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 Using 50% Tissue-culture Infectious Dose (TCID50)
<ol>
	<li>Prepare 1 x 106 HEK293A cells in 10 mL of DMEM + 10% FBS media.</li>
	<li>Seed 100 &#181;L of cell suspension into each well of a 96-well flat-bottom cell culture plate.</li>
	<li>Prepare 1 mL of a 1:100 dilution of virus stock in complete medium. Add 11 &#181;L of diluted virus to each well of column 1.</li>
	<li>Perform serial 10-fold dilutions directly in the 96 well plate by mixing 11 &#181;L of diluted virus with 100 &#181;L of cell suspension until a final dilution of 1 x1012 is reached. Although cells are non-adherent at this stage, the number of cells transferred between wells remains constant.</li>
	<li>Pipette 11 &#181;L of each dilution into each column starting with the highest dilution of 104. Replace pipette tips with each dilution to limit cross-over of viral particles.</li>
	<li>Add 11 &#181;L of virus-free complete media to column 12 as a negative control.</li>
	<li>Incubate plate for 7 - 10 days at 37 &#176;C with 10% CO2.</li>
	<li>Using a microscope, score the number of wells in each column displaying signs of cytopathic or cytotoxic effects. Consider a well positive if any sign of infection is present.</li>
	<li>Calculate the fraction of positive wells for each dilution using the Reed and Muench (1938) statistical method14.</li>
	<li>Calculate the proportional distance (PD) using: (A-50)/(A-B), where A is the percentage response above or equal to 50% and B is the percentage response below 50%.</li>
	<li>Calculate TCID50 using: 10X-PD, where X is the dilution giving a response greater than 50%.</li>
	<li>Calculate viral titer from using the following: 0.69/(0.011 x TCID50) PFU/mL</li>
</ol>
2. Adenovirus MOI Determination
<ol>
	<li>Seed 1.0 - 1.5 x 105 MDA-MB-231 cells with 500 &#956;L of DMEM culture media containing 5% FBS in each well of a 12 well plate (35% cell confluency, 6 wells in total).</li>
	<li>Calculate the volumes of Ad-CMV-Td-Tom stock required for 0, 250, 500, 2500, and 5000 MOI using following formula: MOI = (volume [&#956;L] x PFU/&#956;L)/number of cells. For Ad-CMV-Td-Tom with a titer of 1 x 1010 (PFU/mL), the volumes of virus infection required MOIs are 0, 3.75, 7.5, 37.5, and 75 &#956;L respectively.</li>
	<li>Infect the cells with the calculated volumes of Ad-CMV-Td-Tom in Section 2.2 by directly pipetting viral stock into the wells with thorough mixing.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Remove the media from the wells and immediately fix the cells with 500 &#956;L of 10% formalin for 5 min. &#160;</li>
	<li>Aspirate the formalin from wells and wash wells with 500 &#956;L of PBS once.</li>
	<li>Stain the cell nucleus with 500 &#956;L of Hoechst solution for 5 min.</li>
	<li>Remove the Hoechst solution and wash the wells with 500 &#956;L of PBS 3 times.</li>
	<li>Image the Td-Tomato protein and cell nucleus signal using 200X magnification on a fluorescence microscope. Excite Td-Tomato protein at 554 nm and the Hoechst dye at 352 nm.</li>
	<li>Analyze images using ImageJ to determine the working MOI required for 100% infection of Td-Tomato15.</li>
</ol>
3. Dual-adenovirus Transduction in Live Single Cells
<ol>
	<li>Seed 1.0 - 1.5 x 105 MDA-MB-231 cells with 500 &#956;L of DMEM culture media containing 5% FBS in each well of a 12 well plate (35% cell confluency, 2 wells in total).</li>
	<li>Infect cells with 75 &#956;L of Ad-CMV-GFP (titer = 5 x 109 PFU/mL) and 7.5 &#956;L of Ad-CAGA12-Td-Tom (titer = 5 x 1010 PFU/mL) to obtain a 2,500 MOI, which can achieve 100% infection positivity.</li>
