We thank the members of the Jha laboratory for their helpful discussion and comments. S.J. was supported by grants from the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiative to the Cancer Science Institute of Singapore (R-713-006-014-271), the National Medical Research Council (CBRG-NIG; BNIG11nov001 and CS-IRG; R-713-000-162-511), the Ministry of Education Academic Research Fund (MOE AcRF Tier 1 T1-2012 Oct -04). Y.Z., G.S.C., and C.Y.T. were supported by a post-graduate fellowship awarded by the Cancer Science Institute of Singapore, National University of Singapore.

1. Preparation
<ol>
	<li>Prepare MCF10A culture medium: Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 (1:1) with 5% horse serum, 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 &#181;g/mL insulin.</li>
	<li>Prepare serum-free medium: DMEM/F12 (1:1) with 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 &#181;g/mL insulin.</li>
	<li>Use the following siRNA sequence: 
	siControl: 
	Forward: 5&#39;-CGUACGCGGAAUACUUCGAdTdT-3&#39; 
	Reverse: 5&#39;-UCGAAGUAUUCCGCGUACGdTdT-3&#39; 
	siTIP60: 
	Forward: 5&#39;-UGAUCGAGUUCAGCUAUGAdTdT-3&#39; 
	Reverse: 5&#39;-UCAUAGCUGAACUCGAUCAdTdT-3&#39;</li>
	<li>Use 10-cm cell culture dishes and 24-well plates.</li>
</ol>
2. Transient Depletion of TIP60 Using siRNA
<ol>
	<li>On day 1, aliquot 15 &#181;L of transfection reagent (e.g., Lipofectamine RNAiMAX) and 2 mL of reduced serum medium (e.g., Opti-MEM) and mix them with 10 &#181;L of each siRNA (10 &#181;M stock), respectively.</li>
	<li>Incubate the mixture at room temperature for 20 min.</li>
	<li>Seed 1 x 106 MCF10A cells together with 3 mL of culture medium in a 10 cm dish. Count the cells using a hemocytometer.</li>
	<li>Add the siRNA mixture drop by drop to the 10 cm dish.</li>
	<li>Shake the dish to ensure that the cells are equally distributed before putting it into the 37 &#176;C incubator.</li>
	<li>After 6 h of incubation, replace the siRNA-containing medium with 8 mL of fresh culture medium.</li>
	<li>On day 2, prepare another batch of siRNA mixtures and incubate them at room temperature for 20 min.</li>
	<li>Aspirate out 8 mL of the culture medium added on day 1 and replace it with 3 mL of fresh culture medium.</li>
	<li>Add the second batch of siRNA mixture to the dishes drop by drop. Shake the dishes to ensure that the reagents are fully mixed before putting them into the incubator.</li>
	<li>After 6 h, replace the siRNA-containing medium with 8 mL of fresh culture medium.</li>
</ol>
3. Seed Cells for the Wound-healing Assay
<ol>
	<li>On day 3, discard the medium and wash the cells once with 2 mL of phosphate-buffered saline (PBS) buffer. Discard the PBS buffer.</li>
	<li>Trypsinize the siControl and siTIP60-treated cells by adding 1 mL of 1x&#160;trypsin-EDTA buffer to the plate. Put it back in the incubator for 20 min.</li>
	<li>Add 4 mL of complete culture medium to each plate, resuspend the cells by pipetting, and transfer them to a 15-mL tube.</li>
	<li>Centrifuge at 200 g for 5 min and remove the supernatant.</li>
	<li>Re-suspend the cells with 5 mL of serum-free medium and count the cells using a hemocytometer.</li>
	<li>Prepare a cell suspension of 6 x 105 cells/mL for both the siControl and siTIP60; ensure that the cells are well-mixed. Add 500 &#181;L of the cell suspension to one well of a 24-well plate to ensure 100% confluency. Seed 5 to 6 wells each for siControl and siTIP60.</li>
	<li>Gently shake the plate back and forth and then side-to-side to ensure even distribution. Avoid a circular motion. Leave the plate in the incubator for 24 h.</li>
	<li>Harvest the remaining cells to check the knockdown efficiency of TIP60. To do so, centrifuge the remaining cell suspension at 200 g for 5 min and remove the supernatant.</li>
	<li>Isolate RNA using a commercial reagent, following the manufacturer&#39;s protocol, and proceed to real-time quantitative PCR to evaluate the knockdown efficiency.</li>
</ol>
4. Create the Wound
<ol>
