Name: monitoring hippo signaling pathway activity using a luciferase-based large tumor suppressor (lats) biosensor
Uploaded: 2018-09-13T15:00:00.000+00:00
Duration: 7 min 16 s
Description: Watch this Scientific Journal Video about Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor at JoVE.com

This work was supported by grants from the Canadian Institute of Health Research (CIHR#119325, 148629) and the Canadian Breast Cancer Foundation (CBCF) to XY. TA is supported by the Vanier Canada Graduate Scholarship and the Ontario International Graduate Scholarship. HJJVR is supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology.

1. Investigation of a Putative Regulator of Hippo Signaling Using the LATS-BS
<ol>
	<li>Plating and transfection of cells
	<ol>
		<li>Preparation of cell culture
		<ol>
			<li>Warm 1x PBS, DMEM containing 10% FBS and 1% penicillin/streptomycin, and 0.25% trypsin-EDTA to 37 &#176;C in a water bath for approximately 30 min.</li>
			<li>Thoroughly clean a biosafety cabinet with 70% ethanol.</li>
			<li>Place tissue culture dishes, Pasteur pipettes, serological pipettes, and pipette tips into the tissue culture hood as required.</li>
			<li>Take out 1x PBS, the media, and the trypsin-EDTA from the water bath, clean them with 70% ethanol, and place them in the hood.</li>
		</ol>
		</li>
		<li>Maintenance and plating of the cells for transfection
		<ol>
			<li>Choose a cell line with a high transfection efficiency (e.g., HEK293) for the experiment. Check for the expression of Hippo pathway components in this cell line by western blot to make sure Hippo signaling is active.</li>
			<li>Grow HEK293 cells in DMEM in 10 cm Petri dishes in an incubator at 37 &#176;C and 5% CO2. Monitor the cells under a microscope every day. Passage the cells as described below when 80&#8211;90% confluency is observed.</li>
			<li>Wash the cells by adding 5 mL of 1x PBS into a plate, gently swirling the plate, and aspirating the media completely.</li>
			<li>Add 1 mL of trypsin-EDTA to the plate. Swirl the plate to ensure that the trypsin evenly covers the cells.</li>
			<li>Incubate the cells at 37 &#176;C in a CO2 incubator for 5 min or until all the cells are detached.</li>
			<li>Add 4 mL of media (with 10% FBS and 1% penicillin/streptomycin) to neutralize the trypsin, resuspend the cells by pipetting up and down several times, and dispense one-tenth (0.5 mL) of the cells into new 100 mm plates (for a passing ratio of 1:10) containing 9.5 mL of complete media.</li>
			<li>For LATS-BS transfection, count the cells using a hemocytometer and plate 2 x 105 cells into each well of a 12-well plate 24 h before transfection. 
			NOTE: Using higher cell numbers may increase the background bioluminescent signal as Hippo signaling activity is regulated by cell confluency. Using lower cell numbers may increase cell sensitivity to transfection reagents and increase cell death24.</li>
		</ol>
		</li>
		<li>Transfection of the LATS-BS alone or together with the candidate gene
		<ol>
			<li>Miniprep LATS-BS plasmids (NLuc-YAP15-pcDNA3.1-Hygro and 14-3-3-CLuc-pcDNA3.1-Hygro) fresh from DH5a bacteria liquid culture (necessary to achieve an optimum LATS-BS expression).</li>
			<li>1 h before transfection, aspirate the medium from the plate, add 500 &#181;L of complete growth medium to each well of the 12-well plates and return the cells to the incubator. 
			NOTE: The following protocol describes transfection methods using a commercially available polymer-based transfection reagent. Other transfection reagents may also be suitable for transfection; however, we have not yet tested them.</li>
			<li>Make solutions A and B as described in Table 1. For N transfections, make (N + 0.5) x solution B premix.</li>
			<li>Immediately add the diluted polymer-based transfection reagent (solution B) to DNA (solution A). Pipette up and down gently to mix.</li>
			<li>Incubate the sample at room temperature for 15 min to allow transfection complexes to form.</li>
			<li>Add the total volume (76 &#181;L) of transfection complexes dropwise to each well of the 12-well plate with gentle swirling.</li>
			<li>Incubate the cells overnight (18&#8211;24 h) at 37 &#176;C. 
