We thank Dr. Maria Reichenbach and Dr. Christian Hiepen for their support on the flow-set up system and Eleanor Fox and Yunyun Xiao for critically reading the manuscript. P-L.M. was funded by the international Max Planck Research School IMPRS-Biology and Computation (IMPRS-BAC). PK received funding by the DFG-SFB1444. Figure 1 was created using BioRender.

1. Cell culture and fluid shear stress exposure
NOTE: Human umbilical vein ECs (HUVECs) were used as an example to study SS induced interaction of SMADs. The protocol described below can be applied to every SS responsive cell type.
<ol>
	<li>Coat 6-channel slide with 0.1% porcine skin gelatin in PBS for 30 min at 37 &#176;C.</li>
	<li>Seed HUVECs in pre-coated 6-channel slides at a density of 2.5 x 106 cells per mL in 30 &#181;L of M199 full medium. 
	NOTE: For further information on how to seed cells in the flow slide, see reference24.</li>
	<li>Let cells adhere for 1 h at 37 &#176;C in a humidified incubator.</li>
	<li>Add 60 &#181;L of pre-warmed M199 full medium to each of the reservoirs.</li>
	<li>Culture for 2 days, with a gentle medium exchange once a day, at 37 &#176;C in a humidified incubator.
	<ol>
		<li>Aspirate the reservoirs completely, add 120 &#181;L of pre-warmed M199 full medium in one of the reservoirs, and aspirate from the other side.</li>
		<li>Add 60 &#181;L of pre-warmed M199 full medium to both reservoirs.</li>
	</ol>
	</li>
	<li>Assemble and start the flow set-up as detailed in the reference25.
	<ol>
		<li>Mount tubing on fluidic units. Here, silicone tubing with a diameter of 0.8 mm and 1.6 mm are used to apply shear stress of 1 dyn/cm2 and 30 dyn/cm2, respectively. 
		NOTE: The material and tubing length should remain constant, as changes could influence the resulting shear stress. In general, other combinations of pump systems and tubing can be used, as long as the resulting shear stress is known, and the pump creates a steady laminar flow.</li>
		<li>Fill the reservoirs with an appropriate amount of pre-warmed M199 full medium (minimum 10 mL).</li>
		<li>Connect fluidic units with the tubing to the pump system and perform a pre-run without cells to equilibrate the medium and to remove any remaining air25.</li>
		<li>Serially connect the single channels on the 6-channel slide to one another by using serial connection tubing. The first and the last channel on the slide will be connected to the tubing assembled in 1.6.1 (see Figure 1A for a scheme). Be careful not to introduce any air into the system as this could severely harm the cells. Further information on the serial connection can be found in reference26.</li>
		<li>For the exposure of cells to high levels of shear stress (&#62;20 dyn/cm2), use a ramp phase, i.e., increase the shear stress stepwise with adaptation phases. Steps can be set in increments of 5 dyn/cm2 per 30 min.</li>
	</ol>
	</li>
</ol>
2. Fixation
<ol>
	<li>Detach slides from the pumps after the fluid SS exposure. Use clamps on the tubing when detaching, to avoid the medium spill in the incubator.</li>
	<li>Immediately transfer flow slides on ice, while the remaining tubing is detached sequentially. When removing the tubing from reservoirs, the reservoir on the other side should be kept closed with a finger to avoid trapping air bubbles in the channel. This might interfere with fixation steps.</li>
	<li>Keeping the cells on ice, aspirate the medium carefully from the reservoirs but not from the channel where the cells reside. Subsequently, wash samples with cold sterile PBS (4 &#176;C) with three times the channel volume (90 &#181;L). Add PBS in one reservoir and aspirate carefully from the other reservoir. Repeat this step in each of the 6 channels per slide. 