	<li>Check under a phase contrast microscope to ensure that the cells are fully attached. Observe the cells under the microscope (4X, 10X, and 20X&#160;magnification) to ensure that the cells are fully attached that a confluent monolayer of cells has formed. 
	NOTE: siControl cells usually attach better than siTIP60 cells. This is expected because the depletion of TIP60 makes the cells more mesenchymal-like20. Here, a normal benchtop white-light phase contrast microscope is used.</li>
	<li>To generate the wound, gently scratch a straight line across the center of the well with a 200 &#181;L pipette tip; the length of the wound generated is same as the diameter of the well. Repeat this step for all remaining wells. Use a new pipette tip for each well.</li>
	<li>Gently wash each well 5 times using serum-free medium to remove the detached cells.</li>
	<li>Add 1 mL of serum-free medium to each well and proceed to live-cell imaging.</li>
</ol>
5. Setup of Live-cell Imaging
<ol>
	<li>Turn on the microscope and the temperature control at least 1 h before setting up live-cell imaging to ensure that the temperature in the chamber reaches 37 &#176;C.</li>
	<li>Turn on the carbon dioxide (CO2) gas supply before starting the live-cell imaging. Set the CO2 concentration to 5%.</li>
	<li>Place the 24-well plate on the stage of the microscope and select the objective lens with 10X&#160;magnification for imaging. Direct the emitted light to the eyepieces instead of the camera and adjust the focus to locate the wound and the cells around it. 
	NOTE: In this case, white light with phase contrast was used.</li>
	<li>Use a live-cell observer system to mark the position of the wound in each well. 
	NOTE: The automated stage of the live-cell imaging system allows for image acquisition at multiple positions. Images are taken on every marked position once every interval.</li>
	<li>Set up the time condition: 1 h interval and 72 h duration; according to this setting, the system will take pictures at each marked position every hour for 72 h. Set up any desired time interval and period for monitoring. 
	NOTE: Here, a duration of 72 h is best because these cells start dying after 72 h in serum-free medium.</li>
</ol>
6. Tracking Single-cell Movement
<ol>
	<li>Export the data after 72 h. Export the data both as images in JPEG format and as videos in AVI format. 
	NOTE: This is important because ImageJ software can only recognize videos in AVI format. The different cell migration speeds between siTIP60 and siControl cells can be clearly observed in the video.</li>
	<li>Open the AVI video in the ImageJ software.</li>
	<li>Open the MTrackJ plugin (the plugin must be downloaded and installed separately): Plugins &#62; MtrackJ.</li>
	<li>Add a track for single cells chosen from the video by clicking the &#34;Add&#34; tab in the MtrackJ toolbar. Choose a cell at the tip of the invasive front and a cell that can migrate on its own (single-cell migration). Follow the movement of the chosen cells on the video and trace them using MtrackJ.</li>
	<li>Measure the distance of movement by clicking the &#34;measure&#34; tab on the toolbar. 
	NOTE: Here, almost no cells in the siControl moved as single cells, making it difficult to measure single-cell movement, whereas many of the siTIP60 cells moved as single cells. However, the movement of cells located at the invasive front can still be traced and calculated in both siControl and siTIP60 cells.</li>
	<li>Save the video with the tracks in AVI format.</li>
</ol>
7. Quantification of Cell Migration Ability
<ol>
	<li>For quantification, select images at 0 h and images at &#34;n&#34; h, where &#34;n&#34; refers to the timepoint when either the siTIP60 or siControl cells have completely closed the wound. 
	NOTE: Here, 48 h was chosen, as the wounds in siTIP60 cells were almost completely closed.</li>
	<li>Quantify the percent migration using ImageJ using the following formula: (area of wound at 0 h - area of wound at &#34;n&#34; h) / area of wound at 0 h x 100.</li>
</ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+functions+of+TIP60.">Cellular functions of TIP60.</a> Int J Biochem Cell Biol. 38 (9), 1496-1509 (2006).</li><li>Squatrito, M., Gorrini, C., Amati, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip60+in+DNA+damage+response+and+growth+control:+many+tricks+in+one+HAT.">Tip60 in DNA damage response and growth control: many tricks in one HAT.</a> Trends Cell Biol. 16 (9), 433-442 (2006).</li><li>Sun, Y., Xu, Y., Roy, K., Price, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-induced+acetylation+of+lysine+3016+of+ATM+activates+ATM+kinase+activity.">DNA damage-induced acetylation of lysine 3016 of ATM activates ATM kinase activity.</a> Mol Cell Biol. 27 (24), 8502-8509 (2007).</li><li>Sykes, S. 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=Acetylation+of+the+p53+DNA-binding+domain+regulates+apoptosis+induction.">Acetylation of the p53 DNA-binding domain regulates apoptosis induction.</a> Mol Cell. 24 (6), 841-851 (2006).</li><li>Tang, Y., Luo, J., Zhang, W., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip60-dependent+acetylation+of+p53+modulates+the+decision+between+cell-cycle+arrest+and+apoptosis.">Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis.</a> Mol Cell. 24 (6), 827-839 (2006).</li><li>Chen, G., Cheng, Y., Tang, Y., Martinka, M., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Tip60+in+human+melanoma+cell+migration,+metastasis,+and+patient+survival.">Role of Tip60 in human melanoma cell migration, metastasis, and patient survival.</a> J Invest Dermatol. 132 (11), 2632-2641 (2012).</li><li>Gorrini, 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=Tip60+is+a+haplo-insufficient+tumour+suppressor+required+for+an+oncogene-induced+DNA+damage+response.">Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response.</a> Nature. 448 (7157), 1063-1067 (2007).</li><li>Jha, S., 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=Destabilization+of+TIP60+by+human+papillomavirus+E6+results+in+attenuation+of+TIP60-dependent+transcriptional+regulation+and+apoptotic+pathway.">Destabilization of TIP60 by human papillomavirus E6 results in attenuation of TIP60-dependent transcriptional regulation and apoptotic pathway.</a> Mol Cell. 38 (5), 700-711 (2010).</li><li>Kim, J. H., 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=Transcriptional+regulation+of+a+metastasis+suppressor+gene+by+Tip60+and+beta-catenin+complexes.">Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes.</a> Nature. 434 (7035), 921-926 (2005).</li><li>Sakuraba, 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=Down-regulation+of+Tip60+gene+as+a+potential+marker+for+the+malignancy+of+colorectal+cancer.">Down-regulation of Tip60 gene as a potential marker for the malignancy of colorectal cancer.</a> Anticancer Res. 29 (10), 3953-3955 (2009).</li><li>Subbaiah, V. 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=E3+ligase+EDD1/UBR5+is+utilized+by+the+HPV+E6+oncogene+to+destabilize+tumor+suppressor+TIP60.">E3 ligase EDD1/UBR5 is utilized by the HPV E6 oncogene to destabilize tumor suppressor TIP60.</a> Oncogene. , (2015).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Zijl, F., Krupitza, G., Mikulits, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+steps+of+metastasis:+cell+invasion+and+endothelial+transmigration.">Initial steps of metastasis: cell invasion and endothelial transmigration.</a> Mutat Res. 728 (1-2), 23-34 (2011).</li><li>Lamouille, S., Xu, J., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial-mesenchymal+transition.">Molecular mechanisms of epithelial-mesenchymal transition.</a> Nat Rev Mol Cell Biol. 15 (3), 178-196 (2014).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat Protoc. 2 (2), 329-333 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIP60+inhibits+metastasis+by+ablating+DNMT1-SNAIL2-driven+epithelial-mesenchymal+transition+program.">TIP60 inhibits metastasis by ablating DNMT1-SNAIL2-driven epithelial-mesenchymal transition program.</a> J Mol Cell Biol. , (2016).</li><li>Riahi, R., Yang, Y., Zhang, D. D., Wong, P. 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The authors declare that they have no competing financial interests.