	<li>Treat cells with or without 0.25 &#956;L of TGF-&#946; per well (10 mg/mL in stock, 5 ng/&#956;L as the final concentration) immediately after adenovirus infection.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Image live cells with phase contrast fluorescence microscope. Excite GFP at 470 nm and Td-Tomato protein at 554 nm.</li>
	<li>Fix cells and stain cell nucleus as described in Steps 1.6 - 1.9.</li>
	<li>Image the Td-Tomato protein and Hoechst stain using a fluorescence microscope. Excite the Hoechst dye at 352 nm.</li>
	<li>Using ImageJ, calculate the percentage of GFP and Td-Tomato positive cells15.</li>
</ol>
4. Live Single Cell Signaling in Wound Healing Assay
<ol>
	<li>Seed 4 - 5 x 105 MDA-MB-231 cells with 1 mL of DMEM culture media containing 5% FBS in each well of a 6 well plate (50% cell confluency, 2 wells in total).</li>
	<li>Infect the cells with 22.5 &#956;L of Ad-CAGA12-Td-Tom (titer = 5 x 1010 PFU/mL, MOI = 2,500).</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Scratch two crossed lines on the cell layer of each well using a P200 pipette tip.</li>
	<li>Remove the old media from wells and replace with 2 mL of fresh 5% FBS media with or without 0.5 &#956;L of TGF-&#946; per well (10 mg/mL in stock, 5 ng/&#956;L as final concentration).</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 5 min and take images of selected wound areas using 100X magnification on a phase contrast microscope.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Take images of the wound closure at the same areas as before.</li>
	<li>Fix the cells, stain cell nucleus as described in Steps 1.6 - 1.9 and visualize Td-Tomato signal in the wound area and non-wound area using 200X magnification on a fluorescence microscope.</li>
	<li>Quantify the percentage of Td-Tomato positive cells in the wound and non-wound area using ImageJ software15.</li>
</ol>
5. In Vitro Luciferase Assay
<ol>
	<li>Prepare a cell suspension of 3 - 5 x 104 MDA-MB-231 cells in 1 mL of DMEM media containing 5% FBS.</li>
	<li>Infect cell suspension with 2.5 &#956;L of Ad-CAGA12-Luc (titer = 5 x 1010 PFU/mL, MOI = 2,500).</li>
	<li>Seed the infected cells into a 96 well plate at 3,000 cells/100 &#181;L/well.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Remove old media from wells and replace with 100 &#181;L of fresh 5% FBS media with or without 0.1 &#956;L of TGF-&#946; per well (1 mg/mL in stock, 1 ng/&#956;L as the final concentration) and in the presence or absence of 0.17 &#956;L of TGF-&#946; inhibitor per well (7 mg/mL in stock, 12 ng/&#956;L as final concentration) (a novel TGF-&#946; ligand trap protein, Chen et al., unpublished work).</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Thaw out the luciferase assay system substrate and prepare 1x cell culture lysis reagent in double distilled water (DDW).</li>
	<li>Remove media from the 96 wells and lyse wells with 50 &#956;L of 1x cell culture lysis buffer on ice using an orbital shaker with gentle agitation for 30 min.</li>
	<li>Transfer 30 &#956;L of lysate from each well into an opaque luciferase plate and allow the plate to warm to room temperature.</li>
	<li>On the luminometer software, create a new protocol. Select for 1 injector with measurements before and after injection. Set measurements before injection at 1 s for both Delay in Measurement and Integration Time. For after injection measurement, set the Injection Volume to 40 &#181;L with a Delay in Measurement of 1 s after injection and 5 s Integration Time. Confirm settings by selecting OK.</li>