			Note: For cells that are sensitive to polymer-based transfection reagents, the incubation time can be reduced to 5 h.</li>
			<li>The day after transfection, remove the transfection-complex-containing media and replace it with fresh complete media. Culture the cells at 37 &#176;C for another day to perform luciferase assay.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Luciferase assay 
	NOTE: The Luciferase Reporter Assay System detecting firefly and Renilla together in one assay is used for the luciferase assays. If Renilla was not used as an internal control, perform one-step luciferase assay by adding LARII without adding the Renilla detection reagent.
	<ol>
		<li>Preparation of the cell lysates
		<ol>
			<li>Dilute 5x passive lysis buffer (PLB) with distilled water as follows:</li>
			<li>Total volume of 1x PLB required = N x (50 &#956;L of 5x PLB + 200 &#956;L of dH2O) = N x 250 &#956;L of 1x PLB per well of the 12-well plates (N = number of samples).</li>
			<li>Aspirate the media completely. Add 1 mL of 1x PBS into each well. Swirl the plate gently to remove detached cells and residual growth media. Aspirate the 1x PBS completely. Make sure no residual 1x PBS remains prior to the addition of 1x PLB.</li>
			<li>Add 250 &#956;L of 1x PLB/well.</li>
			<li>Place the culture plates on a rocking platform with gentle rocking to ensure complete and even coverage of the cell monolayer with 1x PLB. Rock the culture plates at room temperature for 15 min to allow the cells to lyse.</li>
			<li>Transfer the lysate to a 1.5 mL microcentrifuge tube.</li>
		</ol>
		</li>
		<li>Measuring the bioluminescent signal
		<ol>
			<li>Take out the LARII (20 &#956;L/sample) from -80 &#176;C and equilibrate it to room temperature.</li>
			<li>Prepare Renilla detection reagent for N assays: add (N + 1) x 0.4 &#956;L of 50x Substrate to (N + 1) x 19.6 &#956;L of its Buffer.</li>
			<li>Predispense 10 &#956;L of each cell lysate to 1.5-mL tubes.</li>
			<li>Program a luminometer to perform a 2-s premeasurement delay, followed by a 10-s measurement period for each dual luciferase assay. 
			NOTE: The first and second measurements will be corresponding to the LATS-BS signal and the Renilla internal control signal, respectively. Any luminometer compatible with a single or dual luciferase can be used.</li>
			<li>Carefully transfer 20 &#956;L of LARII reagent into one tube and quickly pipette up and down 3x to mix it.</li>
			<li>Immediately place the tube in the luminometer and initiate the reading.</li>
			<li>Remove the sample tube from the luminometer, immediately add 20 &#956;L of Renilla Reagent, and mix by pipetting up and down 3x. Bring the sample in the luminometer and initiate the next reading. Record the measurements.</li>
			<li>Discard the reaction tube and proceed to the next sample.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. A screen to Identify Hippo Pathway Regulators Using the LATS-BS
<ol>
	<li>Cell culture and transfections
	<ol>
		<li>Culture HEK293AD cells as described in section 1.1. 
		NOTE: HEK293AD is more adherent than other HEK293 cell lines, which makes it a good cell line for use in screens.</li>
		<li>Detach cells as described in section 1.1.2. Count and plate 2 x 106 cells in 100 mm plates 24 h before transfection.</li>
		<li>1 h before transfection, aspirate the media from the plate and replace them with 5 mL of fresh complete growth media.</li>
		<li>Make solutions A and B as described in Table 2.</li>
		<li>Immediately add the diluted polymer-based transfection reagent (solution B) to DNA (solution A). Pipette up and down to mix.</li>
		<li>Incubate the sample at room temperature for 15 min to allow transfection complexes to form.
		<ol>
			<li>Add the total volume (500 &#181;L) of transfection complexes dropwise to cells with gentle swirling.</li>
			<li>Incubate the cells overnight at 37 &#176;C.</li>
			<li>The next day, trypsinize and count the cells. Plate 1 x 104 to 2 x 104 cells into each well of a 96-well plate in a total volume of 100 &#181;L of media per well. Incubate for another day.</li>
		</ol>
		</li>
