We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

NOTE: The throughput of such assay systems depends on the available HTS lab equipment. This protocol is facilitated by a Tecan Freedom EVO 200 robot, which performs liquid handling in 96-well format. Miniaturization to 384-well format is also possible. The robotic liquid handling system is positioned under a laminar flow hood in order to maintain aseptic conditions during all experimental steps. If no liquid-handling automation is available, the protocol can be readily adapted to low-throughput format.
Primary Screening
1. Day 1: Prepare and Seed Cells
NOTE: Seed cells to yield 80% confluence at the time of assaying luciferase activity, i.e., 48 hr after seeding.
<ol>
	<li>Resuspend trypsinized CHP134-mycn3UTR neuroblastoma cells to a concentration of 2 &#215; 105 cells/ml in RPMI containing 10% FBS and 1% L-glutamine. For 27 plates prepare at least 250 ml of cell suspension taking into account liquid handling, troughs dead volumes and repeated pipetting steps. Pour the cell suspension into sterile reservoir and place it on the robot desk.</li>
	<li>Place 9 barcoded white flat-bottom 96-well plates and a box of 200 &#956;l sterile pipet tips on the robot desk. Mix the cell suspension by pipetting before each dispensation step to avoid cells settling under gravity. Dispense 75 &#956;l of cells into each well of 9 plates to get a final density of 1.5 &#215; 104 cells per well.</li>
	<li>Cover the plates with the lids and leave them outside of the incubator for 30 min to allow the cells to sediment to the bottom of the well. This will minimize edge effects.</li>
	<li>Repeat steps 1.2 and 1.3 twice in order to prepare a total number of 27 plates.</li>
	<li>Place the plates at 37 &#176;C and 5% CO2 and incubate for 24 hr.</li>
</ol>
2. Day 2: Treat the Cells with Library Compounds
NOTE: The Spectrum Collection small molecule library consists of 2,000 compounds arranged in 8 rows and 10 columns in 25 96-well plates at the concentration of 10 mM in DMSO. The wells on the first and last columns are left empty for controls. Compound screening assays are typically performed at 1&#8211;10 &#181;M compound concentration8. The screening protocol described here fixes 2 &#956;M as the working concentration and 24 hr as the assay end point.
<ol>
	<li>Thaw 9 compound library plates at room temperature.</li>
	<li>Prepare two troughs with sterile PBS and PBS-0.5% DMSO solutions and place them on the robot desk. For each library plate, prepare and label accordingly one dilution plate &#8212; a polypropylene U-bottom 96-well plate with a working volume of at least 400 &#956;l. Fill each well of columns 2 to 11 of dilution plates with 398 &#956;l of sterile PBS using the liquid handler&#8217;s fixed tips. Fill columns 1 and 12 with 50 &#956;l of sterile PBS with 0.5% DMSO in order to reproduce the vehicle concentration in the control wells (0.02%).</li>
	<li>Place two library plates, the correspondingly labeled dilution plates, two boxes of 50 &#956;l sterile pipet tips and six cells plates on the robot desk. Uncover cells plates.</li>
	<li>Pipet 2 &#956;l of the 10 mM stock solution from one of two library plates in the corresponding dilution plate and mix well. This will result in an intermediate compounds concentration of 50 &#956;M. Using the same set of tips, dispense 3 &#956;l of the 50 &#956;M dilution in 3 barcoded white 96-well plates with cells. This dilution scheme results in 2 &#956;M working concentration of each tested compound. 
	NOTE: To avoid unnecessary manipulations with the tips, use a full set of 96 tips from the beginning, even though the library plates contain only 80 compounds. This is possible since the first and the last columns of the library plates are empty.</li>
	<li>Replace the pipet tips and repeat Step 2.4 for the second library plate.</li>
	<li>Cover the cells plates and return them to the incubator for 24 hr.</li>
	<li>Repeat Steps 2.3-2.6 two more times for the remaining library plates.</li>
</ol>
3. Day 3: Perform Luciferase Assay
NOTE: Before performing the luciferase assay, it is possible to multiplex it with a cell viability assay based on reduction of resazurin in order to obtain a cell-viability index from each well9. Multiplexing luciferase and viability assay assays results in a reduction of luciferase signal that, however, can be ignored if the cells provide high enough level of luciferase activity. Luciferase activity can be detected by a variety of commercial and homemade10,11 luciferase assay reagents with stable luminescent signal.
<ol>
	<li>Equilibrate the reconstituted luciferase reagent of choice to room temperature. Make sure the volume is enough for all the assayed plates. For 27 plates 170 ml of luciferase reagent will be required taking into account liquid handling, troughs dead volume and repeated pipetting steps. Pour the luciferase reagent into a reservoir and place it on the robot desk.</li>
