We would like to thank Emily Norton and Mikaelan Cucciarre-Stuligross for assisting in the preparation of shRNA vectors. This work was supported in part by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

NOTE: A schematic summary of this protocol is shown in Figure 1.
1. Lentiviral vector library preparation
NOTE: The demonstrated screen used an arrayed shRNA library purchased as glycerol stocks in 96-well plates, but libraries can also be assembled manually based on a list of candidates. Appropriate controls should be considered and included in any library. This includes a non-targeting control shRNA (shNTC), a control shRNA targeting the transcription factor being investigated, and if possible, an shRNA targeting firefly luciferase.
<ol>
	<li>Add 1.3 mL of Luria Broth (LB) (1% bacto-trypton, 0.5% yeast extract, 1% NaCl, pH 7.5) containing 100 &#181;g/mL of ampicillin to each well of a 96-well deep well plate. Inoculate each well with 2 &#181;L of glycerol stock and grow at 37 &#176;C overnight with constant agitation at 225 rpm.</li>
	<li>Transfer each bacterial culture into a 1.5 mL centrifuge tube and pellet the bacteria by centrifugation at 21,000 x g at 4 &#176;C for 10 min.</li>
	<li>Purify each vector using a bacterial mini-prep kit by following the manufacturer&#39;s protocol.</li>
	<li>Determine the concentration of each vector using a spectrophotometer.</li>
	<li>Store the plasmids at -20 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Packaging of the arrayed lentiviral library
NOTE: All work involving lentivirus, including packaging, infection, and subsequent culturing of infected cells should strictly follow the institutional biosafety rules and regulations.
<ol>
	<li>Expand the 293FT cells using complete growth media (Dulbecco&#39;s modified Eagle medium (DMEM) containing 4 mM L-glutamine, 4,500 mg/L glucose and sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, 100 &#181;g/mL streptomycin antibiotic and 2 mM L-glutamine).</li>
	<li>For each vector in the library from step 1.4, seed one 24-well with 1 x 105 293FT cells. 
	NOTE: It is recommended that some extra wells of a control viral vector be packaged and used to test the titer of the virus prior to proceeding to step 3 (see below).</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h. 
	NOTE: A general protocol for packaging lentivirus that was described previously14 has been scaled down to 24 wells for this protocol. It uses psPAX2 for lentiviral packaging and VSVG as a coat protein. If ectropic virus is desired, a vector delivering Eco can be used instead of VSVG. It is highly recommended that this protocol is optimized to achieve a viral titer that gives between 30%-70% infection efficiency of the target cells (see Discussion). See Supplemental Table 1 for a list of all vectors used.</li>
	<li>Set up a transfection mixture for each viral vector from Step 1.4 as described below. Each transfection should contain 250 ng of the viral vector, 125 ng of psPAX2, 125 ng of VSVG, 1.25 &#181;L of transfection reagent 1, and 23.75 &#181;L of transfection buffer (see Table of Materials).
	<ol>
		<li>Dilute each lentiviral vector to 50 ng/&#181;L with nuclease-free water, and then transfer 5 &#181;L (250 ng) into a well of a 96-well PCR plate.</li>
		<li>Make the transfection super mix by mixing 1.25 &#181;L * X of transfection reagent 1 and 23.75 &#181;L * X of pre-warmed transfection buffer where &#34;X&#34; is the total number of transfections plus several extra to account for volume loss during pipetting.</li>
		<li>Incubate the transfection super mix at room temperature for 5 min.</li>
		<li>Add 125 ng * X of psPAX2 and 125 ng * X of VSVG to the tube of transfection super mix from Step 2.4.3 and gently pipet up and down to mix. Rapidly proceed to Step 2.4.5.</li>
		<li>Immediately aliquot the mixture from Step 2.4.4 into each tube of a PCR strip, and then use multi-channel pipette to transfer 25 &#181;L the mixture into each well containing viral vector from Step 2.4.1.</li>
		<li>Incubate at room temperature for 20 min.</li>
		<li>Transfer all 30 &#181;Ls from each 96-well from Step 2.4.6 into a well of the 24-well containing 293 FT cells from Step 2.3.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h and then replace the media in every well of the 24-well plate with 500 &#181;L of fresh complete growth media. Incubate the cells at 37 &#176;C with 5% CO2 for another 24 h.</li>
	<li>Using a multi-channel pipette, collect the viral supernatant from each well and aliquot 220 &#181;L (enough for 1 infection in Step 3 plus some extra volume) into two 96-wells each. These are the arrayed viral supernatant plates.</li>
	<li>Store the arrayed viral supernatant plates at -80 &#176;C. 
