The authors thank Drs. Sueyoshi and Wang at the National Institute of Environmental Health Sciences (NIEHS) for their critical reading of the manuscript, and&#160;Tanner Jefferson for performing procedures on the video.&#160;This work was supported by the National Institutes of Health Grant 1ZIAES070065 (to K.S.K.) from the Division of Intramural Research of the NIEHS.

1. Preparation of Plasmids for the Mammalian Two-hybrid Assay
<ol>
	<li>Use the following plasmids: pG5-Luc, pBIND-ER&#945;EF, and pACT-ER&#945;EF. 
	NOTE: The plasmids are available from the authors upon request and are sent on filter paper. pG5-Luc is the reporter plasmid that contains five repeats of GAL4 responsive elements fused with the luciferase expression unit (Figure 1A). pBIND-ER&#945;EF is the protein expression plasmid for GAL4DBD-fused ER&#945; LBD proteins. pBIND-ER&#945;EF contains a Renilla luciferase expression unit for normalizing the transfection efficiency (Figure 1B). pACT-ER&#945;EF is the protein expression plasmid for the VP16AD-fused ER&#945; LBD proteins (Figure 1C). The diagram of the expressed protein structure from the plasmids is shown in Figure 2.</li>
	<li>Prepare plasmid DNAs.
	<ol>
		<li>Cut out the plasmid-loaded area from the paper using clean scissors and put it into a 1.5 mL tube. Wear gloves and use clean tweezers to pick up the plasmid-loaded paper. Add 100 &#181;L of Tris-EDTA (TE) buffer (pH 8.0) and vortex well.</li>
		<li>Add 1 &#181;L of the plasmid DNA solution from step 1.2.1 to competent Escherichia coli cells (see Table of Materials) and keep the tube on ice for 15 min.</li>
		<li>Heat-shock the plasmid-added competent cells; incubate the tube(s) in 42 &#176;C water bath for 30 s and, then, keep them on ice for 2 min.</li>
		<li>Add 250 &#181;L of super optimal broth with catabolite repression (SOC) medium (room temperature) to the tube(s) and culture with shaking at 37 &#176;C for 30 min.</li>
		<li>Plate 100 &#181;L of the cultured E. coli onto a prewarmed Luria broth (LB) plate with 100 &#181;g/mL ampicillin (10 cm-diameter dish) and culture at 37 &#176;C overnight.</li>
		<li>Pick one colony from the plate and add it into a 14 mL tube containing 2 mL of terrific broth (TB) with 100 &#181;g/mL ampicillin. Culture with shaking at 37 &#176;C until the medium is faintly clouded.</li>
		<li>Add 1 mL of the cultured medium from step 1.2.6 to an autoclaved 250 mL glass flask containing 50 mL of TB with 100 &#181;g/mL ampicillin. Culture with shaking at 37 &#176;C for 20 - 24 h.</li>
		<li>Transfer the cultured E. coli to a 50 mL conical tube and centrifuge at 3,450 x g at room temperature for 30 min using the swing-bucket rotor. Discard the medium. Save the E. coli pellet at -80 &#176;C.</li>
		<li>Add 3 mL of cell resuspension solution (see Table of Materials) to the E. coli pellet and suspend it completely. Add 3 mL of cell lysis solution (see Table of Materials), mix the tube gently by inverting it 10x, and keep it on ice for 5 min (keep the time punctually). Add 6 mL of neutralization solution (see Table of Materials), mix the tube steadily with tapping, and then, keep it on ice for 10 min.</li>
		<li>Centrifuge the tube at 3,450 x g at 4 &#730;C for 30 min using the swing-bucket rotor. Transfer the DNA-containing solution to a new 50 mL conical tube and add 10 mL of DNA purification resin (see Table of Materials). Suspend the resin gently.</li>
		<li>Centrifuge the tube at 625 x g for 1 min using the swing-bucket rotor. Discard the solution and keep the resin at the bottom.</li>
		<li>Add 15 mL of column wash solution (80 mM potassium acetate, 8.5 mM Tris-HCl [pH 7.5], 40 &#181;M EDTA, and 55% ethanol) to the 50 mL conical tube and shake it gently to suspend the resin. Repeat step 1.2.11. 
		NOTE: The column wash solution must be prepared using diethyl pyrocarbonate (DEPC)-treated water or nuclease-free water.</li>
		<li>Add 5 mL of column wash solution to the 50 mL conical tube and suspend the resin with a 5 mL pipet. Apply the resin to the column (see Table of Materials) that is set on the vacuum manifold. Vacuum the column until the resin dry. Add 6 mL of column wash solution to the column and vacuum.</li>