		<li>The next day, add agents/small molecules regulating the Hippo pathway at a final concentration of 10 &#181;M to each well, 1&#8211;4 h before performing the luciferase assay. 
		Note: For most small molecules/agents, a 4 h treatment at 10 &#181;M does not affect cell viability. If a lower concentration is used, treatment may be extended for a longer period of time.</li>
	</ol>
	</li>
	<li>Luciferase assay
	<ol>
		<li>In vitro luciferase assay
		<ol>
			<li>Aspirate the media from the cells completely. Wash the cells with 100 &#181;L of 1x PBS in each well. Aspirate completely.</li>
			<li>Add 20 &#181;L of 1x PLB (prepared as described in step 1.2.1.1) into each well of the 96-well plates.</li>
			<li>Place the culture plates on a rocking platform with gentle rocking at room temperature for 15 min.</li>
			<li>Carefully transfer 20 &#181;L of LARII reagent into each well and pipette up and down 3x to mix.</li>
			<li>Immediately place the plate in a plate-reading luminometer and initiate the reading.</li>
		</ol>
		</li>
		<li>Living cell luciferase assay
		<ol>
			<li>Make 11x D-luciferin stock as follows: dissolve 1.5 mg of D-luciferin powder in 1 mL of normal culture media.</li>
			<li>Transfer 10 &#181;L of 11x D-luciferin into each well and mix it gently with the media which is already in the well.</li>
			<li>Replace the cells into the incubator for 10 min.</li>
			<li>Place the plate in the luminometer and initiate the reading. 
			Note: If the signal intensity is not strong enough at this concentration of D-luciferin, another 10 &#181;L of the D-luciferin stock may be added to the cells.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Justice, R. W., Zilian, O., Woods, D. F., Noll, M., Bryant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+tumor+suppressor+gene+warts+encodes+a+homolog+of+human+myotonic+dystrophy+kinase+and+is+required+for+the+control+of+cell+shape+and+proliferation.">The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.</a> Genes &amp; Development. 9 (5), 534-546 (1995).</li><li>Xu, T., Wang, W., Zhang, S., Stewart, R. A., Yu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+tumor+suppressors+in+genetic+mosaics:+the+Drosophila+lats+gene+encodes+a+putative+protein+kinase.">Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase.</a> Development. 121 (4), 1053-1063 (1995).</li><li>Taha, Z., Janse van Rensburg, H. J., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+Pathway:+Immunity+and+Cancer.">The Hippo Pathway: Immunity and Cancer.</a> Cancers. 10 (4), 94 (2018).</li><li>Maugeri-Sacc&#224;, M., De Maria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+in+normal+development+and+cancer.">The Hippo pathway in normal development and cancer.</a> Pharmacology &amp; Therapeutics. 186, 60-72 (2018).</li><li>van Rensburg, H. J. J., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+the+Hippo+pathway+in+cancer+metastasis.">The roles of the Hippo pathway in cancer metastasis.</a> Cellular Signalling. 28 (11), 1761-1772 (2016).</li><li>Yeung, B., Yu, J., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+Hippo+pathway+in+lung+development+and+tumorigenesis.">Roles of the Hippo pathway in lung development and tumorigenesis.</a> International Journal of Cancer. 138 (3), 533-539 (2016).</li><li>Zhao, Y., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+in+chemotherapeutic+drug+resistance.">The Hippo pathway in chemotherapeutic drug resistance.</a> International Journal of Cancer. 137 (12), 2767-2773 (2015).</li><li>Yu, F. -. X., Guan, K. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway:+regulators+and+regulations.">The Hippo pathway: regulators and regulations.</a> Genes &amp; Development. 27 (4), 355-371 (2013).</li><li>Yang, X., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+size+control+in+development+and+human+diseases.">Molecular mechanism of size control in development and human diseases.</a> Cell Research. 21 (5), 715 (2011).</li><li>Visser, S., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LATS+tumor+suppressor:+a+new+governor+of+cellular+homeostasis.">LATS tumor suppressor: a new governor of cellular homeostasis.</a> Cell Cycle. 9 (19), 3892-3903 (2010).