	NOTE: For all washing and incubation steps the respective solution is added in one of the reservoirs which leads to an exchange of solutions in the channel. To allow for complete substitution of solutions in the channel, the excess solution is then aspirated from the other reservoir. Solution on the top of the cells in the channel is not removed. Cells should not dry at any time. Therefore, it is important to wash carefully without any air bubble insertion into the slides.</li>
	<li>Fix the cells by adding 90 &#181;L of buffered 4% PFA solution in the same reservoir where the PBS was added beforehand and similarly aspirate the liquid from the other reservoir. Repeat this step in each channel in each slide. After the addition of PFA solution, transfer the samples from ice to room temperature (RT) and incubate for 20 min. 
	CAUTION: PFA is toxic and should be handled carefully. Use gloves and work under a fume hood.</li>
	<li>Wash cells 3x with PBS (RT) by adding it in one reservoir and aspirating carefully from the other reservoir. Empty just the reservoirs, ensuring not to dry out the channel. Repeat this step for each of the 6 channels per slide.</li>
	<li>Quench the PFA-fixation by adding 90 &#181;L of ambient 50 mM ammonium chloride in PBS in one of the reservoirs. Aspirate excess solution from the other reservoir. Repeat for each channel in the slide. Incubate the samples for 10 min at RT.</li>
	<li>Wash as described in step 2.5. 
	&#8203;NOTE: At this point, the samples may be stored at 4 &#176;C overnight, or the protocol can be immediately continued with blocking and primary antibody incubation (see step 3).</li>
</ol>
3. Blocking and primary antibody incubation
<ol>
	<li>To permeabilize the cells, add 90 &#181;L of 0.3% Triton-X-100 in PBS in an emptied reservoir, and aspirate from the other reservoir for each channel. Incubate for 10 min at RT.</li>
	<li>Wash as described in step 2.5.</li>
	<li>Add 90 &#181;L of sterile PLA blocking solution in one reservoir of a channel and aspirate from the other side. Repeat this step for each channel. Block for 1 h at 37 &#176;C in a humidified chamber.
	<ol>
		<li>To make a humidified chamber, use a 10 cm dish with wet tissue sealed with wax film and place the dish in the incubator. Alternatively, other humidity chamber formats can be used that supply a humid atmosphere. 
		NOTE: Alternatively, self-made blocking solution can be used (e.g., 3% (w/v) BSA in PBS, sterile filtered).</li>
	</ol>
	</li>
	<li>Prepare primary antibodies (1:100) in PLA antibody diluent. Prepare 30 &#181;L of the solution per channel. Add both primary antibodies simultaneously and vortex. 
	NOTE: Alternatively, self-made antibody diluent can be used (e.g., 1% (w/v) BSA in PBS). Antibodies used here are combinations of SMAD1-SMAD2/3, SMAD2/3-SMAD4 and phospho-SMAD1/5-SMAD4. Detailed information can be found in the Table of Materials.</li>
	<li>Before the application of primary antibodies, aspirate the blocking solution from the reservoirs and, also, carefully from the channel. Pipette 30 &#181;L of the primary antibody solution immediately into the empty channel by tilting the channel while adding the solution. 
	NOTE: Perform the removal of the blocking solution and addition of the antibody solution channel-by-channel to ensure cells do not dry out in between.</li>
	<li>Incubate samples with the primary antibodies overnight in humidified chambers at 4 &#176;C. 
	&#8203;NOTE: The incubation can also be performed for 1 h at room temperature, if interested in continuing with the following steps on the same day.</li>
</ol>
4. PLA probe incubation
NOTE: For all steps in section 4.1-7.3, the washing buffers A and B are stored at 4 &#176;C and need to be warmed to RT prior to the use.