		<li>Once the resin dries visually, detach the reservoir from the column. Transfer the bottom section of the column (resin) to a 1.5 mL tube. Centrifuge the column at maximum speed for 2 min using a microcentrifuge to dry the resin completely.</li>
		<li>Transfer the bottom section of the column (resin) to a new 1.5 mL tube. Add 300 &#181;L of nuclease-free water to the column and wait until the whole resin is soaked.</li>
		<li>Centrifuge the column at maximum speed for 1 min to collect the plasmid DNA-containing solution (200 - 250 &#181;L).</li>
		<li>Treat the DNA-containing solution with phenol-chloroform (1:1), then with chloroform as follows.
		<ol>
			<li>Add the same volume of phenol-chloroform (1:1) to the DNA-containing solution (200 - 250 &#181;L), shake well, and centrifuge at maximum speed for 2 min using a microcentrifuge. Collect the upper layer (DNA-containing solution) and add it to a new 1.5 mL tube. Repeat this step 1x.</li>
			<li>Add the same volume of chloroform to the DNA-containing solution (200 - 250 &#181;L), shake well, and centrifuge at maximum speed for 1 min. Collect the upper layer (DNA-containing solution) and add it to a 1.5 mL tube. 
			NOTE: Endotoxin (lipopolysaccharide) induces cellular signaling in some cells. To avoid such an unpredicted effect, step 1.2.17 is recommended, which can reduce endotoxin contamination.</li>
		</ol>
		</li>
		<li>Precipitate the plasmid DNA using the ethanol precipitation technique as follows.
		<ol>
			<li>Add 1/9 of DNA mixture volume&#160;of 3 M sodium acetate (pH 5.5) and 20x of 3M sodium acetate volume&#160;of 100% ethanol to the DNA-containing solution. For example, add 27.8 &#181;L of 3 M sodium acetate and 278 &#181;L of 100% ethanol to 250 &#181;L of the DNA solution (the final volume is 555.8 &#181;L).</li>
			<li>Keep the solution at -80 &#176;C for 20 min at least. Centrifuge at maximum speed for 20 min at 4 &#730;C. Discard the solution and keep the pellet. Add 200 &#181;L of 75% ethanol and rinse the inside of the tube. Centrifuge for 20 min at 4 &#176;C, discard the solution, and keep the pellet. Dry the DNA pellet using a vacuum concentrator.</li>
			<li>Add 100 - 250 &#181;L of TE buffer (pH 8.0) to the tube. Dissolve the DNA pellet and check the DNA concentration using a spectrophotometer at a wavelength of 260 nm. Keep the DNA concentration around 0.5 mg/mL.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Mammalian Two-hybrid Assay
<ol>
	<li>Maintain the cells.
	<ol>
		<li>Culture human hepatocellular carcinoma HepG2 cells in a 750 mL, 175 cm2 tissue culture flask with the phenol red-free minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS; heat-inactivated), 2 mM L-glutamine, and 1% penicillin-streptomycin solution (antibiotics) for maintaining the cells.</li>
		<li>Culture the cells in a 5% CO2-supplemented, 37 &#176;C incubator until the cells are approximately 90% confluent. 
		NOTE: To reduce estrogenic background activity, phenol red-free medium should be used for all M2H experiments. This step should be performed inside a clean bench/biological safety cabinet. The cells should be maintained following the general mammalian cell culture protocol7.</li>
	</ol>
	</li>
	<li>Prepare the cells for transfection. 
	NOTE: The following procedural steps should be performed inside the clean bench/biological safety cabinet. The cells are cultured/incubated in a 5% CO2-supplemented, 37 &#176;C incubator in this step every time.
	<ol>
		<li>Replate the 90% confluent cells to the 24-well plates; rinse the cells 1x with phosphate-buffered saline (PBS).</li>
		<li>Add 7 mL of 0.05% trypsin-EDTA solution to the culture flask and soak the cells for 1 - 2 min while keeping the culture flask in a clean bench at room temperature. Remove a part of the trypsin-EDTA solution and leave 1 - 2 mL of the solution in the culture flask. Incubate the culture flask in the incubator for 1 - 2 min.</li>
		<li>Smack the bottom of the flask to detach the cells and check the cells under the microscope. 
		NOTE: If the cells are still attached to the flask, incubate for another 1 - 2 min and, then, repeat step 2.2.3.</li>