	<li>Remove 9 plates with cells from the incubator, place them on the robot desk, uncover and wait until equilibrated to room temperature. Add 50&#160;&#956;l of the luciferase reagent per well plate by plate. Between the plates, introduce a delay equal to the luminescence reading time of one plate. This will assure that all the plates are measured within equal time interval from the reagent addition.</li>
	<li>After a 30 min incubation, place the first plate in the luminometer and measure luminescence after a brief shaking step. Repeat for all plates. Record the barcode of each plate and associate it to the corresponding data file to avoid mistakes arising from a large number of handled plates.</li>
	<li>Repeat Steps 3.2-3.3 twice for the remaining 18 plates with cells.</li>
	<li>Collect the remaining luciferase reagent and store it at -20 &#176;C.</li>
</ol>
4. Day 4: Data Analysis
<ol>
	<li>Process experimental data using normalization of all samples to the average of on-plate vehicle-treated controls. For each library compound, calculate the mean value x&#773; and SD over triplicates.</li>
	<li>Select up-regulating and down-regulating hits by setting a threshold of x&#773; &#177; k &#215; SD, where the mean value x&#773; and SD are computed over all assay processed values, and k is a defined constant.</li>
	<li>Select an arbitrary threshold cut-off &#60; 20% of vehicle-treated controls when a reduction of luciferase signal is most probably associated with compound toxicity. Discarding the hits below this threshold will significantly reduce a number of false-positive hits in the counter-screening. 
	NOTE: Instead of control-based normalization, experimental data can be processed applying Z score or its robust analog B score12. In addition, alternative statistical approaches for hit selection are available12.</li>
</ol>
5. Counter-screening
NOTE: Compounds identified in the primary screen (labeled &#8216;hits&#8217;) are confirmed and evaluated for the specificity by a counter-screening.
<ol>
	<li>Cherry pick the selected hits from the aliquots stored in single 2D barcoded tubes and create new &#8220;hit plates&#8221;, saving the corresponding layout. Do not forget to leave free positions for controls. 
	NOTE: In the absence of an efficient cherry-picking system, one might test both the control and 3&#8217; UTR-bearing cells in parallel in the primary screening. In this case, specific selection of compounds with effects dependent only on the introduced 3&#8217; UTR will be possible after the primary screening eliminating the need of counter-screening. However, the parallel screening in two cell lines will double the assay costs.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 1: Prepare and Seed Cells&#8221;), for each &#8220;hit plate&#8221; prepare three 96-well plates of each CHP134-mycn3UTR and CHP134-CTRL stable cells.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 2: Treat the Cells with Library Compounds&#8221;), use each hit plate to treat three CHP134-mycn3UTR and three CHP134-CTRL plates at 2 &#956;M working concentration. 
	NOTE: While the counter-screen described here is performed in triplicates at the single dose of 2 &#956;M, it might be beneficial to substitute the standard replicates and test the hits in 3 concentrations as 10-fold dilutions ranging from 2 &#956;M to 20 nM. Applying this approach would not only allow to confirm the data of the primary screen, but also to obtain preliminary dose-response data on the primary hits, minimizing false-positive rates. In this case, a different analysis pipeline should be applied to process the experimental data and confirm the hits.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 3: Perform Luciferase Assay&#8221;), measure luciferase activity in all CHP134-mycn3UTR and CHP134-CTRL plates.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 4: Data Analysis&#8221;), normalize experimental data from treated wells to the average of on-plate vehicle-treated controls and calculate the mean and SD over replicates. For each compound tested, compute the fold change of luciferase activity in CHP134-mycn3UTR vs. CHP134-CTRL cells. Select arbitrary fold change cut-offs of &#62;1.5 and significance p-values of &#60;0.01. 
	NOTE: In the described assay settings the determinant parameter for hit specificity is a significant fold change of luciferase activity in CHP134-mycn3UTR vs. CHP134-CTRL cells. If needed, one might also assess compound toxicity by performing concurrently a cell viability assay on the selected compounds. This might be especially beneficial if the primary hits are counter-screened in a dose response analysis. For a single dose, our experience indicates a strong correlation of reduced luciferase activity with decreased cell viability6. Therefore, decreased luciferase activity in target cells as well as control cells can be considered as an indicator of compound toxicity at the tested concentration.</li>
</ol>