<ol>
	<li>Dilute PLA-probes (+)-mouse and (-)-rabbit to 1:5 in PLA antibody diluent (or 1% BSA) solution. Prepare 30 &#181;L per channel.</li>
	<li>Wash samples 2x for 5 min using 90 &#181;L of the wash buffer A at RT by adding it in one of the reservoirs and aspirating carefully from the other reservoir. Repeat this step for each of the 6 channels per slide.</li>
	<li>Aspirate the wash buffer A carefully and add 30 &#181;L of PLA probe solution (prepared in step 4.1), similar to the addition of primary antibodies in step 3.5.</li>
	<li>Incubate samples for 1 h at 37 &#176;C in a humidified chamber.</li>
</ol>
5. Ligation
<ol>
	<li>Wash samples 2x for 5 min using 90 &#181;L of the wash buffer A at RT, as described in 4.2.</li>
	<li>Prepare a 1:5 dilution of the ligation buffer in deionized water. Use this buffer to dilute the ligase enzyme to 1:40 (on ice). Use 30 &#181;L per channel.</li>
	<li>Aspirate the wash buffer A completely and add the ligation solution as described in 3.5.</li>
	<li>Incubate samples for 30 min at 37 &#176;C in a humidified chamber.</li>
</ol>
6. Amplification
<ol>
	<li>Wash samples 2x for 2 min using 90 &#181;L of wash buffer A at RT, as described in 4.2.</li>
	<li>Prepare the amplification buffer by diluting it 1:5 in deionized water and use it to dilute the polymerase enzyme to 1:80 (on ice). Protect from light. Prepare 30 &#181;L per channel.</li>
	<li>Aspirate the wash buffer A completely and immediately add the prepared amplification solution into the empty channel, as described in 3.5. Incubate samples for 100 min at 37 &#176;C in a humidified chamber.</li>
</ol>
7. Mounting
<ol>
	<li>Wash samples 2x for 10 min using 90 &#181;L of Wash Buffer B at RT as described in 4.2. Add DAPI (1:500) from 1 mg/mL stock solution (in deionized water) in the first wash to stain nuclei. Do not dry the channel.</li>
	<li>Dilute the wash buffer B in deionized water (1:10) and wash 1x with 90 &#181;L of 0.1x buffer B solution as described in 4.2.</li>
	<li>Aspirate the wash buffer B completely and immediately add 2-3 drops of the liquid mounting medium into one reservoir. Distribute it in the channel by tilting the slide. Store samples at 4 &#176;C in a humidified environment until imaging.</li>
</ol>
8. Image acquisition
<ol>
	<li>Acquire images using a fluorescence microscope. Ensure that the respective filters fitting the fluorescent PLA probes are available. 
	NOTE: It is beneficial to make use of a confocal microscope, if possible, as obtained PLA spots are more defined. This also supports further image processing and data analysis.</li>
</ol>
9. Image analysis and quantification using ImageJ/FIJI
<ol>
	<li>Process exported images (.tiff) with an image processing program, such as ImageJ27. 
	NOTE: All scripts used within this study and that are necessary for the automatic counting of cellular, nuclear, and all PLA events (per cell) can be found in a GitHub repository: https://github.com/Habacef/Proximity-Ligation-Assay-analysis. Perform statistical analysis using any suitable program or tool.</li>
</ol>

<ol>
	<li>Yadin, D., Knaus, P., Mueller, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+BMP+receptors:+Specificity,+activation+and+inhibition.">Structural insights into BMP receptors: Specificity, activation and inhibition.</a> Cytokine and Growth Factor Reviews. 27, 13-34 (2016).</li><li>Sieber, C., Kopf, J., Hiepen, C., Knaus, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+BMP+receptor+signaling.">Recent advances in BMP receptor signaling.</a> Cytokine and Growth Factor Reviews. 20 (5-6), 343-355 (2009).</li><li>Hiepen, 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=BMPR2+acts+as+a+gatekeeper+to+protect+endothelial+cells+from+increased+TGF&#946;+responses+and+altered+cell+mechanics.">BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGF&#946; responses and altered cell mechanics.</a> PLoS Biology. 17 (12), 3000557 (2019).</li><li>Hildebrandt, 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=ActivinA+induced+SMAD1/5+Signaling+in+an+iPSC+derived+EC+model+of+Fibrodysplasia+Ossificans+Progressiva+(FOP)+can+be+rescued+by+the+drug+candidate+saracatinib.">ActivinA induced SMAD1/5 Signaling in an iPSC derived EC model of Fibrodysplasia Ossificans Progressiva (FOP) can be rescued by the drug candidate saracatinib.