		<li>Suspend the cells in the phenol red-free MEM supplemented with 10% charcoal-stripped FBS (heat-inactivated), 2 mM L-glutamine, and 1% penicillin-streptomycin solution. 
		NOTE: Charcoal-stripped FBS must be used to reduce the effect of serum-derived endogenous estrogen.</li>
		<li>Count the cell number and seed cells in a 24-well plate at a density of 1.2 x 105 cells/well. Assign three wells for each data point (triplicate).</li>
		<li>Culture the cells overnight. Continue to step 2.4.1.</li>
	</ol>
	</li>
	<li>Prepare a DNA mixture for transfection. 
	NOTE: The following plasmid DNAs are transfected in one well: 100 ng of pG5-Luc reporter plasmid, 50 ng of expression plasmids for GAL4DBD fusion proteins (pBIND), and 50 ng of expression plasmids for VP16AD fusion proteins (pACT).
	<ol>
		<li>To analyze the ligand-dependent ER&#945; LBD dimerization activity, prepare the following combinations (Figure 3). Combination (i): pG5-Luc + pBIND-hER&#945;EF + pACT, and pG5-Luc + pBIND-mER&#945;EF + pACT. Combination (ii): pG5-Luc + pBIND-hER&#945;EF + pACT-hER&#945;EF, and pG5-Luc + pBIND-mER&#945;EF + pACT-mER&#945;EF. Combination (iii): pG5-Luc + pBIND + pACT-hER&#945;EF, and pG5-Luc + pBIND + pACT-mER&#945;EF. 
		NOTE: Combination (i) represents the basal experimental level of human ER&#945; LBD and mouse ER&#945; LBD, respectively. If the result shows a remarkably high level with ligand alone, this method is unfeasible for use with that ligand (e.g., diethylstilbestrol [DES]). Combination (ii) represents the levels of dimerized human ER&#945; LBD and dimerized mouse ER&#945; LBD, respectively. Combination (iii) represents negative controls; use this set only when the experiment is performed for the first time to evaluate the background level of the system.</li>
		<li>Before mixing, calculate the total DNA amount required based on the number of wells for transfection. For experiments conducted in triplicates and at five data points, mix the following plasmid DNAs in two 1.5 &#181;L tubes for combinations (i) and (ii): pG5-Luc, 5 (data points) x 3 (wells) x 100 ng = 1,500 ng (15 &#181;L); pBIND-mER&#945;EF, 5 (data points) x 3 (wells) x 50 ng = 750 ng (7.5 &#181;L); pACT (for combination i) or pACT-mER&#945;EF (for combination ii), 5 (data points) x 3 (wells) x 50 ng = 750 ng (7.5 &#181;L). 
		NOTE: The concentration of each plasmid DNA solution is 0.1 &#181;g/&#181;L.</li>
		<li>Precipitate the DNA mixture using the ethanol precipitation technique. Add 1/9 of DNA mixture volume of 3 M sodium acetate (pH 5.5) and 20x of 3M sodium acetate volume of 100% ethanol to the DNA mixture. Then, vortex the tube. 
		NOTE: For example, add 3.4 &#181;L of 3 M sodium acetate, and 34 &#181;L of 100% ethanol to the 30 &#181;L of plasmid DNA mixture exemplified in step 2.3.2. This step helps to keep an invariable transfection condition, and it is not necessary to perform this step in a biological safety cabinet.</li>
		<li>Keep the mixture overnight at -20 &#176;C. Centrifuge it at maximum speed for 20 min at 4 &#176;C. Discard the solution (the pellet is invisible). Add 120 &#181;L of 75% ethanol to the tube; then, rinse the inside of the tube. Centrifuge for 20 min at 4 &#176;C. Discard the solution and dry the DNA mixture using a vacuum concentrator for 30 min.</li>
	</ol>
	</li>
	<li>Perform the transfection. 
	NOTE: The following steps should be performed in a clean bench or a biological safety cabinet.
	<ol>
		<li>The following morning, rinse the cells seeded in a 24-well plate (from step 2.2.6) 2x with 0.5 mL of PBS (room temperature).</li>
		<li>Add warm (37 &#176;C) fresh phenol red-free MEM supplemented with 10% charcoal-stripped FBS (heat-inactivated) and 2 mM L-glutamine without antibiotics to each well (0.5 mL medium/well). Keep the cells in a 5% CO2-supplemented, 37 &#176;C incubator.</li>
		<li>Suspend the dried plasmid DNA mixture (from step 2.3.4) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) (no serum, phenol red-free, without glutamine; 13 &#181;L/well). Calculate the amounts of DMEM from the number of wells for transfection. 
		NOTE: 15 wells for combination (i) x 13 &#181;L = 195 &#181;L, and 15 wells for combination (ii) x 13 &#181;L = 195 &#181;L.</li>