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+YAP+oncoprotein+by+the+Hippo+pathway+is+involved+in+cell+contact+inhibition+and+tissue+growth+control.">Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.</a> Genes &amp; Development. 21 (21), 2747-2761 (2007).</li><li>Hao, Y., Chun, A., Cheung, K., Rashidi, B., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+LATS1+is+a+negative+regulator+of+oncogene+YAP.">Tumor suppressor LATS1 is a negative regulator of oncogene YAP.</a> Journal of Biological Chemistry. 283 (9), 5496-5509 (2008).</li><li>Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+Luciferase+as+an+Optical+Probe+for+Detecting+Protein.">Split Luciferase as an Optical Probe for Detecting Protein.</a> Protein Interactions in Mammalian Cells Based on Protein Splicing. Analytical Chemistry. 73 (11), 2516-2521 (2001).</li><li>Hashimoto, T., Adams, K. W., Fan, Z., McLean, P. J., Hyman, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+oligomer+formation+of+amyloid-&#946;+peptide+using+a+split-luciferase+complementation+assay.">Characterization of oligomer formation of amyloid-&#946; peptide using a split-luciferase complementation assay.</a> Journal of Biological Chemistry. 286 (31), 27081-27091 (2011).</li><li>Decock, 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=Analysis+by+a+highly+sensitive+split+luciferase+assay+of+the+regions+involved+in+APP+dimerization+and+its+impact+on+processing.">Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.</a> FEBS Open Bio. 5 (1), 763-773 (2015).</li><li>Azad, T., Tashakor, A., Rahmati, F., Hemmati, R., Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillation+of+apoptosome+formation+through+assembly+of+truncated+Apaf-1.">Oscillation of apoptosome formation through assembly of truncated Apaf-1.</a> European Journal of Pharmacology. 760, 64-71 (2015).</li><li>Br&#252;ckner, A., Polge, C., Lentze, N., Auerbach, D., Schlattner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+two-hybrid,+a+powerful+tool+for+systems+biology.">Yeast two-hybrid, a powerful tool for systems biology.</a> International Journal of Molecular Sciences. 10 (6), 2763-2788 (2009).</li><li>Pattnaik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance.">Surface plasmon resonance.</a> Applied Biochemistry and Biotechnology. 126 (2), 79-92 (2005).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer.">Fluorescence resonance energy transfer.</a> Current Opinion in Biotechnology. 6 (1), 103-110 (1995).</li><li>Azad, T., Tashakor, A., Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-luciferase+complementary+assay:+applications,+recent+developments,+and+future+perspectives.">Split-luciferase complementary assay: applications, recent developments, and future perspectives.</a> Analytical and Bioanalytical Chemistry. 406 (23), 5541-5560 (2014).</li><li>Hu, C. -. D., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+of+multiple+protein+interactions+in+living+cells+using+multicolor+fluorescence+complementation+analysis.">Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.</a> Nature Biotechnology. 21 (5), 539-545 (2003).</li><li>Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+enigma+of+multicolor+bioluminescence+of+firefly+luciferase.">Molecular enigma of multicolor bioluminescence of firefly luciferase.</a> Cellular and Molecular Life Sciences. 68 (7), 1167-1182 (2011).</li><li>Paulmurugan, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+library+screening+for+developing+an+improved+split-firefly+luciferase+fragment-assisted+complementation+system+for+studying+protein-protein+interactions.">Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions.</a> Analytical Chemistry. 79 (6), 2346-2353 (2007).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+LATS+biosensor+screen+identifies+VEGFR+as+a+regulator+of+the+Hippo+pathway+in+angiogenesis.">A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis.</a> Nature Communications. 9 (1), 1061 (2018).</li><li>Moroishi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+YAP/TAZ-induced+feedback+mechanism+regulates+Hippo+pathway+homeostasis.">A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis.</a> Genes &amp; Development. 29 (12), 1271-1284 (2015).</li></ol>

