This work was supported by grants from the National Health and Medical Research Council (NHMRC) to H-JZ. TMBW is a recipient of an Australia Postgraduate Award from the Australian Government and the Ann Henderson Top-Up Scholarship from Australian Rotary Health in partner with Rotary of Templestowe and Dine for a Cure.

All animal experiments were approved by the University of Melbourne Animal Ethics Committee.
Note: The sequence, construction and generation protocol for the adenoviral vectors pAd-CMV-Td-Tom, pAd-CMV-GFP and pAd-CAGA12-Luc/Td-Tom have been previously described11,12,13. All vectors are commercially available.
1. Virus Titer Determination ...

<ol>
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The authors report no conflicts of interest.

We have developed a technique to allow for real-time imaging of TGF-&#946;/Smad3 signaling in single live cells. Using this novel method, we have previously identified a sub-population of cells with dynamic TGF-&#946;/Smad3 transcriptional activity that was associated with enhanced invasion and migration8. This method improves on traditional assays for TGF-&#946; signaling, such as western blots of Smad3 phosphorylation and TGF-&#946; targeted gene expression, by capturing the heterogeneity of TGF...

Transforming Growth Factor &#946; (TGF-&#946;) is an essential cytokine implicated in human development that signals through a heterodimeric complex consisting of type II and type I receptors1. Binding to type II receptor results in the recruitment and phosphorylation of type I receptor, which in turn phosphorylates downstream Smad2/3 proteins2,3. These activated Smad2/3 proteins bind to Smad4, forming a complex that translocates into the nucleus and regulates gene transcription4. Under homeostatic conditions TGF-&#946;/Smad signaling is tightly regulated; however, in many diseases, the signaling pathway is deregulated and often overexpressed leading to progression of disease5,6,7. Recent studies have demonstrated that cellular response to TGF-&#946; is heterogenous and subpopulations of TGF-&#946;/Smad active cells are responsible for biological function in a time-dependent manner8,9. Common cellular analysis of TGF-&#946;/Smad signaling involves the use of fixed endpoint assays that provide only a snapshot of cellular activity and often quantitate the average TGF-&#946;/Smad effect10. These methods, however, may not accurately represent the molecular behaviors of TGF-&#946;/Smad signaling in the physiological state during disease progression. Image-based analysis of live cells capture the dynamics of cellular and biological processes with both a spatial and temporal understanding.
Our goal was to develop a sensitive high-throughput method for live cell imaging of TGF-&#946;/Smad signaling using adenovirus-based reagents. Here, we infected the human breast cancer cell line MDA-MB-231 with an adenovirus expressing the Smad3 CAGA motif binding sequence and a luciferase (Luc) or Td-Tomato (Td-Tom) reporter gene. Adenoviral reporter systems provide a quick and cheap method for plasmid introduction that can result in a 100% infection rate in cancer cell lines. Adenoviral reporter systems have also been successfully applied to cell lines that are difficult to transfect with conventional plasmid11. In this protocol we will describe a reproducible and noninvasive process to achieve live cell imaging of the TGF&#946;/Smad signaling pathway both in vivo and in vitro.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>1881024</td>
			<td>Warm in 37 &#176;C waterbath before use</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Scientifix Life</td>
			<td>FBS500-S</td>
			<td>Heat inactivated before use</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-&#946;1</td>
			<td>PEPROTECH</td>
			<td>100-21</td>
			<td>Aliquot in DDW to make final concentration at 10 mg/mL</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Tocris Bioscience</td>
			<td>5117</td>
			<td>Dilute in to PBS to make final concentration at 1 &#956;L/mL</td>
		</tr>
		<tr>
			<td>Luciferase Reporter Assay Kit</td>
			<td>Promega</td>
			<td>197897</td>
			<td>Dilute 5x in PBS before use</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>9100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast fluorescence microscopy</td>
			<td>OLYMPUS</td>
			<td>IX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGl Luciferin</td>
			<td>Promega</td>
			<td>P1041</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10x)</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td>Diltue to 0.05% (1x) in PBS</td>
		</tr>
		<tr>
			<td>Cell Culture Lysis 5x Reagent</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute to 1x in DDW</td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>F5554-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293A</td>
			<td>ThermoFisher</td>
			<td>R70507</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>PRKDC-SCID</td>
			<td>Animal Resources Centre</td>
			<td>SCIDF6</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Chem-supply</td>
			<td>EA043-10L-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Refresh Night Time</td>
			<td>Allergan</td>
			<td>1750D</td>
			<td>Lubricating Eye Ointment</td>
		</tr>
		<tr>
			<td>Solution</td>
			<td></td>
			<td></td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>NaH2PO4.2H2O (4 mM); NaHPO4 (16 mM); NaCl (0.12M)</td>
		</tr>
		<tr>
			<td>FBS-DMEM&#160;</td>
			<td></td>
			<td></td>
			<td>5% heat inactivated FBS; 10 &#956;g/mL penicillin; 100 &#956;g/mL streptomycin</td>
		</tr>
	</tbody>
</table>