		<li>Suspend the transfection reagent (see Table of Materials; 1.5 &#181;L/well) in DMEM (12.5 &#181;L/well) in another 1.5 mL tube. Calculate the amounts of transfection reagent and DMEM from the number of wells for transfection. 
		NOTE: 30 [15 wells for combination (i) and 15 wells for combination (ii)] x 1.5 &#181;L of transfection reagent + 30 x 12.5 &#181;L of DMEM = 420 &#181;L.</li>
		<li>Add an equal amount of the transfection reagent medium (from step 2.4.4) to each tube (from step 2.4.3). Incubate the tubes for 5 - 10 min at room temperature. 
		NOTE: 200 &#181;L of transfection reagent medium + 195 &#181;L of combination (i) DNA mixture = 395 &#181;L, and 200 &#181;L of transfection reagent medium + 195 &#181;L of combination (ii) DNA mixture = 395 &#181;L. It is important to add an equal volume of transfection reagent medium to the tubes of combination (i) and (ii). It is not necessary to use up the medium in this step.</li>
		<li>Add the combined mixture (from step 2.4.5) to each well (25 &#181;L/well) of the 24-well plate (from step 2.4.2) and incubate the cells for 6 h in the 5% CO2-supplemented, 37 &#176;C incubator.</li>
		<li>Remove the transfection medium from the 24-well plate. Add warm (37 &#176;C) fresh phenol red-free MEM supplemented with 10% charcoal-stripped FBS (heat-inactivated), 2 mM L-glutamine, 1% penicillin-streptomycin solution, and ligand (suspended in ethanol) (0.5 mL medium/well). Culture the cells for 16 - 18 h in the 5% CO2-supplemented, 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Harvest the samples.
	<ol>
		<li>Rinse the cells with 1 mL of PBS and, then, add 100 &#181;L of 1x passive lysis buffer (see Table of Materials).</li>
		<li>Vigorously shake the 24-well plates with a vortex mixer to detach the cells.</li>
		<li>Freeze the plates (cell lysates) 1x by liquid nitrogen or save them at -80 &#176;C before analysis. Immediately before the assay, vigorously shake the plates on a shaker until they are at room temperature. 
		NOTE: This process prevents irregularly appearing anomalous values of luciferase activity.</li>
	</ol>
	</li>
	<li>Perform a dual-luciferase assay.
	<ol>
		<li>Prepare the reagents for the dual-luciferase assay system (see Table of Materials).
		<ol>
			<li>For making the luciferase assay substrate (LAS) buffer, add all luciferase assay buffer into the luciferase assay substrate which is in a glass brown bottle. Save the LAS buffer at -20 &#176;C. 
			NOTE: The LAS buffer should be warmed to room temperature before use.</li>
			<li>For making the Renilla luciferase substrate (RLS) buffer, mix Renilla luciferase substrate and Renilla luciferase buffer in a ratio of 1:50. Make fresh RLS buffer for every assay and use a black tube or a tube shaded by tinfoil. Calculate the amount of RLS buffer making a fresh buffer. 
			NOTE: The RLS buffer should be warmed to room temperature before use.</li>
		</ol>
		</li>
		<li>Set the reagents in the microplate reader.
		<ol>
			<li>Wash the injection lines 2x with 70% ethanol; then, wash the lines 2x with water. 
			NOTE: This is important to prevent disturbing the assay results.</li>
			<li>Set the LAS buffer to the P-injector line (first injection) and set the RLS buffer to the M-injector line (second injection). Do not mix these buffers. RLS buffer inhibits luciferase activity.</li>
		</ol>
		</li>
		<li>Apply 10 &#181;L of harvested samples (from step 2.5.3) to the 96-well white plastic plate.</li>
		<li>Apply the following settings to the microplate reader. For the P-injector, use a volume of 50 &#181;L, a delay of 2 s, and a speed of 50 &#181;L/s. For the M-injector, use a volume of 50 &#181;L, a delay of 2 s, and a speed of 50 &#181;L/s. Set an integration time of 15 s. Set the temperature to 25 &#176;C.</li>
		<li>Run the microplate reader.</li>
		<li>Record the values of the luciferase activity (IPValue) and the values of the Renilla luciferase activity (IMValue) using the data acquisition program (see Table of Materials).</li>
		<li>Wash the injection lines after the data collection (see step 2.6.2.1).</li>
	</ol>
	</li>
	<li>Perform data analysis.
	<ol>