	<li>Place the injector tube into DDW and click the Injector 1 button under the Prime settings to clean the injector tube before use. Next remove the injector tube from the DDW and place in air followed by two further Primes of injector 1 to flush all solution from the tube. Place the injector tube into Firefly substrate and again Prime twice to prepare the substrate.</li>
	<li>Place the opaque luciferase plate with the samples into the luminometer and select Options on the software. Highlight the wells of interest and click Apply. Press Start to measure luciferase signal.</li>
	<li>Once measurements are complete, remove and rinse the plate with water. Recover any remaining substrate into the injector tube by placing the tube into air and Prime injector 1 back into firefly substrate tube. Place the injector tube into DDW and perform two further Primes to clean the injector tube of any remaining substrate.</li>
</ol>
6. Cell Preparation for Live Signaling Imaging In Vivo
<ol>
	<li>48 h before tumor implantation, seed 2 - 3x106 MDA-MB-231 cells into 175 cm2 flasks (10 flasks in total). Culture cells in 20 mL of DMEM media containing 5% FBS at 37 &#176;C, 10% CO2.</li>
	<li>24 h later, add 300 &#956;L of Ad-CAGA12-Luc (titer = 5 x 1010 PFU/mL, MOI = 2,500) into each flask. Keep cells in culture for another 24 h.</li>
	<li>On the day of implantation, remove the old media and wash the adenovirus infected cells once with 20 mL of PBS, pH 7.4. Add 2 mL of 0.05% trypsin-EDTA into the flask and shake the flask slightly to allow trypsin to cover all the flask surface. Place the flask in to the incubator 37 &#176;C, 10% CO2 for 3 min.</li>
	<li>Quench the trypsin by adding 8 mL of FBS-containing DMEM media into flasks. Transfer all cell suspensions into a glass reagent bottle.</li>
	<li>Count the cell number using a hemocytometer and transfer 4.8 x 107 cells in two 50ml centrifuge tubes.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at RT to remove trypsin and resuspend cells in 720 mL of fresh FBS-DMEM media.</li>
	<li>Transfer the cell suspensions into a sterile 5 mL tube and add 160&#160;&#956;L Matrigel (10% total matrigel suspension).</li>
	<li>Keep cells on ice until implantation.</li>
</ol>
7. Orthotopic Implantation
<ol>
	<li>Weigh 12 SCID mice and randomly allocate mice into control and treatment groups (6 mice/group).</li>
	<li>Perform the implantation in a sterile bio-cabinet to maintain a sterile environment. Anaesthetize the mouse using 2.5 -&#160;4.5% isoflurane with an oxygen flow rate of 1 L/min. Place the mouse on a heat pad and dispense a drop of lubricating eye ointment onto both eyes to avoid corneal damage. Fix the mouse to the heat pad in a supine position while maintaining anaesthesia by placing the snout into a nose cone connected to the isoflurane.</li>
	<li>Confirm the success of anaesthesia by the lack of reaction to toe pinch. Reduce isoflurane to a maintenance dose of 1.5 -&#160;2.5% with 1 L/min oxygen for the reminder of implantation.</li>
	<li>Clean the skin from the fourth nipple on both sides to the midline by using the cotton swab dipped into 80% ethanol.</li>
	<li>Find the 4th mammary fat pad through palpation and squeeze the fat pad to further expose the tissue.</li>
	<li>Mix the cell suspension well by pipetting up and down. Gently aspirate 50 &#181;L of cell mixture into a 27G insulin syringe and inject into the mammary fat pad on both sides of the mouse.</li>
