhibition.">Autophagy-regulating protease Atg4: structure, function, regulation and inhibition.</a> The Journal of Antibiotics. 71 (1), 72-78 (2017).</li><li>Ketteler, R., Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+autophagy+by+luciferase+release+assay.">Quantitation of autophagy by luciferase release assay.</a> Autophagy. 4 (6), 801-806 (2008).</li><li>Ketteler, R., Sun, Z., Kovacs, K. F., He, W. -. W., Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pathway+sensor+for+genome-wide+screens+of+intracellular+proteolytic+cleavage.">A pathway sensor for genome-wide screens of intracellular proteolytic cleavage.</a> Genome Biology. 9 (4), 64 (2008).</li><li>Luft, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Gaussia+luciferase+in+bicistronic+and+non-conventional+secretion+reporter+constructs.">Application of Gaussia luciferase in bicistronic and non-conventional secretion reporter constructs.</a> BMC Biochemistry. 15, 14 (2014).</li><li>Ketteler, R., Glaser, S., Sandra, O., Martens, U. M., Klingm&#252;ller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+transgene+expression+in+primitive+hematopoietic+progenitor+cells+and+embryonic+stem+cells+efficiently+transduced+by+optimized+retroviral+hybrid+vectors.">Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors.</a> Gene Therapy. 9 (8), 477-487 (2002).</li></ol>

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>
		</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&#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>
		</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.

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

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

The main aim of research in my lab is to understand the regulation of autophagy and in particular of the ATG4 cysteine protean and use this knowledge to develop small molecule inhibitors that target this pathway in particular for diseases such as pancreatic cancer. We use high content, high throughput screening technologies to identify drug targets as well as drug candidates and we focus on the ATG4 cysteine protease family because we think it's a very good drug target for various forms of cancer. We have been studying the ATG4 family members for quite some time now.We made significant discoveries into dysfunction of this protein family and cells. We've also identified novel post-translational mechanisms of regulation of the ATG4 family members and currently we are developing small molecule inhibitors and also activators of the ATG4 families for diseases such as cancer and neuro degeneration. There are not many assays to monitor ATG4 protease activity in cells.So we were one of the first to develop a luciferase based assay to monitor this activity. And our assay is quite unusual because it uses secreted, let's say phrase, therefore we don't need to license the cells and we can adapt this protocol easily to high throughput screening methods and lab automation. We have focused on the ATG4 B protein protease for quite some time now.We have generated knockout cell lines of the ATG4 family members. We have identified novel post-translational regulation mechanisms of ATG4, and ultimately we have also identified several novel small molecule compounds that could inhibit and even activate the ATG4 B protease. In the future, we'd like to continue our work on the ATG4 family members.We think that's quite a lot that is currently unknown and still remains to be uncovered. For example, how these proteins regulate autophagy in neurons is currently completely not understood. We'd also like to focus our work on validation of these small molecule compounds that we've identified and potentially to use activators in neurodegenerative diseases.

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

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

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

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

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474 Views.  University College London. The main aim of research in my lab is to understand the regulation of autophagy and in particular of the ATG4 cysteine protean and use this knowledge to develop small molecule inhibitors that target this pathway in particular for diseases such as pancreatic cancer. We use high content, high throughput screening technologies to identify drug targets as well as drug candidates and we focus on the ATG4 cysteine protease family because we think it's a very good drug target for various forms of ca...