		<li>Use the normalized values (IPValue/IMValue) for data analysis.</li>
		<li>Set the normalized value of combination (i) [pG5-Luc + pBIND-ER&#945;EF + pACT] with vehicle (0 nM ligand) as 1 for the calculation of the basal level and the homodimerized ER&#945; LBD level. The levels are represented as &#34;Relative activity&#34;.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Arao, Y., Korach, K. 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+F+domain+of+estrogen+receptor+&#945;+is+involved+in+species-specific,+tamoxifen-mediated+transactivation.">The F domain of estrogen receptor &#945; is involved in species-specific, tamoxifen-mediated transactivation.</a> Journal of Biological Chemistry. 293 (22), 8495-8507 (2018).</li><li>Brzozowski, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+agonism+and+antagonism+in+the+oestrogen+receptor.">Molecular basis of agonism and antagonism in the oestrogen receptor.</a> Nature. 389 (6652), 753-758 (1997).</li><li>Shiau, A. K., 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=The+structural+basis+of+estrogen+receptor/coactivator+recognition+and+the+antagonism+of+this+interaction+by+tamoxifen.">The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen.</a> Cell. 95 (7), 927-937 (1998).</li><li>Yang, J., Singleton, D. W., Shaughnessy, E. A., Khan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-domain+of+estrogen+receptor-alpha+inhibits+ligand+induced+receptor+dimerization.">The F-domain of estrogen receptor-alpha inhibits ligand induced receptor dimerization.</a> Molecular and Cellular Endocrinology. 295 (1-2), 94-100 (2008).</li><li>Montano, M. M., M&#252;ller, V., Trobaugh, A., Katzenellenbogen, B. 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+carboxy-terminal+F+domain+of+the+human+estrogen+receptor:+role+in+the+transcriptional+activity+of+the+receptor+and+the+effectiveness+of+antiestrogens+as+estrogen+antagonists.">The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists.</a> Molecular Endocrinology. 9 (7), 814-825 (1995).</li><li>Koide, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Regions+within+the+F+Domain+of+the+Human+Estrogen+Receptor+&#945;+that+Are+Important+for+Modulating+Transactivation+and+Protein-Protein+Interactions.">Identification of Regions within the F Domain of the Human Estrogen Receptor &#945; that Are Important for Modulating Transactivation and Protein-Protein Interactions.</a> Molecular Endocrinology. 21 (4), 829-842 (2011).</li><li>Mitry, R. R., Hughes, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cell Culture Protocols. , (2011).</li><li>Arao, Y., Coons, L. A., Zuercher, W. J., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivation+Function-2+of+Estrogen+Receptor+&#945;+Contains+Transactivation+Function-1-regulating+Element.">Transactivation Function-2 of Estrogen Receptor &#945; Contains Transactivation Function-1-regulating Element.</a> Journal of Biological Chemistry. 290 (28), 17611-17627 (2015).</li><li>Huang, H. -. J., Norris, J. D., McDonnell, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+negative+regulatory+surface+within+estrogen+receptor+alpha+provides+evidence+in+support+of+a+role+for+corepressors+in+regulating+cellular+responses+to+agonists+and+antagonists.">Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists.</a> Molecular Endocrinology. 16 (8), 1778-1792 (2002).</li><li>Chang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+LXXLL+nuclear+receptor-coactivator+interaction+motif+using+combinatorial+peptide+libraries:+discovery+of+peptide+antagonists+of+estrogen+receptors+alpha+and+beta.">Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.</a> Molecular and Cellular Biology. 19 (12), 8226-8239 (1999).</li><li>Arao, Y., Hamilton, K. J., Coons, L. A., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+&#945;+L543A,+L544A+mutation+changes+antagonists+to+agonists,+correlating+with+the+ligand+binding+domain+dimerization+associated+with+DNA+binding+activity.">Estrogen receptor &#945; L543A, L544A mutation changes antagonists to agonists, correlating with the ligand binding domain dimerization associated with DNA binding activity.</a> The Journal of Biological Chemistry. 288 (29), 21105-21116 (2013).</li></ol>