<ol>
	<li>Schwanh&#228;usser, B., 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=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Tebaldi, T., 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=Widespread+uncoupling+between+transcriptome+and+translatome+variations+after+a+stimulus+in+mammalian+cells.">Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells.</a> BMC Genomics. 13 (1), 220 (2012).</li><li>Mitchell, S. F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+properties+of+eukaryotic+mRNPs.">Principles and properties of eukaryotic mRNPs.</a> Mol. Cell. 54 (4), 547-558 (2014).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nat. Rev. Genet. 15 (5), 293-306 (2014).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3&#8217;+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3&#8217; untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA Biol. 9 (5), 563-576 (2012).</li><li>Sidarovich, V., Adami, V., Quattrone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cell-Based+High-Throughput+Screen+Addressing+3&#8217;+UTR-Dependent+Regulation+of+the+MYCN+Gene.">A Cell-Based High-Throughput Screen Addressing 3&#8217; UTR-Dependent Regulation of the MYCN Gene.</a> Mol. Biotechnol. 56 (7), 631-643 (2014).</li><li>Cheung, N. -. K. V., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroblastoma:+developmental+biology,+cancer+genomics+and+immunotherapy.">Neuroblastoma: developmental biology, cancer genomics and immunotherapy.</a> Nat. Rev. Cancer. 13 (6), 397-411 (2013).</li><li>Hughes, J. P., Rees, S., Kalindjian, S. B., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+early+drug+discovery.">Principles of early drug discovery.</a> Br. J. Pharmacol. 162 (6), 1239-1249 (2011).</li><li>Herbst, 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=Multiplexing+a+high-throughput+liability+assay+to+leverage+efficiencies.">Multiplexing a high-throughput liability assay to leverage efficiencies.</a> Assay Drug Dev. Techn. 7 (3), 294-303 (2009).</li><li>van Olst, E. S. i. e. b. r. i. n. g. -., 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=Affordable+luciferase+reporter+assay+for+cell-based+high-throughput+screening.">Affordable luciferase reporter assay for cell-based high-throughput screening.</a> J. Biomol. Screen. 18 (4), 453-461 (2013).</li><li>Baker, J. M., Boyce, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+functional+screening+using+a+homemade+dual-glow+luciferase+assay.">High-throughput functional screening using a homemade dual-glow luciferase assay.</a> J. Vis. Exp. (88), (2014).</li><li>Malo, N., Hanley, J. A., Cerquozzi, S., Pelletier, J., Nadon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+practice+in+high-throughput+screening+data+analysis.">Statistical practice in high-throughput screening data analysis.</a> Nat. Biotechnol. 24 (2), 167-175 (2006).</li><li>Hitti, 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=A+versatile+ribosomal+protein+promoter-based+reporter+system+for+selective+assessment+of+RNA+stability+and+post-transcriptional+control.">A versatile ribosomal protein promoter-based reporter system for selective assessment of RNA stability and post-transcriptional control.</a> RNA. 16 (6), 1245-1255 (2010).</li><li>Oikonomou, P., Goodarzi, H., Tavazoie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+regulatory+elements+in+conserved+3&#8217;+UTRs+of+human+transcripts.">Systematic identification of regulatory elements in conserved 3&#8217; UTRs of human transcripts.</a> Cell Rep. 7 (1), 281-292 (2014).</li><li>Gandin, V., 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=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J. Vis. Exp. (87), (2014).</li><li>Conne, B., Stutz, A., Vassalli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3&#8217;+untranslated+region+of+messenger+RNA:+A+molecular+&#8220;hotspot&#8221;+for+pathology.">The 3&#8217; untranslated region of messenger RNA: A molecular &#8220;hotspot&#8221; for pathology.</a> Nat. Med. 6 (6), 637-641 (2000).</li><li>Cheneval, D., Kastelic, T., Fuerst, P., Parker, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+methods+to+monitor+the+modulation+of+mRNA+stability:+a+novel+approach+to+drug+discovery+and+therapeutic+intervention.">A review of methods to monitor the modulation of mRNA stability: a novel approach to drug discovery and therapeutic intervention.</a> J. Biomol. Screen. 15 (6), 609-622 (2010).</li><li>Pascale, A., Govoni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+world+of+post-transcriptional+mechanisms:+is+their+deregulation+a+common+link+for+diseases?+Focus+on+ELAV-like+RNA-binding+proteins.">The complex world of post-transcriptional mechanisms: is their deregulation a common link for diseases? Focus on ELAV-like RNA-binding proteins.</a> Cell. Mol. Life Sci. 69 (4), 501-517 (2012).</li><li>Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+pre-mRNA+processing+and+altered+architecture+of+3&#8217;+untranslated+regions+contribute+to+the+development+of+human+disorders.">Impaired pre-mRNA processing and altered architecture of 3&#8217; untranslated regions contribute to the development of human disorders.</a> Int. J. Mol. Sci. 14 (8), 15681-15694 (2013).</li><li>Bhattacharyya, A., Trotta, C. R., Peltz, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+the+GEMS--a+novel+platform+technology+targeting+post-transcriptional+control+mechanisms.">Mining the GEMS--a novel platform technology targeting post-transcriptional control mechanisms.</a> Drug Discov. Today. 12 (13-14), 553-560 (2007).</li><li>Peltz, S. W., Welch, E. M., Trotta, C. R., Davis, T., Jacobson, 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+post-transcriptional+control+for+drug+discovery.">Targeting post-transcriptional control for drug discovery.</a> RNA Biol. 6 (3), 329-334 (2009).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