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

Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening

Begin by thawing the Selleckchem Small Molecule Compound Library plates at room temperature. Open the software and click on New Protocol. On the Protocol tab, click on Sample Plate Format, followed by 384PP.Then on Sample Plate Type, select 384PP_DMSO2. Next, choose Cell Carrier 384 Ultra PN as the Destination Plate Type. Click on Pick List and select the import options to import the spreadsheet containing the dispensing program.Click on the Run Protocol option to verify the accuracy of the displayed information and then click on Run. Next press Start on the new window titled Run status, and then follow the steps displayed on the prompt windows. Use a nanoscale acoustic liquid dispenser and dispense 50 nanoliters per well of the Small Molecule Compound Library into a 384 well assay plate.To begin cellular supernatant harvesting, start by configuring the deck of the lab automation workstation. Place a disposable 96 tip rack on the P1 position and place the assay plates on positions P2 and P4.Finally, place the empty, solid black 384 well plates at positions P3 and P5.Using the tips from P1, aspirate 10 microliters of the supernatant from the plate at P2 and then transfer it into the empty solid black plate on P3.Discard the tips at position P6.Repeat the transfer of supernatant from P4 to P5.

Luciferase Assay for the Detection of Autophagy Protease ATG4B

Begin by opening the program and initialize the injector pump by clicking on the Dispenser Control, and select Pump 2, and then clicking on Init. To rinse the tubing with the ionized water, click on Rinse, then press Next. Select Pump 2 and set the tip mount to the desired tip.Then click on Next again. Click Start to begin tube rinsing. Repeat rinsing with methanol.Meanwhile, prepare a working substrate solution by diluting the substrate stock by 1 to 100. Finally, rinse the tubing with the working dilution of the substrate stock in PBS. Load the plate into the plate reader.And then, on Measurement settings, choose the aperture to 384 plate US luminescence aperture, followed by the distance between plate and detector to 0.6 millimeters, and the measurement time as 0.5 seconds. Once the measurement settings have been settled, on the Protocol-General settings tab, select the plate type as 384 Nunc, and choose the By columns bi-directional option as the measurement mode. Input 1 into the number of assay repeats tab.Now, on the Dispense tab, select the desired dispense measurement. Then set the measurement time for each well as one second and select the desired used pump. Dispensing speed of 200 microliters per second.And the dispensing volume to 50 microliters. Choose Dispensing volume as the syringe filling volume for Pump 2. And then set the Start dispensing at measurement number to 1.On the Well selection tab, use the Add/remove samples dropdown menu and choose the desired sample type, and select the wells to be measured. After that, start the measurement by clicking Run. The basal luminescence signal from DMSO is seen in column 1, and the decrease of luminescence signal on the presence of ATG4B inhibitor, FMK-9a, is seen in column 24.

Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay

Begin by launching the microscope operating software. Under the setup tab, choose the correct predefined plate type. Press eject to access the plate stage to position the plate and then press load to load the plate into the microscope.Next, choose the 20 times air objective with a numerical aperture of 0.4. Adjust the correction collar of the 20x air objective for the plate thickness of the chosen plate type. Set the channel selection to Hoechst 33342, and adjust the imaging parameters for the Hoechst channel.Test the chosen settings by selecting a representative well within the plate. On defined layout, select all plate wells and for fields. Then choose the corresponding folder under online jobs to transfer the data to the online analysis software.The experiment settings are saved. Under the run experiment tab, the plate which is going to be imaged is named, and the imaging process is started. To begin image analysis, first launch the software, choose the images for analysis, and then click on the image analysis tab to start image segmentation.Next, click on the plus sign in the input image tab to add a new building block. From the list, click on find nuclei. Choose the most appropriate segmentation method after inspecting the segmented objects on the image.Adjust the detection parameters within the segmentation method. Click on define results and choose standard output as the method. Click on save analysis to database to save the analysis pipeline.Under the batch analysis tab, click on the saved pipeline which will appear under the method option. Then select the data to be analyzed. Start the analysis by clicking on start analysis to generate the dataset for cell numbers.The described cell-based assay relies on a reporter readout that indirectly reflects changes in the ATG4B activity, enabling the detection of both inhibitors and activators of ATG4B activity. The ratio of the ATG4B activity of each compound to its cell viability is used as a cutoff value for the identification of ATG4B inhibitors. Ratios greater than one indicate possible ATG4B activators and ratios less than one indicate possible ATG4B inhibitors.