The authors have nothing to declare.

Herein, we described the protocol for the M2H assay, focusing on the assay conditions for detecting the homodimerization activity of ER&#945; LBD as an example. In general, the M2H assay is not popular for the assessment of ligand-dependent ER&#945; LBD dimerization activity. This is due to the ER&#945; LBD possessing a transcriptional activation function; the activity of which disturbs, in some cases, the results of the M2H assay. However, as we demonstrate here, the M2H assay can be used for analyzing the LBD dimerizat...

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The amino acid sequenceof ER&#945; is highly conserved among species. Because of the higher homology between human and mouse ER&#945;, the function of these receptors is thought of as identical, and the differential activity of estrogenic substances (e.g., tamoxifen) in those species is caused by the species&#39; differences in chemical metabolisms rather than by the structural differences of ER&#945;. ER&#945; has highly conserved domain structures that are common among the nuclear receptor (NR) superfamily, designated A to F domains. The E domain or ligand-binding domain (LBD) includes the ligand-binding pocket and the transcriptional activation function 2, named AF-2. The F domain is localized immediately adjacent to the E domain and is the most variable domain among the NRs. Even between human and mouse ER&#945;, the homology of the F domain is significantly lower than that of the other domains1. The ligand-bound LBD of ER&#945; enhances the homodimerization of the ER&#945; protein to bind the specific estrogen-responsive DNA element directly for regulating the ligand-dependent gene transcription (classical action of ER&#945;). Crystallographic studies have revealed the differential positioning of helix 12 (AF-2 core domain) with estradiol (E2)- or 4-hydroxy-tamoxifen (4OHT)-bound LBD dimers2,3. The ER&#945; F domain (45 amino acids) connect helix 12 directly. However, there is no information regarding the effect of this extension of 45 amino acids (F domain) from the helix 12 on the ER&#945; LBD dimerization. In this study, we demonstrate the contribution of the F domain to the species-specific 4OHT-dependent ER&#945; LBD homodimerization using a mammalian two-hybrid (M2H) assay.
The M2H assay is a method to demonstrate protein-protein interactions in mammalian cells introducing the three different plasmid DNAs: two protein expression plasmids, which express GAL4 DNA-binding domain (DBD) fusion ER&#945; LBD and VP16AD fusion ER&#945; LBD, and a GAL4RE-fused luciferase expression reporter plasmid. When the GAL4DBD fusion ER&#945; LBD and the VP16AD fusion ER&#945; LBD interact (make a dimer) in the cells, this protein complex binds to the GAL4RE and, then, activates luciferase gene expression through the VP16AD-dependent transactivation function. The level of LBD homodimerization can be evaluated by the luciferase activity.
The yeast two-hybrid (Y2H) assay is an alternative method based on the same principle that uses yeast as the host environment. Previous reports using the Y2H system demonstrated that F-domain-truncated human ER&#945; LBD increases E2-dependent coactivator recruitment, concluding that the F domain prevents E2-mediated transcription4. This result is inconsistent with other reports which have demonstrated the attenuated transcriptional activity of F-domain-truncated human ER&#945; in the mammalian cells5,6. Recently, our study, using the M2H system, demonstrated that the E2-dependent coactivator recruitment activity of human ER&#945; LBD is decreased by F domain truncation in mammalian cells and is consistent with transcription activity1. These observations suggest that the physiological role of protein-protein interaction differs in a cell type-specific manner and context. The M2H assay can demonstrate protein-protein interaction activity in the same cellular context that is used for determining the transcriptional activity. This provides an advantage of the M2H assay compared to the Y2H or other in vitro protein-protein interaction analyses.
There remain questions regarding the molecular mechanisms of the partial agonist activity of selective estrogen receptor modulators (SERMs) (e.g., 4OHT) to regulate ER&#945;-mediated transcription. ER&#945; LBD-based M2H assays may be applied to study the mechanism of the partial agonist activity of SERMs in various mammalian cell types.

