This work was supported by National Institutes of Health grants R01-DK080834 and R01-DK093045. We thank J. Denise Wetzel, CCHMC Medical Writer, for editing of the manuscript.

1. Ingenuity Pathway Analysis
<ol>
	<li>Uploading Protein-interactome Dataset
	<ol>
		<li>Insert the proteins/genes of interest in a spreadsheet with their unique gene identifiers (preferably gene symbols and gene identifier numbers as obtained with mass spectrometric data).</li>
		<li>Assign one column in the spreadsheet for Gene identifier number and one column for observational value (e.g., Fold-change or p-value). Select the &#39;contains column header&#39; option to view the column headings.</li>
		<li>Upload the dataset on the IPA by clicking on the upload dataset tab and selecting the spreadsheet mentioned above. Choose the flexible format tab, and select the appropriate category of gene identifier. 
		Note: A flexible format of the dataset overcomes the formatting specifications for uploading.</li>
		<li>After the dataset appears, select ID and observation columns. Ensure that all of the proteins (MRP4 interactome as identified by mass-spectrometry) are mapped by checking on the dataset summary tab. The dataset is now ready for the desired analysis.</li>
	</ol>
	</li>
	<li>Data Analysis 
	Note: Described are the partial uses of IPA features that were utilized for this study.
	<ol>
		<li>After uploading MRP4 interactome with their gene names as the unique identifiers, click on new and choose core analysis in the top left menu of IPA software.</li>
		<li>Set the expression cutoffs value for intensity in accordance with the experiment type (Intensity value of 100 is recommended for analysis).</li>
		<li>Calculate the fold-change in expression while preparing the dataset if control and experimental conditions are involved and include it as part of the dataset file if a comparison analysis needs to be performed (A cut-off value of 1.5 fold change is usually recommended for analysis). 
		Note: After completion of the core analysis, multiple sections of analyses can be obtained. The first tab shows the summary of the total analyses and suggests top canonical pathways, upstream regulators, molecular and cellular functions, and networks among others; all calculated based on the confidence level (p value) and arranged in ascending order of p value.</li>
		<li>Open the major canonical pathways (using the open pathway option) as large molecular networks that pictorially show cross talking, overlapping, and affected signaling pathways. 
		Look at the list of the canonical pathways that are majorly involved based on the p-value (p &#60;0.05 is considered as significant). 
		Note: The significance values for the canonical pathways are calculated by Fisher&#39;s exact test right-tailed. The significance indicates the probability of association of molecules from the dataset with the canonical pathway by random chance alone.</li>
		<li>Confirm the input proteins (MRP4 interactome as identified by mass-spectrometry) among the list of proteins involved in each pathway. 
		Note: Using this option, it was observed that the cellular actin cytoskeleton signaling network is the major affected pathway and how the input proteins fit into it (Figure 1). This approach helps identify which molecular networks would be closely associated and affected by the test proteins.</li>
		<li>Use the network tab in the analyses to identify the top diseases and functions that are potentially linked to a particular protein network based on the number of focus molecules. 
		Note: A score value is given based on the known proteins that are determined as part of a network e.g., cellular assembly and organization network, through literature knowledge and experimental evidences and focus molecules that are identified from the dataset and become part of the network. Henceforth, the network gets filtered in the first place based on the number of focus molecules.</li>
	</ol>
	</li>
</ol>
2. High-content Microscopy
<ol>
	<li>Preparation of Cells 
	Note: All cell culture work was done under the horizontal-flow cell culture hood.