live cell imaging of the tgf-	&beta;/smad3 signaling pathway in vitro and in vivo using an adenovirus reporter system

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

Live single cell imaging in vitro
To accurately assess the activation of TGF-&#946;/Smad signaling in single cells using adenovirus based reagents, it is important to first determine the optimal Multiplicity of Infection (MOI) for each cell line. The optimal MOI is determined when 100% of cells are positive for adenoviral infection with no present cytopathic or cytotoxic effects. To determine this, we used a constitutively...

Watch this Scientific Journal Video about Live Cell Imaging of the TGF-	Î²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System at JoVE.com

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

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

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

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

This method can hep answer key questions in the cell signaling field by noninvasively tracking cell signaling in real time. The main advantage of this technique is that adenovirus systems provide a quick and easy method for cell signaling detection that can achieve a 100%infection rate in cancer cell lines. The implications of this technique extend toward therapeutic development for cancers and can be expanded to other signaling pathways.Though this method can provide insight into breast cancer models, it can also be applied to other cancer types, such as brain, bone, and lung. On the day of adenovirus infection, seed the wells of a 12-well plate with approximately 1.25 times 10 to the fifth MDA-MB-231 cells in 500 microliters of medium for a confluency of 35%Infect cells in all wells with 75 microliters of adenovirus-CMV-GFP and 7.5 microliters of adenovirus CAGA12-Td-Tomato to obtain a 2, 500 MOI which can achieve 100%infection positivity. Then add TGF-beta to the treatment well immediately after adenovirus infection.Incubate at 37 degrees Celsius with 10%carbon dioxide for 24 hours. The next day, image live cells with a phase-contrast fluorescence microscope. Then after fixing the cells and staining nuclei with Hoechst dye, image the TD-Tomato protein and Hoechst stain using a fluorescence microscope.After imaging all wells, calculate the percentage of GFP and Td-Tomato positive cells using ImageJ. For the wound healing assay, begin by seeding the wells of a six-well plate with approximately 4.5 times 10 to the fifth MDA-MB-231 cells in one milliliter of medium for 50%confluency on the day of infection. Infect the cells with 22.5 microliters of adenovirus CAGA12-Td-Tomato, then place the plate in the incubator at 37 degrees Celsius with 10%carbon dioxide for 24 hours.After 24 hours, use a P200 pipette tip to scratch two crossed lines on the cell layer of each well. Remove the medium from the wells and treat with TGF-beta as previously shown, then place the plate in the incubator at 37 degrees Celsius with 10%carbon dioxide for five minutes. After five minutes of incubation, take images of selected wound areas using 100X magnification on a phase-contrast microscope before returning the plate to the incubator.24 hours later, take phase-contrast images of the same areas to assess wound closure. After fixing the cells and staining nuclei, visualize the Td-Tomato signal in the wound area and non-wound area using 200X magnification on a fluorescence microscope. 48 hours before tumor implantation, seed approximately 2.5 times 10 to the sixth MDA-MB-231 cells into 10 175-square-centimeter flasks.After 24 hours in culture, add 300 microliters of adenovirus CAGA12-luciferase into each flask before returning the cells to the incubator for another 24 hours. On the day of implantation, remove the old media and wash the adenovirus-infected cells once with 20 milliliters of PBS. Add two milliliters of 0.05%Trypsin-EDTA into the flask and shake the flask slightly to allow Trypsin to cover all the flask surface.Incubate at 37 degrees Celsius for three minutes, then quench the Trypsin by adding eight milliliters of FBS-containing DMEM media into the flasks. Transfer all cell suspensions into a glass