<table border="0" cellpadding="0" cellspacing="0" style="width:891px;" width="891">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">pG5-Luc</td>
			<td style="width:112px;">Promega</td>
			<td style="width:119px;">E249A</td>
			<td style="width:360px;">Component of CheckMate Mammalian Two-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">pBIND</td>
			<td style="width:112px;">Promega</td>
			<td>E245A</td>
			<td>Component of CheckMate Mammalian Two-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">pACT</td>
			<td style="width:112px;">Promega</td>
			<td>E246A</td>
			<td>Component of CheckMate Mammalian Two-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One Shot TOP10 Chemically Competent E. coli</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Rotor</td>
			<td>SORVALL</td>
			<td>75006445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swing Buckets</td>
			<td>SORVALL</td>
			<td>75006441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Resuspension Solution (CRA)</td>
			<td style="width:112px;">Promega</td>
			<td>A7112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Lysis Solution (CLA)</td>
			<td style="width:112px;">Promega</td>
			<td>A7122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neutralization Solution (NSA)</td>
			<td style="width:112px;">Promega</td>
			<td>A7131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Column Wash Solution (CWB)</td>
			<td style="width:112px;">Promega</td>
			<td>A8102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Midipreps DNA Purification Resin</td>
			<td style="width:112px;">Promega</td>
			<td>A7701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Midicolumns</td>
			<td style="width:112px;">Promega</td>
			<td>A7651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td style="width:119px;">51200038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">0.5% Trypsin-EDTA (10x)</td>
			<td>Gibco</td>
			<td style="width:119px;">15400054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin (100x)</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BenchMark fetal bovine serum (FBS)</td>
			<td>Gemini-Bio</td>
			<td>100-106</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Charcoal:dextran stripping fetal bovine serum</td>
			<td>Gemini-Bio</td>
			<td>100-119</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>31053028</td>
			<td>for transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Passive Lysis 5X Buffer</td>
			<td style="width:112px;">Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-Luciferase Reporter Assay System</td>
			<td style="width:112px;">Promega</td>
			<td>E1980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax L microplate reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">SoftMax Pro Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting the ligand-binding domain dimerization activity of estrogen receptor alpha using the mammalian two-hybrid assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

Figure 3&#160;displays the scheme of possible responses in the combination (i) and combination (ii) plasmid-transfected cells. The experimental results are shown in Figure 4. The activity of combination (i) (pG5-Luc + pBIND-mER&#945;EF + pACT) shows stimulation by 10 nM E2 (Figure 4A), because the ER&#945; LBD contains the ligand-dependent transactivation functional domain, AF-2. Ago...

Watch this Scientific Journal Video about Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay at JoVE.com

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

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Research

JoVE Journal

Cancer Research

6.3K Views.  National Institutes of Health (NIH). We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

Video: Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

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 ...