	NOTE: The protocol can be paused here. It is also recommended to test some of the extra control virus that was packaged (see above) on the cells to be infected before proceeding to Step 3. This is to ensure that the titer is sufficient to achieve at least 30% infection efficiency.</li>
</ol>
3. Infection of the cells for the screen
NOTE: Human melanoma cells (A375) were used to demonstrate this approach, but this method can be applied to any adherent cells that infect with lentivirus. However, cell culture and plating conditions should be optimized for each cell line (see Discussion).
<ol>
	<li>Expand the cells to be infected in complete growth media.</li>
	<li>Seed 24-well plates with 1 x 105 cells in 0.5 ml of complete growth media per well. Seed one well for each viral vector to be tested (including controls) and include an extra well that will not be infected which will serve as a control for drug selection in Step 3.7.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
	<li>Infect each well from Step 3.2 with a different viral supernatant from the frozen arrayed lentiviral supernatant as follows.
	<ol>
		<li>Prepare complete growth media that contains 20 &#181;g/mL polybrene.</li>
		<li>Thaw the arrayed lentiviral library supernatants from Step 2.7 to room temperature.</li>
		<li>Aspirate the growth media from the 24-well plates from Step 3.2 and immediately add 200 &#181;L of polybrene-containing growth media to each well.</li>
		<li>Using a multi-channel pipette, transfer 200 &#181;L of viral supernatant from each 96-well from Step 3.4.2 to the 24 wells from Step 3.4.3.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 - 48 h. 
	NOTE: Some cell lines may require longer than 24 h to express the shRNAs and become puromycin resistant. The viral vector used here delivers a Turbo-GFP-IRES-puroR with a miR30-based shRNA in the 3&#39;UTR of puroR (Figure 1). Infection efficiency and expression of the puromycin resistance gene and the shRNA were monitored by green fluorescent protein.</li>
	<li>Prepare complete growth media that contains 2.5 &#181;g/mL puromycin. 
	NOTE: The selection concentration of puromycin varies between cell lines. It is recommended that an antibiotic kill curve be performed for each cell line to be assayed prior to the screen.</li>
	<li>Aspirate the media from each well and replace with 500 &#181;L of puromycin-containing complete growth media. 
	NOTE: Be sure to also add puromycin to a control non-infected well that can be used in subsequent steps to ensure the puromycin selection is complete.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 48 h. 
	NOTE: It is best to select for 48 h, so plating density and viral titer should be optimized so that the cells are not overconfluent prior to 48 h.</li>
	<li>Ensure that the infected cells are green under the fluorescent microscope, and that cells on a control non-infected well treated with puromycin are all dead before proceeding to Step 4.</li>
</ol>
4. Seeding cells for transfection of dual-luciferase reporter
NOTE: A test transfection should be done to determine the optimal seeding density for each new cell line.
<ol>
	<li>Trypsinize each well from Step 3.9 and transfer roughly 1 x 105 cells into wells at the corresponding position on the new 24-well plate as follows. 
	NOTE: This protocol is designed for the screening of libraries with hundreds of shRNAs so it is not feasible to count every well of infected cells. Therefore, the steps below were used to estimate cell numbers in each well to help ensure roughly equal plating density.
	<ol>
		<li>Group the wells from Step 3.9 into 3 - 4 groups such that all wells in a group have a similar cell density.</li>
		<li>Trypsinize 1 representative well from each group with 200 &#181;L of trypsin-EDTA (1x PBS supplemented with 0.5 mM EDTA and 0.1% trypsin) for 5 min at 37 &#176;C. Then neutralize the trypsin-EDTA by adding 400 &#181;L of puromycin containing complete growth media.</li>
		<li>Count each representative well to determine the total cell number and dilute the cell suspension from each representative well to 2 x 105 cells/mL of using complete growth media.</li>
		<li>Seed 0.5 mL (1 x 105 cells) of each well from Step 4.1.3 into the corresponding position on a new 24-well plate and incubate this new 24-well plate at 37 &#176;C with 5% CO2 for 24 h.</li>
		<li>For each group from Step 4.1.1, use the total cell number determined in Step 4.1.3 to calculate the volume of trypsin-EDTA to add to each well so that the resulting cell suspension will be 1 x 106 cells/mL.</li>
		<li>Add the appropriate volume of trypsin-EDTA to each well and incubate for 5 min at 37 &#176;C. 
		NOTE: For larger screen it is recommended that the 24-well plates be trypsinized and replated in groups rather than all at once to ensure the viability of the cells.</li>