	<li>Confirm a successful injection by feeling for swelling of the fat pad. Release the fat pad gently and place the mouse back into the cage.</li>
	<li>Leave the cages on the heat pad for at least 30 min to allow the mice to gain consciousness and then place the cages back in the holding room for 3 days.</li>
</ol>
8. IVIS Imaging
<ol>
	<li>Open the Living Image software.</li>
	<li>Initialize the IVIS system by clicking the Initialize IVIS System button on the control panel. Wait until the temperature sign turns green.</li>
	<li>Select the Luminescent Imaging Mode on the control panel.</li>
	<li>Turn on isoflurane anaesthesia to the chamber and IVIS system.</li>
	<li>Anaesthetize the mice by using 2.5 -&#160;4.5% isoflurane with 1 L/min oxygen. Once anaesthetized, set isoflurane at 1.5 -&#160;2.5% for maintenance. Inject 150 mg/kg body weight of D-luciferin into mice by intraperitoneal (i.p.) injection.</li>
	<li>Immediately transfer mice into the IVIS system, place them onto the temperature-controlled imaging platform and place their nose into the nose cone.</li>
	<li>Click on the Sequence Setup button on the control panel and select Medium Binning.</li>
	<li>Set up the imaging exposure time as follows: 10 s, 20 s, 30 s, 60 s, and 120 s. Start imaging approximately 3 min after the luciferin injection and continue to image until the signal starts to drop (normally this will take around 20 min).</li>
	<li>Remove the mice from IVIS while maintaining isoflurane anaesthetic through a nose cone and treat the mice with 50 &#181;L PBS or 50 &#181;L TGF-&#946; signaling inhibitor (50 &#956;g/tumor) via intra-tumor injection.</li>
	<li>Put the mice back in the cage and leave them on the heat pad for at least 30 min. Then place the cage back in the holding room.</li>
	<li>Repeat IVIS imaging procedure as described above the day following treatment.</li>
</ol>
9. Data Analysis
<ol>
	<li>Open saved files in Living Image software.</li>
	<li>Find the image information using View | Image Information.</li>
	<li>Select photos taken at a similar time point with the same exposure time. Load photos as a group.</li>
	<li>Uncheck the Individual box in Image Adjust to normalize the intensity.</li>
	<li>Select Photon mode to analyze the intensity. Quantify the intensity of the signal by selecting the Region of Interest (ROI) button in ROI Tools.</li>
	<li>Drag the ROI circle to the region containing the bioluminescent signal and click the Measure button to acquire the signal intensity.</li>
</ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+adenovirus+vectors+with+modified+fibers+for+altering+viral+tropism.">Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.</a> Journal of Virology. 70, 6839-6846 (1996).</li><li>Wickham, T. J., 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=Increased+in+vitro+and+in+vivo+gene+transfer+by+adenovirus+vectors+containing+chimeric+fiber+proteins.">Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.</a> Journal of Virology. 71, 8221-8229 (1997).</li><li>Smith, F., Jacoby, D., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+vectors+for+gene+delivery+to+the+nervous+system.">Virus vectors for gene delivery to the nervous system.</a> Restorative neurology and neuroscience. 8, 21-34 (1995).</li><li>Zhou, F., 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=Nuclear+receptor+NR4A1+promotes+breast+cancer+invasion+and+metastasis+by+activating+TGF-beta+signalling.">Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling.</a> Nature communications. 5, 3388 (2014).</li></ol>