	<ol>
		<li>Use 96-well microplates for the wound-healing assay.</li>
		<li>Coat the microplate with 1% fibronectin solution (reconstituted in PBS), and keep the plate in a standard CO2 incubator at 37 &#176;C for 2 hr.</li>
		<li>Remove Mrp4-/- and Mrp4+/+ mouse embryonic fibroblast (MEFs) grown in 25 cm&#178; cell culture flasks from the incubator, wash 1 time with PBS, and trypsinize the cells for 5 min using 1 ml of 4x trypsin-EDTA solution.</li>
		<li>Wash the cells with complete medium (DMEM containing 10% FBS and 1% penicillin/streptomycin) and resuspend them in 5 ml of complete medium.</li>
		<li>Aspirate the fibronectin solution from the wells. Count cells using a hemocytometer and seed 30,000 cells (cell number needs to be optimized based on cell type) into each well.</li>
		<li>Grow the cells to 100% confluence in a standard CO2 incubator at 37 &#176;C for 24 hr.</li>
	</ol>
	</li>
	<li>Creating Wound
	<ol>
		<li>Ensure all the wells are filled with solution. Fill unused wells with PBS to prevent damage to the wound making tool (e.g., WoundMaker).</li>
		<li>After confirming 100% cell confluence, place the 96-well plate inside the wound making tool on the metal base plate and press the 96-well pin block down.</li>
		<li>Wash the cells three-times with PBS to remove any dislodged cells and add 100 &#181;l of complete medium.</li>
		<li>Add the test compounds - H-89 (50 &#181;M; 1:1,000 dilution with complete media using 50 mM stock in DMSO) or Latrunculin B (1 &#181;M; 1:1,000 dilution with complete media using 1 mM stock in DMSO) at this stage.</li>
		<li>Place the assay plate inside the reader at 37 &#176;C and monitor migration over the experimental period.</li>
	</ol>
	</li>
	<li>Monitoring Cell Migration 
	Note: Cell migration was monitored using the microscope and software provided with high-content microscopy system. The use of 96-well microplates ensures that the wounds are automatically monitored by the microscope and registered by the software.
	<ol>
		<li>Set the software to scan the assay plate every hour for migration assays using the schedule scan tab . Select a start time at least 15 min after placing the assay plate inside the reader to allow equilibration prior to imaging.</li>
		<li>Select the tray and choose vessel type (96-well microplate) and experiment type (Scratch Wound).</li>
		<li>Select 10X objective and choose phase-contrast imaging parameters. Select the wells that need to be scanned using the edit scan pattern option.</li>
		<li>Specify the treatments groups and any replicates by selecting the properties tab and setting up a plate map. Scanning can be stopped at any time based on the experimental condition.</li>
	</ol>
	</li>
	<li>Data Analysis
	<ol>
		<li>After the scanning is done, select the vessel view tab for the assay plate. Go to the analysis job utilities and select launch new analysis job.</li>
		<li>Set scratch wound as job type and select the time range for the analysis (0 time point to 24 hr time point).</li>
		<li>Select the wells to be chosen for analysis by a single click on the well and click &#39;OK&#39; to initiate the analysis. Once the analysis is done, graphically visualize relative wound density (RWD) data for each well at each time point using export to the graph option.</li>
		<li>View the phase-contrast image set of each well corresponding to different time-points using the view image tab. Select a specific well by clicking on the plate map, select a specific time point from the time range tab and click on the view image tab.</li>
	</ol>
	</li>
</ol>
3. F&#246;rster Resonance Energy Transfer (FRET)
<ol>
	<li>Preparation of Cells
	<ol>
		<li>Coat 35 mm glass-bottomed dishes with 100 &#181;l of 1% fibronectin solution (as described in section 2.1) and keep them in a standard CO2 incubator at 37 &#176;C for 2 hr.</li>
		<li>Aspirate the fibronectin solution from the plates and seed equal numbers of HEK293 cells (10,000 cells/plate) in complete medium.</li>
		<li>Grow the cells to 60-70% confluence in the fibronectin coated glass-bottomed dishes in a standard CO2 incubator at 37 &#176;C for 24 hr.</li>
	</ol>
	</li>
	<li>Transfection
	<ol>
		<li>Perform all transient transfections using a commercial transfection reagent according to manufacturer&#39;s instructions.</li>
		<li>Aliquot 250 &#181;l of complete medium into two separate 1.5 ml&#160;sterile centrifuge tubes for each 35 mm dish.</li>
		<li>Add 2 &#181;g of DNA (pmAKAR3 or pmAKAR3-TA) in one tube and 5 &#181;l (2.5 fold the DNA concentration) of transfection reagent in another tube.</li>
		<li>Incubate the tubes for 20 min at room temperature and then transfer the diluted DNA into the transfection reagent-containing tube.</li>
		<li>Mix thoroughly and incubate at 37 &#176;C for another 30 min.</li>
		<li>Aspirate the medium off the cells and wash them once with PBS. Add 1 ml of antibiotic-free DMEM F-12 medium containing 10% FBS to the cells.</li>
		<li>Add 507 &#181;l of DNA-lipid conjugate with media to the cells in each plate, mix gently, and keep in the CO2 incubator at 37 &#176;C for 48 hr. Use 2 &#181;g of DNA (1 &#181;g/&#181;l stock) and 5 &#181;l lipid for 10,000-50,000 cells in each plate.</li>
	</ol>
	</li>
	<li>Live-cell Imaging
	<ol>
		<li>After 48 hr of transfection, remove growth medium and wash the cells 2 times with Hank&#39;s Balanced Salt Solution (HBSS, pre-warmed at 37 &#176;C). Add a final volume of 1.9 ml HBSS to the washed cells and mount them on an inverted wide-field microscope system for FRET imaging inside a custom-made 37 &#176;C maintained chamber. 