reagent bottle. Count the cell number using a hemocytometer.After this, transfer 4.8 times 10 to the seven cells into 250-milliliter centrifuge tubes and centrifuge the cells at 400 times g for five minutes at room temperature. Following the centrifugation, re-suspend the cells in 720 microliters of fresh medium and transfer the suspension to a five-milliliter tube. Then add medium containing 10%Matrigel to dilute the cell suspension to three times 10 to the sixth cells per 50 microliters.Transfer the cell suspension into a sterile five-milliliter tube and keep on ice until implantation. Weigh 12 SCID mice and randomly allocate mice equally between control and treatment groups. After anesthetizing the mouse, dispense a drop of lubricating eye ointment onto both eyes to avoid corneal damage, then fix the mouse to a heat pad in a sterile bio-cabinet and maintain anesthesia by placing the snout into a nosecone connected to the isoflurane.Confirm the success of anesthesia by the lack of reaction to a toe pinch. Reduce the isoflurane to a maintenance dose for the remainder of the implantation. Next, use a cotton swab dipped into 80%ethanol to clean the skin from each fourth nipple to the midline.Find the fourth mammary fat pad through palpation and squeeze the fat pad to further expose the tissue, then mix the cell suspension by pipetting up and down. Gently aspirate 50 microliters of cell mixture into a 27-gauge insulin syringe and inject into the mammary fat pad. Confirm a successful injection and gently release the fat pad.Place the mouse back into the home cage. After the mice have regained consciousness, return the cage to the holding room for three days before proceeding to IVIS imaging. Live MDA-MB-231 cells presented varied levels of Td-Tomato expression despite 100%infection as shown by GFP expression, which indicates heterogeneous activation of TGF-beta-dependent Smad3 transcription across cells.Analysis of fixed cells again showed 100%GFP expression while only around 26%of cells displayed detectable TGF-beta/Smad3 transcriptional activity 24 hours after TGF-beta stimulation without TGF-beta compared to 0%positivity treatment. In the wound healing assay, MDA-MB-231 cells infected with adenovirus CAGA12-Td-Tomato exhibited normal migration behavior and cells treated with TGF-beta showed enhanced wound closure. Notably, about 62%of cells in the wound area presented positive TGF-beta-dependent Smad3 transcriptional activity, which is significantly higher than observed in the non-wounded area after TGF-beta treatment.The adenovirus CAGA12-luciferase reagent was tested in a breast tumor orthotopic implantation animal model. Both the control and TGF-beta-inhibitor-treated groups demonstrated similar CAGA12-luciferase reporter activity before treatment. However, for the control group, the luciferase signal did not change after PBS treatment, whereas mice treated with the TGF-beta inhibitor showed around threefold signal reduction.Once mastered, this technique can be done in less than one hour if it is performed properly. While attempting this procedure, it's important to remember that excessively high volumes of adenovirus can have cytotoxic and cytopathic effects which may affect the accuracy of your results. Following this procedure, this system can be expanded to analyze multiple signaling pathways simultaneously in order to answer additional questions related to cell signaling cross talk.After its development, this technique paved the way for researchers in the field of cancer cell signaling to explore tumor progression behavior in breast cancer models.

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10.3K Görüntüleme.  University of Melbourne. This method can hep answer key questions in the cell signaling field by noninvasively tracking cell signaling in real time. The main advantage of this technique is that adenovirus systems provide a quick and easy method for cell signaling detection that can achieve a 100%infection rate in cancer cell lines. The implications of this technique extend toward therapeutic development for cancers and can be expanded to other signaling pathways.Though this method can provide insight into brea...