		<li>During the above incubation, use a multi-channel pipette to add 400 &#181;L of puromycin-containing complete growth media to the corresponding wells on a new 24-well plate.</li>
		<li>Transfer 100 &#181;L of cell suspension (approximately 1 x 105 cells) from each well to the corresponding position on the new 24-well plates prepared in Step 4.1.7.</li>
		<li>Repeat Steps 4.1.6 through 4.1.8 for all plates of grouped wells from Step 4.1.1, one plate at a time.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
</ol>
5. Transfection of dual-luciferase reporter
<ol>
	<li>Transfect each well from Step 4.2 with the dual-luciferase reporter constructs as follows. 
	NOTE: The total amount of DNA and the optimal ratio of firefly luciferase reporter vector to control Renilla luciferase vector should be determined prior to starting this assay. Here, 400 ng of a DNA mixture that contains 20 parts firefly luciferase reporter and 1 part control Renilla luciferase was used.
	<ol>
		<li>Make the transfection dilution mixture (Tube A) and the reporter dilution mixture (Tube B) by mixing the indicated volumes of each reagent (Table 1) multiplied by the total number of transfections (plus several extra). 
		NOTE: This protocol is optimized for transfection reagent 2 (see Table of Materials). If a different transfection reagent is used, the transfection should be optimized prior to this step.</li>
		<li>Mix the transfection dilution mixture (Tube A) with reporter dilution mixture (Tube B) and incubate at room temperature for 15 min to produce transfection mixture.</li>
		<li>During the above incubation, rinse each 24-well from Step 4.2 with 0.25 mL of phosphate buffer saline (PBS), and add 447 &#181;L of complete growth media to each well.</li>
		<li>After the 15 min incubation, use a multi-channel pipette to distribute 53 &#181;L of transfection mix to each well of the 24-well plates.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
</ol>
6. Quantification of dual-luciferase activity
<ol>
	<li>Measure luciferase activity using a plate reader and a dual-luciferase reporter assay kit as described below. 
	NOTE: This protocol is optimized for the indicated reporter assay kit (see Table of Materials) and follows the manufacturer&#39;s recommended protocol.
	<ol>
		<li>Prepare enough 1x passive lysis buffer for all wells plus several extra (75 &#181;L is needed per well) by diluting 5x passive lysis buffer (provided in kit) 1 to 5 with deionized water. Also thaw reagent A and reagent B Buffer (provided in kit, 100 &#181;L of each is needed for each well).</li>
		<li>Aspirate the media from each well of the 24-well plate from Step 5.2.</li>
		<li>Add 75 &#181;L of 1x passive lysis buffer to each well and incubate at room temperature for 30 min with occasionally shaking.</li>
		<li>Prepare reagent B by diluting 50x reagent B substrate (provided in kit) 1:50 with thawed reagent B buffer.</li>
		<li>Add 30 &#181;L of 1x passive lysis buffer to 4 wells for blanking (see Supplemental Table 2).</li>
		<li>Transfer 30 &#181;L of lysate from Step 6.1.3 into duplicate wells of a 96-well flat bottom white assay plate.</li>
		<li>Use a multi-channel pipette to add 50 &#181;L of reagent A to each well and read the firefly luciferase signal with a plate reader.</li>
		<li>Use a multi-channel pipette to add 50 &#181;L of reagent B from Step 6.1.4 to each well and read the Renilla luciferase signal with a plate reader.</li>
	</ol>
	</li>
	<li>Process the raw data as follows (for a detailed description, see Supplemental Table 2).
	<ol>
		<li>Exclude samples with very low Renilla luciferase signal as low values indicate that the viral construct was toxic or that too few transfected cells were assayed. 
		NOTE: As explained in the Discussion, significantly &#34;low&#34; Renilla luciferase signal can result in anomalous results. Here, wells in which the Renilla luciferase signal was more than 1 standard deviation below the mean were excluded (see Supplemental Table 2). This was based on previous studies performed using this reporter system in these cells14, but may differ in other cell lines.</li>
		<li>Normalize the raw firefly luciferase value of each well to the raw Renilla luciferase value of the same well to obtain the firefly/Renilla ratio.</li>
		<li>Average the firefly/Renilla ratios of all replicate control wells and then divide the firefly/Renilla ratio of every other well by that number to get a fold change.</li>
		<li>Assign the control sample and set its firefly/Renilla ratio value to 1.</li>
		<li>Average the firefly/Renilla ratios of the duplicate wells and plot with the standard deviation. 
		NOTE: The standard deviation is not used for statistical analysis, but instead as a means to identify wells where the replicates differ significantly.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