		Note: In this system, the excitation light is provided by a 300 W Xenon lamp attenuated with a Neutral Density filter with 50% light transmission.</li>
		<li>Use 60X objective. Manually focus on the cells and set an optimal field of view using the microscope. Activate the selected field of view on the computer screen using the Focus &#34;F&#34; option on the software. For performing FRET measurements, select the proper filter set (CFP/YFP filter set with a 430/25 nm excitation filter, a double dichroic beam splitter, and two emission filters (470/30 nm for CFP and 535/30 nm for FRET) manually.</li>
		<li>Visualize fluorescence by first checking on the channel option CFP/YFP/FRET followed by clicking open fluor tab. Improve the signal intensity by using the intensification tool in the camera tab.</li>
		<li>Select cells expressing pmAKAR3 or pmAKAR3-TA avoiding saturation of the signal intensity. Use the capture window to initiate FRET measurement.</li>
		<li>Select the time-lapse option and setup a time-scan for 30 min with 30 sec interval. Check the FRET option and set the exposure time at 100 msec. Enter the image label and press start to initiate the measurement.</li>
		<li>After five time points and establishment of a baseline, add 25 &#181;M of forskolin (5 &#181;l of 10 &#181;M stock in ethanol, diluted with 100 &#181;l of HBSS) to the cells without disturbing the plate and monitor the cells for a total of 30 min.</li>
	</ol>
	</li>
	<li>Data Analysis 
	Note: Use ratiometric calculation module for data analysis.
	<ol>
		<li>Click on the select tool on the left-hand side of the image window and choose a solid rectangle from the drop down menu. Drag the rectangle on the image field in a cell free area and right click on it to set it as background for the purpose of background subtraction.</li>
		<li>Go to the mask and use &#39;create a new mask&#39; option. Go to the select tool and choose a pen from the drop down menu to manually draw a mask around the cell to select it for a measurement. Select at least 4-6 cells per condition.</li>
		<li>Select the Mask tab and perform mask statistics.</li>
		<li>Check &#39;Entire mask&#39;, expand cross-channel option and select donor normalized FRET (N-FRET) (FRET/CFP). The N-FRET value is representative of PKA activity at the plasma membrane.</li>
		<li>Add time-stamps, look-up table and scale bar using the annotations.</li>
	</ol>
	</li>
</ol>

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>Arora, 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=Compartmentalization+of+cyclic+nucleotide+signaling:+a+question+of+when,+where,+and+why?.">Compartmentalization of cyclic nucleotide signaling: a question of when, where, and why?.</a> Pflugers Arch. 465 (10), 1397-1407 (2013).</li><li>Howe, A. K., Baldor, L. C., Hogan, B. 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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=Spatial+distribution+of+protein+kinase+A+activity+during+cell+migration+is+mediated+by+A-kinase+anchoring+protein+AKAP+Lbc.">Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc.</a> J Biol Chem. 284 (9), 5956-5967 (2009).</li><li>Weaver, A. M., Young, M. E., Lee, W. L., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+signals+to+the+Arp2/3+complex.">Integration of signals to the Arp2/3 complex.</a> Curr Opin Cell Biol. 15 (1), 23-30 (2003).</li><li>Le Clainche, C., Carlier, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+actin+assembly+associated+with+protrusion+and+adhesion+in+cell+migration.">Regulation of actin assembly associated with protrusion and adhesion in cell migration.</a> Physiol Rev. 88 (2), 489-513 (2008).