The authors have nothing to disclose.

While the Hippo pathway plays critical roles in various biological processes, and dysregulation of the Hippo pathway leads to cancer6, how the Hippo pathway is regulated in response to various stimuli is not completely understood. In addition, there has been no quantitative and real-time system to assess the activity of core Hippo components. Recently, we developed and validated a novel biosensor for measuring LATS kinase activity in the Hippo pathway24. This biosensor can ...

The Hippo signaling pathway was first identified in Drosophila as a novel regulator of cell growth and animal size1,2. Since its initial discovery, mounting evidence has convincingly shown that the Hippo pathway plays critical roles in the development (e.g., early embryonic development, organ size control, and three-dimensional [3-D] morphology), tumorigenesis (e.g., tumor development, metastasis, angiogenesis, immune evasion, genomic instability, stress response, and drug resistance), and tissue homeostasis (e.g., stem cell renewal and differentiation and tissue regeneration after injury)3,4,5,6,7,8,9,10. Hippo signaling is frequently dysregulated in various cancers7,8,9,10,11,12. Therefore, elucidating functions of the Hippo pathway in cancer biology and therapeutics and regenerative medicine has become one of the hottest areas in biomedical research.
In brief, in the Hippo pathway, upon activation by upstream regulators (e.g., cell-cell contact, nutrient stress, and extracellular matrix [ECM]), MST1/2 (MST; mammalian homologs of Drosophila Hippo) serine/threonine (S/T) kinases phosphorylate/activate adaptor proteins hMOB1 and WW45, as well as LATS1/2 (LATS) kinases which, subsequently, phosphorylate transcriptional co-activator YAP and its paralog TAZ at conserved HX(H/R/K)XX(S/T) (H, histidine; R, arginine; K, lysine; S, serine; T, threonine; X, any amino acids) motifs, including YAP-S127 and TAZ-S8911,12. S127-phosphorylated YAP (YAP-pS127) and S89-phosphorylated TAZ (TAZ-pS89) are degraded by ubiquitination or bind to cytoplasmic protein 14-3-3 and are prevented from interacting with TEAD1-4 transcription factors in the nucleus to transactivate downstream genes involved in the cell proliferation and apoptosis (Figure 1). Despite the tremendous interest in the Hippo pathway, few tools for measuring Hippo signaling exist and those that do have been historically limited to reporters of YAP/TAZ/TEAD transcriptional output. Indeed, until very recently, there were no tools for measuring the dynamics and activity of the Hippo signaling components in a quantitative, real-time, high-throughput, and non-invasive manner both in vitro and in vivo.
Given our emerging understanding of the role of protein-protein interactions in physiology and pathology, there is great interest in the development of tools that can be used to study these interactions in a quantitative and real-time manner13,14,15,16. Indeed, there has been significant progress in the development of bio-analytical strategies, including the yeast two-hybrid (Y2H)17, the surface plasmon resonance (SPR)18, and F&#246;rster resonance energy transfer (FRET)19 assays, to evaluate protein-protein interactions. However, these approaches carry the limitation of requiring significant optimization of the reporter orientation, such that many constructs must be tested to find an efficient one. Further, these approaches also have a relatively low signal-to-noise ratio, such that discerning a true positive signaling can be challenging.
Protein complementation assays were developed to overcome these limitations. The first generation of protein complementation assays was based on split multicolor fluorescence proteins and could not solve the aforementioned limitations20. Multicolor fluorescence proteins consist of only one domain, making it difficult to split them into two separate stable fragments with low affinity and background noise21. Subsequently, firefly luciferase was identified as a new candidate for use in developing split protein complementation assays. In this approach, firefly luciferase is split into two fragments (N-terminal and C-terminal luciferase [NLuc and CLuc]) with each fragment fused to a target protein of interest. If the NLuc and CLuc are brought into proximity upon the interaction of the two target proteins, luciferase activity is reconstituted and bioluminescent light is generated in the presence of luciferin substrate and ATP22. In 2001, by performing a combinatorial screening using a library containing NLuc and CLuc fragments cut at various sites and attached to proteins with different linkers, Paulmurugan and Gambhir at Stanford University developed an optimized split-firefly luciferase fragment-assisted complementation system for protein-protein interactions23. In this system, firefly luciferase is cut at amino acid (aa) 398 to form NLuc and CLuc, which are attached to two proteins of interest using a flexible linker of eight glycine residues and two serine residues.
Using a similar approach, we recently developed a new LATS-BS by fusing NLuc to 15 aa of YAP surrounding the LATS phosphorylation site at S127 (YAP15) and CLuc with 14-3-3. The full-length YAP protein was not used, to avoid confounding signals by post-translational modifications of YAP (e.g., phosphorylation at other sites and ubiquitination) by other upstream regulators. The LATS-BS presented here can non-invasively monitor Hippo signaling activity both in vitro in living cells and in vivo in mice20,24 (Figure 2). Here, we describe a detailed protocol for measuring LATS kinase activity in vitro using the LATS-BS. First, we show how the LATS-BS can be used to investigate the effect of an overexpressed protein on LATS activity. Then, we show how the biosensor can be used to monitor the activity of the Hippo pathway after treatment with agents regulating the Hippo pathway. This protocol could be used to identify and characterize signaling pathways or stimuli regulating LATS kinase activity.