In this study, we demonstrate an approach for medium-throughput screening of arrayed viral libraries in combination with a dual-luciferase-based transcriptional reporter assay that can be used to identify and test novel regulators of transcription factors. It is critical to characterize and optimize the reporter system for each cell line prior to any screen. Experiments should be done to confirm that the reporter is responsive to altered activity of the transcription factor being investigated and the magnitude of change ...

The purpose of this assay is to use viral libraries to identify regulators of transcription factors in a relatively quick and inexpensive manner. Aberrant transcriptional activity is associated with cancer and metastasis1,2,3,4,5,6, so targeting transcription factors in cancer cells is a promising therapeutic approach. However, transcription factors are often difficult to target pharmacologically7 and many are required for normal cellular function in adult tissues8,9,10. Targeting the cancer-associated pathways that aberrantly activate transcription factors to drive disease is a more feasible approach with the potential to have less severe side effects. The commercial availability of arrayed lentiviral and retroviral RNAi, CRISPR/CAS9, cDNA, or ORF libraries allows researchers to test the importance of numerous genes in a single experiment. However, a reliable readout for altered transcriptional activity is required.
Here, we describe the use of a dual-luciferase-based transcriptional reporter assay and arrayed lentiviral libraries to identify proteins that regulate transcription factors in cancer cells. In this assay, shRNAs that target cancer-associated genes are delivered to mammalian cancer cells via lentiviral transduction and cells are selected for stable integration using puromycin. The cells are next transfected with a reporter construct that expresses firefly luciferase driven by a promoter specific to the transcription factor that is being investigated and a control construct that expresses Renilla luciferase from a constitutively active promoter that is not responsive to the transcription factor being investigated. We demonstrate this approach with a proof-of-concept screen for regulators of YAP and TAZ, the critical downstream effectors of the Hippo pathway8,10,11. Abnormal activity of YAP and TAZ promotes several steps of the metastatic cascade11 and is observed in many cancers11,12,13. However, how YAP and TAZ become aberrantly activated in some cancer cells is not yet fully understood. YAP and TAZ do not bind DNA, but instead are recruited to promoters by other transcription factors. Members of the TEA domain (TEAD) family of transcription factors are the major binding partners for YAP and TAZ, and are critical for most YAP and TAZ-dependent functions. Our reporter construct expresses firefly luciferase from a YAP/TAZ-TEAD-responsive promoter and previous studies have demonstrated that it faithfully detects changes in YAP-TEAD and TAZ-TEAD transcriptional activity2,14,15.
Our approach is rapid, medium-throughput, and does not require screening facilities, automated robots, or deep sequencing of pooled libraries. The costs are relatively low and there are numerous commercially available libraries to choose from. The required equipment and reagents are also relatively standard in most laboratories. It can be used to screen for regulators of virtually any transcription factor if a luciferase-based reporter exists or is generated. We use this approach to screen shRNAs in cancer cells, but any cell line that can be transfected with reasonable efficiency could be used with any type of arrayed library.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 ml 96-well deep well polypropylene plate</td>
			<td>USA Scientific</td>
			<td>1896-2000</td>
			<td>For bacterial mini-prep</td>
		</tr>
		<tr>
			<td>Trypsin - 2.50%</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>Component of trypsin-EDTA</td>
		</tr>
		<tr>
			<td>96 well flat bottom white assay plate</td>
			<td>Corning</td>
			<td>3922</td>
			<td>For dual-luciferase assay</td>
		</tr>
		<tr>
			<td>Ampicillin - 100 mg/ml</td>
			<td>Sigma-Aldrich</td>
			<td>45-10835242001-EA</td>
			<td>For bacterial mini-prep</td>
		</tr>
		<tr>
			<td>Bacto-tryptone - powder</td>
			<td>Sigma-Aldrich</td>
			<td>95039</td>
			<td>Component of LB broth</td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay system, which include LAR II reagent (reagent A), Stop &#38; Glo substrate (reagent B substrate) and Stop &#38; Glo buffer (reagent B buffer) - Kit</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>For dual-luciferase assay</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline w/o calcium, magnesium and phenol red - 9.6 g/L</td>
			<td>Himedia</td>
			<td>TS1006</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>EDTA - 0.5 M</td>
			<td>VWR</td>
			<td>97061-406</td>
			<td>Component of trypsin-EDTA</td>
		</tr>
		<tr>
			<td>Ethanol - 100%</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td>For bacterial mini-prep</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum - 100%</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide (Polybrene) - 8 mg/ml</td>
			<td>Sigma-Aldrich</td>
			<td>45-H9268</td>
			<td>For virus infection</td>
		</tr>
		<tr>
			<td>HyClone DMEM/High glucose - 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvate</td>
			<td>GE Healthcare life sciences</td>
			<td>SH30243.01</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>I3-P/i3 Multi-Mode Microplate/EA</td>
			<td>Molecular devices</td>
			<td></td>
			<td>For dual-luciferase assay</td>
		</tr>
		<tr>
			<td>L-Glutamine - 200 mM</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 (Transfection Reagent 2) - 100%</td>
			<td>Life technologies</td>
			<td>L3000008</td>
			<td>For transfections</td>
		</tr>
		<tr>
			<td>Molecular Biology Water - 100%</td>
			<td>VWR</td>
			<td>02-0201-0500</td>
			<td>For dilution of shRNA vector for virus packaging</td>
		</tr>
		<tr>
			<td>NaCl - powder</td>
			<td>BDH</td>
			<td>BDH9286</td>
			<td>Component of LB broth</td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo scientific</td>
			<td></td>
			<td>For measuring vector DNA concentration</td>
		</tr>
		<tr>
			<td>Opti-MEM (Transfection Buffer) - 100%</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td>For transfections</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin - 10,000 Unit/ml (Penicillin); 10,000 &#181;g/ml (Streptomycin)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit - Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K210010</td>
			<td>For bacterial mini-prep</td>
		</tr>
		<tr>
			<td>Puromycin - 2.5 mg/ml</td>
			<td>Sigma-Aldrich</td>
			<td>45-P7255</td>
			<td>For antibiotic selection after infection</td>
		</tr>
		<tr>
			<td>TC20 automated cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) - 100%</td>
			<td>Roche</td>
			<td>6365787001</td>
			<td>For virus packaging</td>
		</tr>
		<tr>
			<td>Yeast extract - powder</td>
			<td>VWR</td>
			<td>J850</td>
			<td>Component of LB broth</td>
		</tr>
		<tr>
			<td>P3000 (Transfection Reagent 3) - 100%</td>
			<td>Life technologies</td>
			<td>L3000008</td>
			<td>For transfections</td>
		</tr>
	</tbody>
</table>