</li><li>Raftopoulou, M., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+Rho+GTPases+lead+the+way.">Cell migration: Rho GTPases lead the way.</a> Dev Biol. 265 (1), 23-32 (2004).</li><li>Krause, M., Dent, E. W., Bear, J. E., Loureiro, J. J., Gertler, F. 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D., Wu, J., Cui, J., Chen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnitine+palmitoyltransferase+1A+(CPT1A):+a+transcriptional+target+of+PAX3-FKHR+and+mediates+PAX3-FKHR-dependent+motility+in+alveolar+rhabdomyosarcoma+cells.">Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR-dependent motility in alveolar rhabdomyosarcoma cells.</a> BMC Cancer. 12 (154), (2012).</li><li>Zhang, J., Ma, Y., Taylor, S. S., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+reporters+of+protein+kinase+A+activity+reveal+impact+of+substrate+tethering.">Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering.</a> Proc Natl Acad Sci U S A. 98 (26), 14997-15002 (2001).</li><li>Sinha, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forster+resonance+energy+transfer+-+an+approach+to+visualize+the+spatiotemporal+regulation+of+macromolecular+complex+formation+and+compartmentalized+cell+signaling.">Forster resonance energy transfer - an approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling.</a> Biochim Biophys Acta. 1840 (10), 3067-3072 (2014).</li><li>Sato, M., Ozawa, T., Inukai, K., Asano, T., 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=Fluorescent+indicators+for+imaging+protein+phosphorylation+in+single+living+cells.">Fluorescent indicators for imaging protein phosphorylation in single living cells.</a> Nat Biotechnol. 20 (3), 287-294 (2002).</li><li>Allen, M. D., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+dynamics+of+protein+kinase+A+activity+visualized+by+FRET-based+reporters.">Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.</a> Biochem Biophys Res Commun. 348 (2), 716-721 (2006).</li><li>Ananthanarayanan, B., Ni, Q., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+propagation+from+membrane+messengers+to+nuclear+effectors+revealed+by+reporters+of+phosphoinositide+dynamics+and+Akt+activity.">Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity.</a> Proc Natl Acad Sci U S A. 102 (42), 15081-15086 (2005).</li><li>Yamaguchi, H., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+actin+cytoskeleton+in+cancer+cell+migration+and+invasion.">Regulation of the actin cytoskeleton in cancer cell migration and invasion.</a> Biochim Biophys Acta. 1773 (5), 642-652 (2007).</li><li>Lamalice, L., Le Boeuf, F., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+migration+during+angiogenesis.">Endothelial cell migration during angiogenesis.</a> Circ Res. 100 (6), 782-794 (2007).</li><li>Ghosh, M. 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The authors have nothing to disclose.

Cell migration is an intricate process that plays indispensable roles in many important physiological events including wound healing1,2. Aberrant cell migrations may cause catastrophic events, such as tumor metastasis and angiogenesis24,25. Therefore, fine-tuned regulation of cell migration is required to maintain normal body function.
Using high-content microscopy18, we demonstrated that MRP4-deficient MEFs migrate faster compared to wild-type fibroblasts...

Cell migration is a complicated multi-step process. Studies have shown that during migration cells are polarized into leading and trailing edges. By adhering to the extracellular matrix, the leading edge provides the traction necessary for the cell body to move forward. Finally, the trailing edge releases rear attachments and completes the migration cycle1,2.