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-&#946;/Smad3 signaling within the tumor population and highlighting the importance of single cell biology. Additionally, alternate methods of single cell imaging such as nuclear translocation can be time consuming and expensive if performed in live cells and may not always translate to gene transcription16,17. While these methods are important to examine the upstream pathways of signal transduction, the adenovirus reporter system provides a unique perspective on TGF-&#946;/Smad3 signaling in individual cells in relation to biological function.
Significantly, our method provides a sensitive and reproducible method for in vivo detection of the TGF-&#946;/Smad3 signaling pathway in real-time during tumor progression and treatment. Similar to traditional stable luciferase in vivo imaging, detection of TGF-&#946;/Smad3 signaling within the mouse is dependent on the number of cells and level of promoter activity18. While subcutaneous tissue provides little interference for analysis, increased depth of cells will reduce the sensitivity of signal detection. It is possible to expand the method for use in other organs, such as lung and bone, and other cancers; however, signal optimization or ex vivo imaging is recommended to achieve the sensitivity required for the cancer model of interest.
An additional benefit of using the adenovirus reporter system is that its application is likely to expand to analysis of multiple signaling pathways simultaneously in live cells. In Figure 2A, we demonstrated that MDA-MB-231 cells could be transduced with 2 separate adenovirus vectors, CMV-GFP and CAGA-TOM. This dual-infection system can be further expanded to report two different signaling pathways via both fluorescence reporter proteins and luciferase reporters (using Firefly and Guassia luciferase reporter proteins). Our lab has achieved simultaneous signaling of BMP/Smad1 and TGF-&#946;/Smad3 signaling pathways in a number of live cancer cell lines (data not shown).
Adenovirus reporter systems provide an easy and convenient method for live cell imaging of the TGF-&#946;/Smad signaling pathway both in vitro and in vivo. This system has distinct advantages over other commonly used reporter systems. Firstly, adenovirus vectors are reported to have among the highest viral transduction efficacy, making them an optimal system for cell lines that are difficult to transduce19,20,21,22. Additionally with current advancements in viral technologies, &#39;gutless&#39; adenovirus vectors can be generated by removing most of the viral genome allowing for increased exogenous DNA capacity23. In comparison to non-viral delivery systems, adenovirus vectors represent a cost-effective, quick and easy application for cell transduction that results in far greater delivery efficiency19,20,21,22.
This protocol uses a second-generation adenovirus expression system, which is replication-incompetent in mammalian cells that do not express the E1a and E1b proteins, minimizing biosafety risk24. However, despite these enhanced biosafety features, adenovirus particles can still transduce primary human cells with high efficacy25,26. US Biosafety Level 2 and containment of adenoviral vectors is recommended when handling and disposing of adenovirus contaminated equipment. Large-scale amplification of adenovirus in HEK293A cells can result in a rare generation of replication-competent &#34;wild-type&#34; adenovirus27. PCR screening should be performed to identify wild-type contamination27.
Replication-incompetent adenovirus vectors are transient in expression and do not integrate with host DNA20,21. It is recommended that the experimenter identify the stability in expression of their reporter protein when using the adenovirus reporter system to confirm the validity of long-term experiments. The expression time of the adenovirus vector within a cell is proportional to the division time of the cell and viral MOI. To ensure reproducible results, it is critical that the viral titer is accurately determined for each viral batch. Additionally, excessively high volumes of the adenovirus can result in cytotoxic or cytopathic effects and it is important that an optimal MOI is empirically determined for each cell lined used to ensure best results. Further, in vivo use of adenovirus vectors can induce immune responses; where immune response is not the primary observation of the experiment, immune-compromised animals are recommended20,21. Additional quality control is required when using the adenoviral vectors to target immune responses, though the use of the adenovirus has been validated as an adjuvant to vaccine therapies28,29.
The adenovirus reporter system can be used on a broad range of primary and cultured cell lines of both dividing and non-dividing nature20,22. Using the adenovirus reporter system, we have achieved 100% infection rates in a number of cancer cell lines including brain, breast, colorectal as well Primary Mouse Embryological Fibroblast (MEF) and Canine Kidney Epithelial (MDCK) cells. Additionally, adenoviral vectors can be further modified to increase the affinity for cell-type specific receptors, providing improved organ targeting and efficacy for transduction30,31.
Applications of this method can be expanded to other signaling pathways and cell hosts. The principal advantages of the adenoviral reporter system include the cheap,high transduction efficacy, quick experimental setup and lack of integration with host DNA21,32. This method can be applied to many current assays and in vivo models, including in the evaluation of new targeted therapeutics33. We hope that the use of this technique will enhance our understanding of the relationship between signaling pathways and biological processes leading to new biological discoveries and the development of new therapies.