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.

Our YAP/TAZ-TEAD reporter construct (pGL3-5xMCAT (SV)-492,14,15) contains a minimal SV-49 promoter with 5 repeats of the canonical TEAD binding element (MCAT)15 driving the firefly luciferase gene (Figure 1). It is co-transfected into cells along with the PRL-TK control vector (Promega), which expresses Renilla luciferase from the con...

Watch this Scientific Journal Video about Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter at JoVE.com

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

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 ...

identification-transcription-factor-regulators-using-medium

Research

JoVE Journal

Cancer Research

6.6K Views.  Albany Medical College. 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.

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

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Live Cell Imaging of the TGF-	&amp;beta;/Smad3 Signaling Pathway &lt;em&gt;In Vitro&lt;/em&gt; and &lt;em&gt;In Vivo&lt;/em&gt; Using an Adenovirus Reporter System

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell types, ESCs release small membrane vesicles, such as exosomes, to the extracellular environment. Exosomes serve as essential mediators of intercellular communication and play a basic role in many (patho)physiological processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a cytokine to modulate the immune response. The presence of GM-CSF in exosomes has the potential to boost their immune-regulatory function. Here, GM-CSF was stably overexpressed in the murine ESC cell line ES-D3. A protocol was developed to isolate high-quality exosome-enriched extracellular vesicles (EVs) from ES-D3 cells overexpressing GM-CSF. Isolated exosome-enriched EVs were characterized by a variety of experimental approaches. Importantly, significant amounts of GM-CSF were found to be present in exosome-enriched EVs. Overall, GM-CSF-bearing exosome-enriched EVs from ESCs might function as cell-free vesicles to exert their immune-regulatory activities.

This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...