Cell polarization for efficient cell migration is regulated by spatial segregation of intracellular signaling. Cellular second messengers, such as cAMP, mediate the compartmentalization of the signaling events required for fine-tuned directional cell migration3,4. Preferential accumulations of cAMP and cAMP-dependent kinase PKA activity at the leading edge play key roles in directional cell migration5,6. By phosphorylating small GTPases such as Ras-related C3 botulinum toxin substrate (Rac) and cell division control protein 42 homolog or Cdc42, PKA activates actin-related protein 2/3 (Arp 2/3) at the leading edge and induces the formation of lamellipodia7-9. PKA also phosphorylates an anti-capping agent, vasodilator stimulated phosphoprotein (VASP), thereby regulates the oscillatory cycles of membrane extension and retraction10,11.
In cells, cAMP levels are regulated by three major processes: i) synthesis by adenylate cyclase, ii) degradation by phosphodiesterases, and iii) transportation by membrane-bound efflux transporters3. Multidrug resistance protein 4 (MRP4), a member of ATP-binding cassette (ABC) family of membrane transporters, functions as an endogenous efflux transporter of cyclic nucleotides. Therefore, MRP4 can regulate intracellular cAMP levels and cAMP-dependent cellular signaling11-13. We have previously shown that in Mrp4-/-, fibroblasts contain relatively higher levels of cyclic nucleotides and migrate faster compared to Mrp4+/+ fibroblasts14. We also reported a biphasic effect of cyclic nucleotides on fibroblast migration. Based on previous studies and our finding that Mrp4-/- fibroblasts contain more polarized cAMP during the course of migration, we hypothesized that this MRP4-mediated regulation of fibroblast migration is cAMP dependent. In order to understand the downstream mechanism, we took a direct yet multifaceted approach.
To identify the proteins associated and in interplay with MRP4, we immunoprecipitated MRP4-containing macromolecular complexes from HEK293 cells that over express MRP4. Using mass-spectrometry, we identified multiple MRP4-interacting proteins and analyzed their interconnectivity using Ingenuity Pathway Analysis (IPA). IPA is a useful tool to analyze protein-protein interactions (both structural and functional) and explore their contributions in particular physiological and pathological events based on the literature and experimental evidences15,16. IPA indicated that F-actin is a major downstream target of MRP4 in the context of cell migration where cAMP and cGMP are the key signaling molecules17. These data were further confirmed by high-content microscopy. High-content microscopy can capture and analyze cell behaviors such as cell migration in a more convenient, accurate and high-throughput manner18. The high-content microscopy data demonstrated that the effect of MRP4 on fibroblast migration is completely abolished upon disruption of the actin cytoskeleton or inhibition of PKA17.
Additionally, we used a F&#246;rster resonance energy transfer (FRET)-based PKA sensor to monitor the PKA dynamics in migrating cells in real time. FRET-based kinase sensors usually consist of specific phosphorylation substrate peptides flanked by CFP and YFP fluorophores19-21. pmAKAR3 is an improved and membrane targeted FRET-based PKA sensor that contains forkhead-associated domain 1 (FHA1) and the PKA substrate sequence LRRATLVD5,22. Phosphorylation of pmAKAR3 by the PKA catalytic subunit increases FRET signal between CFP and YFP19. Insertion of a lipid modification domain into the sensor targets it to the plasma membrane for monitoring PKA dynamics, specifically at the membrane compartment23.
Using pmAKAR3, we demonstrated that the leading edge of migrating Mrp4-/- fibroblasts exhibited more polarized PKA activity than Mrp4+/+ fibroblasts, which in turn increased the cortical actin formation at the cell&#39;s leading edge17. Together, these events resulted in better cellular polarization and faster directional cell migration in the absence of MRP4. Our specific and direct approach identified key downstream targets for MRP4 and provides an important, but as of yet unexplored mechanism for MRP4-dependent regulation of fibroblast migration.