It is known that in the process of cell migration, cells can either move in the form of adherent sheets; loose clusters; or single, isolated cells. The mode and dynamic of cell invasion can vary from one condition to another, and the phenotype can change within hours. The traditional wound-healing assay is based on snapshot pictures taken from a microscope with a long time interval, such as 12 or 24 h. This makes it difficult to capture the dynamic of wound closure and the pattern of cell movements.
On the other hand, live-cell imaging systems monitor cell migration over a much shorter interval (30 min or 1 h) and at the exact same position, which is not feasible for images taken manually. The system is automated, and researchers only need to export the data at the end of the experiments. This makes it possible to monitor the pattern of cell movement and the process of wound closure in real time. In this study, the incorporation of a quantitative live-cell imaging system in wound-healing assays allows for the capture of dynamic cell movement, as well as the process of wound closure in control or TIP60-depleted cells. From the video taken using a live-cell imaging system, most of the siControl cells moved as an adherent sheet, whereas TIP60-depleted cells moved as isolated single cells (Figure 3 and Figure 5).
Cell proliferation and cell death might complicate the study of cell migration in a wound-healing assay. Therefore, the wound-healing assay is performed in serum-free medium to minimize the effect of cell proliferation. Also, the knockdown of TIP60 was optimized to reduce its impact on cell survival. However, it is impossible to eliminate the influences of cell proliferation and cell death in wound-healing assays. The snapshots can only capture wound closure at a certain time point, making it impossible to distinguish whether the wound closure was due to cell migration or cell proliferation. Live-cell imaging monitors the dynamics of cell migration and tracks individual cell movement, making it possible for researchers to distinguish cell migration from cell proliferation and cell death. In this study, single-cell tracking revealed the migration path of the isolated single cells in the siTIP60 condition, while siControl cells mostly moved in the form of sheets. The live-cell imaging system also captured an increase in collective cell migration (i.e., the cells migrated as a cohesive group) in siTIP60 cells compared to siControl cells. From the videos, MCF10A siTIP60 showed more single-cell movements and faster migration of epithelial cell sheets compared to siControl (Figure 5).
Various parameters used in this protocol need to be optimized to avoid experimental failure. First, the number of cell deaths after transient transfection should be less than 10% to minimize the effect on cell migration. In this case, the depletion of TIP60 may reduce cell survival; hence, researchers should be aware of this potential phenotype, as it may complicate cell migration. To avoid this problem, researchers should optimize the cell number and amount of siRNA used to deplete their target gene. For MCF10A, 1 x 106 cells with 20 nM siTIP60 was the optimized condition in this study. Second, MCF10A cells are often clustered and difficult to count. Errors in cell counts may lead to a significant difference in initial cell numbers across different conditions, and this could be a confounding variable in wound-healing assays. To avoid this problem, trypsinize MCF10A cells for at least 20 min. Add complete growth medium to stop the trypsinization and mix by pipetting up and down 20 times to disperse the clumps and to make it easier to count. Third, cells are usually in the center of the well after being seeded to the 24-well plate. Re-suspend the cells after seeding to ensure an even distribution. Shake the plate back and forth and side to side before putting it into the incubator. Fourth, the depletion of TIP60 makes the cell more mesenchymal and thus harder to attach. If this happens, leave the plate in the incubator for a longer period of time. After 24 h, the cells are usually attached. If the cells are still not properly attached, researchers could use complete growth medium for cell seeding and replace it with serum-free medium when the cells are fully attached. These cells are usually attached after 12 h in complete medium. Fifth, the edge of the wound may not be smooth. Increase the speed of scratching to create smooth edges. Sixth, the wound may be too big to visualize through the camera of the microscope. Use 200 &#181;L pipette tips with less strength to generate the wound. Alternatively, a needle can be used to generate a smaller gap. Seventh, some cells that are detached during scratching may re-attach to the wound due to insufficient washes. Wash the wells with serum-free medium instead of PBS at least 5 times. Eighth, cells may start to detach from the plate after a few washes. However, the number of washes cannot be compromised in order to ensure that detached cells do not re-attach to the wound. In this case, researchers can wash the cells gently 3 times and leave the plate inside the incubator for a maximum time of 1 h to let the cells settle down before proceeding to the rest of the washes. Ninth, the microscope may lose focus, and the images may become blurry halfway through the experiment. A change in temperature around the microscope may be the reason, as temperature has a minor effect on the adjusted stage distance. Researchers should turn on the microscope and temperature control at least 1 h before starting the experiment to ensure that the incubator is warmed up before adjusting the focus on cells.