For a long period transcriptional regulation of gene expression was thought to play a major if not exclusive role in controlling protein production. Accumulating evidence, however, indicates that post-transcriptional regulation contributes as much as, if not more than, transcriptional regulation to determine cellular protein abundance1,2. Post-transcriptional control of gene expression is much more complex and elaborate than was at first thought. In fact, all the various stages of post-transcriptional control have emerged to be regulated, including mRNA processing, localization, turnover, translation3 as well as the newly described reversible RNA methylation4. From a number of post-transcriptional regulation processes potentially affected, the assay described below focuses on those involving the mRNA 3&#39; untranslated region (3&#8217; UTR). 3&#8217; UTR broadly affects mRNA fate mainly via specific interaction of RNA-binding proteins and non-coding RNAs to its regulatory sequences and/or secondary structures5. Therefore, small molecules that alter these interactions or interfere with upstream signal transduction pathways will shift the balance that regulates protein abundance. This therapeutic approach is of particular importance for human pathologies for which evidence exists showing that disease-relevant genes are subjected to post-transcriptional regulation, which thus represents a potential target for pharmacological intervention. Therefore, systems allowing for screening of mRNA levels&#8217; modulators through interference with post-transcriptional control mechanisms in a high-throughput format can become valuable tools in the identification of potential novel treatments.
The described cell-based reporter gene assay allows for the identification of compounds able to modulate mRNA fate via mechanisms dependent on its 3&#8217; UTR. It consists of several principal components (Figure 1) and requires few preliminary steps to validate the feasibility of the assay. First of all, the reporter construct is designed to include the 3&#8217; UTR from a gene of interest. The 3&#8217; UTR is fused to the end of a reporter gene such as firefly luciferase (Figure 1). Any changes in post-transcriptional control mechanisms exerted through the inserted 3&#8217; UTR will alter the stability and/or translation efficiency of this chimeric transcript, resulting in varied luciferase levels. Therefore, changes in luciferase activity serve as indirect measure of affected post-transcriptional regulation of the gene of interest. The second component of the system is a control reporter construct, an identical expression plasmid that expresses the same reporter gene but does not contain any 3&#8217; UTR. The two reporter plasmids can be designed in the lab using conventional molecular biology protocols or purchased from commercial sources.
The selection of an appropriate cell line is critical for the assay construction. Which cell line is the optimal model depends on numerous factors ranging from clinical requirements like maximal resemblance to pathology to just practical issues like availability, transfectability, and growth characteristics. Importantly, prior to proceed one should make sure that fusion of 3&#8217; UTR to the reporter gene has an expected effect on the level of the chimeric transcript. The absence of significant difference in luciferase signal from the 3&#8217; UTR-bearing and control reporter constructs transiently transfected in the selected cells would indicate that the 3&#8217; UTR is not functional in this cellular model, prompting for search of alternative ones. For the high-throughput format, the 3&#8217; UTR-bearing and control luciferase reporter constructs should be stably integrated in the selected cell line. Using a stably integrated over transiently transfected cell line is preferable for several reasons. This will broaden the choice of a cellular model including hard-to-transfect cell lines, decrease the variability in the data arising from fluctuations in transfection efficiency, subside the costs. Moreover, upon transient transfection cells are very often overloaded with a DNA plasmid leading to the saturation of the system. In these settings a further increase in the reporter expression might be challenging, resulting in decreased sensitivity towards potential upregulating compounds.
Finally, when all the assay components are set up, it is critical before moving to the high-throughput format to determine the feasibility of the assay, i.e., if luciferase activity can be indeed up- and down-regulated specifically via the inserted 3&#8217; UTR. The best controls would be small molecules reported to modulate the stability or translatability of mRNA through the 3&#8217; UTR of interest. If having these is not possible, surrogate controls imitating the desired effect are accepted. These could be miRNAs or RNA-binding proteins known to exert their effects via binding to the 3&#8217; UTR of interest. Evaluation of such controls before the primary screening not only indicates the functionality of the developed assay, but also allows for estimation of its dynamic range, sensitivity and performance in the high-throughput format.
Using the described protocol we screened a 2,000-compound library6. Neuroblastoma, the most common extracranial solid tumor of infancy, was used as a biological model and the 3&#8217; UTR of the MYCN oncogene, whose amplification strongly predicts adverse outcome of neuroblastoma, as a target gene7. Figure 2 outlines the experimental layout. The screening was divided in three runs with the batch size of 27 plates screened in one run. The batch of 27 plates included Spectrum Collection library plates n.1-n.8+n.25 (first run), n.9-n.16+n.25 (second run) and n.16-n.24+n.25 (third run), each plate screened in triplicate. Thus, one library plate (n.25) was assayed within all three runs making possible inter-run comparison. The protocol below describes a single run of 9 library plates tested in triplicate.