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			<td height="21" style="height:21px;width:272px;">Lipofectamine 2000</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
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			<td>Invitrogen (Carlsbad, CA)&#160;</td>
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			<td height="21" style="height:21px;">IncuCyte Zoom</td>
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		<tr height="21">
			<td height="21" style="height:21px;">96-well IncuCyte Image-Lock microplates&#160;</td>
			<td>Essen BioScience</td>
			<td style="width:187px;">4493</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Latrunculin B</td>
			<td style="width:324px;">Sigma-Aldrich (St. Louis, MO).</td>
			<td>L5288</td>
			<td>Stock in DMSO</td>
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			<td height="21" style="height:21px;">H-89</td>
			<td style="width:324px;">Enzo Life Sciences (Farmingdale, NY)</td>
			<td>BML-EI196</td>
			<td>Stock in DMSO</td>
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			<td height="21" style="height:21px;">35-mm glass-bottomed dishes&#160;</td>
			<td style="width:324px;">(MatTek Corporation; Ashland, MA)</td>
			<td style="width:187px;">P35G-1.5-20-C&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fibronectin</td>
			<td style="width:324px;">Sigma-Aldrich (St. Louis, MO).</td>
			<td>F1141</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Opti-MEM Reduced Serum Media</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">31985-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">FRET microscopy system</td>
			<td>Olympus inverted microscope (IX51)</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">CCD camera&#160;</td>
			<td style="width:324px;">Hamamatsu, Japan</td>
			<td style="width:187px;">ORCA285</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">SlideBook software 5.5</td>
			<td style="width:324px;">Intelligent Imaging Innovation ( Denver, CO)</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ingenuity Pathway Analysis software</td>
			<td>IPA, QIAGEN Redwood City,</td>
			<td style="width:187px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Forskolin</td>
			<td>Tocris (Ellisville, MO).&#160;</td>
			<td style="width:187px;">1099</td>
			<td>Stock in100% EtOH</td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM F-12&#160;&#160;</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">11330-057</td>
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			<td height="21" style="height:21px;width:272px;">HBSS</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">14025-134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Excel</td>
			<td style="width:324px;">Microsoft</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">PBS</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">10010-023</td>
			<td></td>
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			<td height="21" style="height:21px;width:272px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td>R-001-100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penicillin-Streptomycin</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
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methods to study mrp4-containing macromolecular complexes in the regulation of fibroblast migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

To study the effect of MRP4 on fibroblast migration, we used a wound-healing assay utilizing high-content microscopy14. Precise wounds were made on confluent monolayers of MEFs isolated from either Mrp4-/- or Mrp4+/+ mice, and images were taken at 1 hr intervals for 24 hr. We observed a higher migration rate for Mrp4-/- MEFs compared to Mrp4+/+ MEFs (Figur...

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Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

The overall goal of this experimental procedure is to observe the cyclic AMP dependent regulation of fibroglast migration by MRP4.High contact microscopy can help answer key questions in the field of cell biology.For example, in addition to conferring germ resistance, MRP4 can also regulate physiological process such as cell migration by modulating intracellular cyclic nucleotide concentration.The main advantage of FRET imaging that we are going to attempt today is to allow monitoring of the intracellular dynamics of cyclic AMP and cyclic AMP dependent kinase in a real-time fashion.The implication of high contact microscopy is it can be used in drug discovery.Once the system is set up, all the instrument is set up, there is no human intervention involved in this plate-based task.Though this method can provide insight into cell migration, it can be applied to other systems, such as lipe cell nonparturbing, high contact imaging of immune cell killing, cooler dilution, angiogenesis, cell proliferation, chemotaxis, and treatese phyllite.To prepare cells for high-contact microscopy under a cell culture hood, use a one percent fibronectin solution in PBS to coat the wells of a 96 well plate.Incubate the plate in a CO2 incubator at 37

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6.7K 조회수.  Cincinnati Children’s Hospital Medical Center. The overall goal of this experimental procedure is to observe the cyclic AMP dependent regulation of fibroglast migration by MRP4.High contact microscopy can help answer key questions in the field of cell biology.For example, in addition to conferring germ resistance, MRP4 can also regulate physiological process such as cell migration by modulating intracellular cyclic nucleotide concentration.The main advantage of FRET imaging that we are going to attempt today is to allow monitoring of the ...

비디오: Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...