HIV-Tat-interactive protein 60 kDa (TIP60) is a lysine acetyltransferase that can acetylate both histone and non-histone proteins1,2. Its functions are found to have implications in multiple signaling pathways, including transcription, DNA-damage repair, and apoptosis1,3,4,5,6,7,8. Furthermore, TIP60 is often downregulated in cancers, and its downregulation is correlated with cancer metastasis and poor survival rates9,10,11,12,13,14. Tumor metastasis is the major cause of cancer-related death. It is a multi-step process, and the initial step of metastasis involves the migration and invasion of tumor cells into adjacent tissues15,16. In order to do so, tumor cells first must detach from the primary tumor mass, either in the form of a collectively invading cell sheet or as detached single cells17. During this process, tumor cells often undergo epithelial to mesenchymal transition (EMT), resulting in changes in morphology and cell adhesion capability17,18.
A few techniques have been developed to study the migration and invasion ability of tumor cells in vitro. Among them, the wound-healing assay is the most efficient and economical19. First, this method involves the creation of an artificial gap on a confluent monolayer of cells, thus allowing cells to migrate and close the gap. Second, images of the gaps can be captured at the beginning and end of the experiment. Finally, a comparison of gap closure is used to determine the rate of cell migration. A phase-contrast microscope is usually used to capture images for conventional wound-healing assays. Another advantage of the wound-healing assay is that it partly resembles in vivo tumor cell migration. Similarly to in vivo tumor metastasis, cell migration in wound-healing assays shows both the collective migration of epithelial sheets and the migration of detached single cells.
However, cell migration is known to be dynamic, and there is a need to document the movement of cells in real time. For instance, a conventional wound-healing assay does not allow a researcher to analyze single-cell movement. On the other hand, cells cultured for wound-healing assays are often serum-starved to inhibit cell proliferation. This is done to rule out the possibility of gap closure due to cell proliferation. Nonetheless, there will still be some proliferation and cell death, and phase-contrast images taken before and after the experiment are unable to differentiate between them.
The live-cell imaging technique addresses this limitation of conventional wound-healing assays. Using a live-imaging microscope combined with a CO2 incubator and appropriate temperature control, researchers can measure the gap size and its rate of closure over time while tracing the movement of actively migrating cells located at the tips of the invasive front. Hence, the implementation of live-cell imaging to monitor cell migration will not only provide better visualization of cell migration, but will also allow for the possibility to differentiate between cell migration and cell proliferation, thus providing a more reliable analysis of cell migration.
In this study, MCF10A breast epithelial cells were used to demonstrate the combination of the wound-healing assay and live-cell imaging to study the role of TIP60 in cell migration. The depletion of TIP60 results in increased migration abilities in MCF10A cells.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">MCF10A cell</td>
			<td style="width:211px;">ATCC</td>
			<td style="width:119px;">CRL-10317TM</td>
			<td style="width:257px;">Breast epithelial cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DMEM/F12 media (1:1)</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">11330-032</td>
			<td>MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Epithelial growth factor (EGF)</td>
			<td style="width:211px;">Peprotech</td>
			<td style="width:119px;">AF-100-15</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cholera Toxin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C-8052</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrocortisone</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">H-0888</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Insulin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-1882</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">House serum</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">16050-122</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Trypsin</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>For MCF10A detach from plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hemacytometer</td>
			<td style="width:211px;">Fisher Scientific</td>
			<td style="width:119px;">267110</td>
			<td>For counting cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Opti-MEM reduced serum media</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>For siRNA mixture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMAX</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">56532</td>
			<td>Transfect reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Trizol reagent</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">15596-026</td>
			<td>For mRNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">iScript cDNA Synthesis Kit</td>
			<td style="width:211px;">Bio-Rad</td>
			<td style="width:119px;">170-8891</td>
			<td>For cDNA generation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">iTaq Universal SYBR Green Supermix</td>
			<td style="width:211px;">Bio-Rad</td>
			<td style="width:119px;">172-5125</td>
			<td>For real time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7500 Fast Real Time PCR system</td>
			<td style="width:211px;">Biosystems</td>
			<td style="width:119px;"></td>
			<td>Real time qPCR mechine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">10-cm dish</td>
			<td style="width:211px;">Greiner bio-one</td>
			<td style="width:119px;">664160</td>
			<td>For Knockdown experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">24-well plate</td>
			<td style="width:211px;">Cellstar</td>
			<td style="width:119px;">662160</td>
			<td>For wound healing assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siControl siRNA</td>
			<td style="width:211px;">Self designed</td>
			<td style="width:119px;">Self designed</td>
			<td>CGUACGCGGAAUACUUCGAdTdT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siTIP60 siRNA</td>
			<td style="width:211px;">Self designed</td>
			<td style="width:119px;">Self designed</td>
			<td>UGAUCGAGUUCAGCUAUGAdTdT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Live cell observer</td>
			<td style="width:211px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Zeiss inverted Cell Observer for live cell experiments</td>
		</tr>
	</tbody>
</table>