<table><tbody><tr><td>&lt;strong&gt;Trypsin-EDTA&amp;nbsp;&lt;/strong&gt;</td><td>Life Technologies</td><td>2520056</td><td>0.25%</td></tr><tr><td>&lt;strong&gt;Fetal Bovine Serum (FBS)&lt;/strong&gt;</td><td>Sigma</td><td>F1051</td><td></td></tr><tr><td>&lt;strong&gt;DMEM&lt;/strong&gt;</td><td>Sigma</td><td>D6429-500ml</td><td>DMEM with high glucose&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Penicillin Streptomycin&lt;/strong&gt;</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>&lt;strong&gt;PolyJet &lt;em&gt;In Vitro&lt;/em&gt; DNA Transfection Reagent&amp;nbsp;&lt;/strong&gt;</td><td>Signagen</td><td>SL100688.5</td><td></td></tr><tr><td>&lt;strong&gt;5x Passive lysis buffer&lt;/strong&gt;</td><td>Promega</td><td>E194A</td><td>30 ml</td></tr><tr><td>&lt;strong&gt;Dual-Glo&amp;reg; Luciferase Assay System&lt;/strong&gt;</td><td>Promega</td><td>E2940</td><td>100 ml kit</td></tr><tr><td>&lt;strong&gt;20/20 luminometer&lt;/strong&gt;</td><td>Turner Biosystems</td><td>998-2036</td><td>Single tube reader luminometer</td></tr><tr><td>&lt;strong&gt;GloMax&amp;reg;Navigator with dual injectors&lt;/strong&gt;</td><td>Promega</td><td>GM2010</td><td>96-well plates reader luminometer</td></tr></tbody></table>

monitoring hippo signaling pathway activity using a luciferase-based large tumor suppressor (lats) biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

The LATS-BS was cotransfected with different genes to evaluate their effect on LATS activity (Figure 3). In this experiment, Renilla was used as an internal control. The transient expression of YAP-5SA, a constitutive active form of YAP, causes increasing levels of YAP transcriptional targets and a subsequent increase in LATS kinase activity through an established feedback pathway25. MST, the upstream activator of LATS in the ...

Watch this Scientific Journal Video about Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor at JoVE.com

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

monitoring-hippo-signaling-pathway-activity-using-luciferase-based

Research

JoVE Journal

Cancer Research

19.6K Views.  Queen's University. Mijn lab heeft de rol van Hippo pad in borst-en longkanker bestudeerd, en onlangs hebben we een nieuwe biosensor ontwikkeld om Hippo signalering activiteit te controleren. Deze methode kan worden gebruikt om belangrijke vragen over cellulaire signalering netwerken te beantwoorden, zoals hoe de Hippo route reageert op factoren en hoe het interageert met andere signalering trajecten. Het belangrijkste voordeel van deze techniek ten opzichte van bestaande methoden is de grote gevoeligheid en ook...

Video: Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Biochemistry

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

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

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

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

Medicine

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Developmental Biology

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Neuroscience

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Biology

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

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

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

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

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

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

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

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

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

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