<table><tbody><tr><td>Culture plate 96-well White</td><td>PerkinElmer</td><td>6005688</td><td></td></tr><tr><td>StorPlate 96-well U bottom (dilution plate)</td><td>PerkinElmer</td><td>6008190</td><td></td></tr><tr><td>100 ml disposable trough for reagents</td><td>Tecan</td><td>10 613 048</td><td></td></tr><tr><td>300 ml disposable robotic reservoir</td><td>VWR</td><td>PB12001301</td><td></td></tr><tr><td>Robot tips DITI 50&amp;nbsp;&amp;mu;l Sterile</td><td>Tecan</td><td>30038607</td><td></td></tr><tr><td>Robot tips DITI 200&amp;nbsp;&amp;mu;l Sterile</td><td>Tecan</td><td>30038617</td><td></td></tr><tr><td>Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td><td>Thermo Scientific</td><td>3711</td><td></td></tr><tr><td>ONE-Glo Luciferase Assay System&amp;nbsp;</td><td>Promega</td><td>E6120</td><td></td></tr><tr><td>RPMI</td><td>Lonza</td><td>BE12-918F</td><td></td></tr><tr><td>PBS-1x, w/o Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Lonza</td><td>BE17-516F</td><td></td></tr><tr><td>L-Glutamine 200&amp;nbsp;mM</td><td>Lonza</td><td>BE17-605E</td><td></td></tr><tr><td>FBS</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>The Spectrum collection (compound library)</td><td>MicroSource Discovery Systems</td><td>N/A</td><td></td></tr><tr><td>Tecan Freedom EVO200 robot&amp;nbsp;</td><td>Tecan</td><td>N/A</td><td></td></tr><tr><td>Tecan F200 multiplate reader&amp;nbsp;</td><td>Tecan</td><td>N/A</td><td></td></tr></tbody></table>

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.

Using the described approach, we screened a 2,000-compound library for potential modulators of post-transcriptional control mechanisms exerted through the 3&#8217; UTR of the MYCN gene. Figure 3 depicts the results of the primary screening exemplified by a single library plate. Luciferase signal displayed as percentage of vehicle-treated controls was obtained by measuring luciferase activity in triplicate plates of CHP134-mycn3UTR cells treated with compounds of a single library plate. As expect...

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

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 ...

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9.4K Views.  University of Trento. 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’ UTR.

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

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genetics

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Developmental Biology

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Immunology and Infection

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...