live-imaging of breast epithelial cell migration after the transient depletion of tip60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

General Schema of Live-cell Imaging-based Cell Migration Assay 
Figure 1A is a schema of this protocol. MCF10A cells transfected with siControl showed typical epithelial morphology. After the depletion of TIP60, MCF10A cells were more mesenchymal compared to control cells (Figure 1B).
Examination of TIP60 Knockdown Efficiency 
Figure 2 shows the TIP60 knockdown efficiency. The TIP60 expression level decreased significantly after siTIP60 treatment. In addition to TIP60, the expressions of several cell migration-related proteins were also examined20. For example, the expression of epithelial adhesion molecule (EpCAM), which is an epithelial marker, was decreased, whereas the expressions of Fibronectin (FN1) and SNAIL2 (SNAI2), which are both EMT drivers, were increased upon depletion of TIP60. These data suggest that the TIP60 levels in the MCF10A cells used for live-cell imaging were sufficiently reduced.
Depletion of TIP60 Changes the Dynamic of Cell Migration 
Figure 3 shows the videos taken for siControl and siTIP60 cells. The videos clearly show a faster cell migration of siTIP60 compared to siControl. Other than this, a difference in the dynamic of cell migration between siControl and siTIP60 cells was also observed (i.e., siControl cells moved as a sheet, whereas more single-cell movement was observed in siTIP60 cells).
Increase in Cell Migration after TIP60 Knockdown 
Figure 4 shows the quantification of cell migration for siControl and siTIP60 cells. The percentage of migrated cells increased significantly after the depletion of TIP60.
TIP60 Depletion Changes the Pattern of Cell Migration 
Figure 5 shows videos tracking single-cell movement. Most of the siControl cells collectively migrated as part of cell sheets, and only two cells showed single-cell migration, whereas most siTIP60 cells showed single-cell migration. The changes in the pattern of cell migration indicate that cells with depleted TIP60 have a more mesenchymal phenotype.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56248/56248fig1.jpg" /> 
Figure 1: Experimental overview and representative images. (A) Schema of the protocol. (B) Representative images of MCF10A cells transfected with siControl or siTIP60. MCF10A cells show a more mesenchymal morphology after the depletion of TIP60. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56248/56248fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56248/56248fig2.jpg" /> 
Figure 2: TIP60 was efficiently depleted after siTIP60 transfection. Real-time quantitative PCR was used to check the expression level of TIP60, EpCAM, FN1, and SNAI2. The total RNA was isolated using Trizol reagent following the manufacture&#39;s protocol. 2 &#181;g of the total RNA of each sample was used to make cDNA. The results are represented as the fold-change. Actin was used as an internal control. Error bar indicates the standard deviation from at least 3 independent experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56248/56248fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56248/56248fig3.jpg" /> 
Figure 3: Live-cell imaging videos of siControl and siTIP60 cells show different patterns of cell movement. The time interval was set to 1 h and the duration was to 72 h. The video was taken using a Zeiss live-cell observer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56248/Figure_3.zip">Please click here to download these videos.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56248/56248fig4.jpg" /> 
Figure 4: Quantification of the percentage of cell migration. (A) Representative images were obtained from live-cell imaging videos. Pictures taken at 0 h and 48 h were used. (B) Quantification of the percentage of cell migration within 48 h was done using ImageJ software using the following formula: (area of wound at 0 h - area of wound at &#34;n&#34; h) / area of wound at 0 h x 100. The error bar indicates the standard deviation from at least three independent experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56248/56248fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56248/56248fig5.jpg" /> 
Figure 5: Live-cell imaging videos show the tracking of single-cell movement. The colored lines show the migratory track of each selected isolated single cell. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56248/Figure_5.zip">Please click here to download these videos.</a>

Watch this Scientific Journal Video about Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60 at JoVE.com

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

live-imaging-breast-epithelial-cell-migration-after-transient

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.8K Views.  National University of Singapore. Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time. 

Video: Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Using the Dot Assay to Analyze Migration of Cell Sheets

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

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

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

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...