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 &amp;deg;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-&amp;beta;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 &amp;mu;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>&lt;strong&gt;Solution&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td></td><td></td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O (4 mM); NaHPO&lt;sub&gt;4 &lt;/sub&gt;(16 mM); NaCl (0.12M)</td></tr><tr><td>FBS-DMEM&amp;nbsp;</td><td></td><td></td><td>5% heat inactivated FBS; 10 &amp;mu;g/mL penicillin; 100 &amp;mu;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 active Ad-CMV-Td-Tom reporter that acted as a marker of positive infection. The percentage of adenovirus infected cells was increased in a MOI dependent manner from 0 to 5,000 MOI. At 2,500 MOI, we observed 100% Td-Tom expression in MDA-MB-231 cells (Figure 1A, 1B). Pixel intensity/cell also increased with MOI, providing enhanced sensitivity and detection of transcriptional output (Figure 1C). Therefore, a working MOI of 2,500 was used to perform a dual infection where cells were infected with Ad-CMV-GFP (served as positive control for cell infection) and Ad-CAGA12-Td-Tom simultaneously. Single cells presented varying levels of Td-Tom expression indicating heterogenous activation of TGF-&#946;/Smad3 transcription across cells in both live and fixed cells. (Figure 2A, 2B). 100% of MDA-MB-231 cells presented with GFP expression while only around 26% of cells displayed detectable TGF-&#946;/Smad3 transcriptional activity 24 h after TGF-&#946; stimulation, compared to 0% positivity without TGF-&#946; treatment (Figure 2C). To use the adenovirus based reagent to investigate the TGF-&#946;/Smad3 transcriptional activity during biological processes, MDA-MB-231 cells were infected with Ad-CAGA12-Td-Tom at 2500 MOI and subjected to a wound healing assay. Infected MDA-MB-231 cells exhibited normal migration behavior and cells treated with 5 ng/mL TGF-&#946; showed enhanced wound closure compared to non-TGF-&#946; treated cells (Figure 3A). Notably, about 62% cells in the wound area presented positive TGF-&#946;/Smad3 transcriptional activity, which is significantly higher than observed in non-wound area (32%) after TGF-&#946; treatment (Figure3B). As such, the adenovirus-based reagents validated their application for imaging of the TGF-&#946; signaling pathway in live single cell in vitro.
Live TGF-&#946; signaling imaging in vivo
The sensitivity of the Ad-CAGA12- Luc reporter was first tested in vitro in response to TGF-&#946; stimulation and the presence of a TGF-&#946; inhibitor. When infected MDA-MB-231 cells were stimulated with 1 ng/mL TGF-&#946;, luciferase reporter activity increased 1,200-fold compared to basal untreated MDA-MB-231 levels. This response was remarkably blocked by 12 &#181;g/mL of TGF-&#946; inhibitor (Figure 4A). The Ad-CAGA12-Luc reagent was subsequently tested in a breast tumor orthotopic implantation animal model. Both control and the TGF-&#946; inhibitor treated groups demonstrated similar 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-&#946; inhibitor showed around 3-fold signal reduction (Figure 4B, 4C). Collectively, these results demonstrate the potential for live and real-time monitoring of TGF-&#946; signaling activity in vivo without the need for animal sacrifice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57926/57926fig1v2.jpg" /> 
Figure 1. Determination of the MOI of adenovirus-based reagent. MDA-MB-231 cells were infected with Ad-CMV-Td-Tom at different MOI and seeded into a 12 wells plate. 24 h after infection, cells were fixed and nuclear stained by Hoechst. (A) Images were taken to visualize Td-Tom positive cells (red) and nucleus (blue) and quantified by ImageJ. (B) Percentage of Td-Tom positive cells. (C) Td-Tom pixel intensity/cell normalized to background. These figures are representative of at least 3 independent experiments. Data shown are means of triplicates &#177; SD. Scale bar = 50 &#181;m (200X). <a href="/files/ftp_upload/57926/57926fig1v2large.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/57926/57926fig2.jpg" /> 
Figure 2. Single cell imaging for dual infection. MDA-MB-231 cells were dual infected with Ad-CMV-GFP and Ad-CAGA12-Td-Tom at MOI (2,500) and seeded into a 12 wells plate. 24 h after infection, cells were treated with or without 5 ng/mL TGF-&#946;. (A) 24 h after treatment, cells were live imaged to visualize GFP positive (green) and Td-Tom positive (red) cells. Cells were then fixed and nuclear stained by Hoechst for quantification. (B) Images were taken to visualize GFP positive (green), Td-Tom positive (red) cells and nucleus (blue). (C) The GFP or Td-Tom positive cells were quantified by ImageJ and the percentage of positive cells was calculated. These figures are representative of at least 3 independent experiments. Data shown are means of triplicates &#177; SD. Statistical significance was determined by Student&#39;s t-test (**** p &#8804;0.0001). Scale bar = 50 &#181;m (200X). <a href="/files/ftp_upload/57926/57926fig2large.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/57926/57926fig3.jpg" /> 
Figure 3. Live cell TGF-&#946; signaling promotes cell migration. MDA-MB-231 cells were infected with Ad-CAGA12-Td-Tom at MOI (2,500) and seeded onto a 6 wells plate. 24 h after infection, cells were scratched using a P200 pipette tip to create a wound and media replaced with or without 5 ng/mL TGF-&#946;. After another 24 h, cells were fixed and nuclear stained by Hoechst for visual representation. (A) Phase contrast of wound closure, Scale bar = 100 &#181;m (100X). (B) Td-Tom positive (red) cells and nucleus (blue). Scale bar = 50 &#181;m (200X) (C) Td-Tom positive cells were quantified by ImageJ and the percentage of positive cells was calculated. These figures are representative of at least 3 independent experiments. Data shown are means of triplicates &#177; SD. Statistical significance was determined by Student&#39;s t-test (**** p &#8804;0.0001). <a href="/files/ftp_upload/57926/57926fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57926/57926fig4.jpg" /> 
Figure 4. In vivo live TGF-&#946; signaling imaging. (A) MDA-MB-231 cells were infected with Ad-CAGA12-Luc and then seeded into 96 wells plate for 24 h. Thereafter, cells were treated with or without 1 ng/mL TGF-&#946; in the presence or absence of 12 &#181;g/mL TGF-&#946; inhibitor overnight. The luciferase activity was measured based on the whole cell lysate. Luciferase activities were presented as the fold of induction normalized to no treatment control. Data shown are means of triplicates &#177; SD. (B) MDA-MB-231 cells were infected with Ad-CAGA12-Luc 24 h before implantation. Cells were implanted into mammary fat pad on both sides of SCID mice. 72 h later, IVIS imaging was performed before and after 24 h treatment (PBS for control group and 50 &#181;g/tumor TGF-&#946; inhibitor for treatment group). (C) Photon flux was quantified by Living Image software. Data shown are means of each treatment group (n = 12) &#177; SD. Statistical significance was determined by Student&#39;s t-test (** p&#160;&#8804;0.01, **** p &#8804;0.0001). p/s refers to photons/s. <a href="/files/ftp_upload/57926/57926fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo 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.

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

live-cell-imaging-tgf-smad3-signaling-pathway-vitro-vivo-using-an

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.3K Views.  University of Melbourne. Hier presenteren we een protocol voor levende cellen beeldvorming van de signalering activiteit TGF-β/Smad3 met behulp van een adenovirus verslaggever systeem. Dit systeem transcriptionele activiteit in real time worden bijgehouden en kunnen worden toegepast op beide afzonderlijke cellen in vitro en in levende diermodellen.

Video: Live Cell Imaging of the TGF-	β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicine

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Immunology and Infection

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

Biochemistry

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Biology

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioengineering

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

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

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