</a> Stem Cell Reviews and Reports. , (2021).</li><li>Goumans, M. 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=Balancing+the+activation+state+of+the+endothelium+via+two+distinct+TGF-beta+type+I+receptors.">Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors.</a> The EMBO Journal. 21 (7), 1743-1753 (2002).</li><li>Goumans, M. 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=Activin+receptor-like+kinase+(ALK)1+is+an+antagonistic+mediator+of+lateral+TGFbeta/ALK5+signaling.">Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling.</a> Molecular Cell. 12 (4), 817-828 (2003).</li><li>Daly, A. C., Randall, R. A., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta-induced+Smad1/5+phosphorylation+in+epithelial+cells+is+mediated+by+novel+receptor+complexes+and+is+essential+for+anchorage-independent+growth.">Transforming growth factor beta-induced Smad1/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorage-independent growth.</a> Molecular and Cellular Biology. 28 (22), 6889-6902 (2008).</li><li>Ramachandran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+uses+a+novel+mode+of+receptor+activation+to+phosphorylate+SMAD1/5+and+induce+epithelial-to-mesenchymal+transition.">TGF-&#946; uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition.</a> eLife. 7, 31756 (2018).</li><li>Flanders, K. 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=Brightfield+proximity+ligation+assay+reveals+both+canonical+and+mixed+transforming+growth+factor-&#946;/bone+morphogenetic+protein+Smad+signaling+complexes+in+tissue+sections.">Brightfield proximity ligation assay reveals both canonical and mixed transforming growth factor-&#946;/bone morphogenetic protein Smad signaling complexes in tissue sections.</a> The Journal of Histochemistry and Cytochemistry : The Official Journal of The Histochemistry Society. 62 (12), 846-863 (2014).</li><li>Miyazono, K., Maeda, S., Imamura, T., Dijke, P. T., Heldin, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Smad Signal Transduction: Smads in Proliferation, Differentiation and Disease. , 277-293 (2006).</li><li>Goumans, M. J., Zwijsen, A., Ten Dijke, P., Bailly, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+proteins+in+vascular+homeostasis+and+disease.">Bone morphogenetic proteins in vascular homeostasis and disease.</a> Cold Spring Harbor Perspectives in Biology. 10 (2), 031989 (2018).</li><li>Cai, J., Pardali, E., S&#225;nchez-Duffhues, G., ten Dijke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+in+vascular+diseases.">BMP signaling in vascular diseases.</a> FEBS Letters. 586 (14), 1993-2002 (2012).</li><li>Cunha, S. I., Magnusson, P. U., Dejana, E., Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulated+TGF-&#946;/BMP+signaling+in+vascular+malformations.">Deregulated TGF-&#946;/BMP signaling in vascular malformations.</a> Circulation research. 121 (8), 981-999 (2017).</li><li>MacCarrick, G., 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=Loeys-Dietz+syndrome:+a+primer+for+diagnosis+and+management.">Loeys-Dietz syndrome: a primer for diagnosis and management.</a> Genetics in Medicine : An Official Journal of the American College of Medical Genetics. 16 (8), 576-587 (2014).</li><li>Baeyens, N., Bandyopadhyay, C., Coon, B. G., Yun, S., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+fluid+shear+stress+sensing+in+vascular+health+and+disease.">Endothelial fluid shear stress sensing in vascular health and disease.</a> The Journal of Clinical Investigation. 126 (3), 821-828 (2016).</li><li>Min, E., 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=Activation+of+Smad+2/3+signaling+by+low+shear+stress+mediates+artery+inward+remodeling.">Activation of Smad 2/3 signaling by low shear stress mediates artery inward remodeling.</a> bioRxiv. , 691980 (2019).</li><li>Zhou, 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=BMP+receptor-integrin+interaction+mediates+responses+of+vascular+endothelial+Smad1/5+and+proliferation+to+disturbed+flow.">BMP receptor-integrin interaction mediates responses of vascular endothelial Smad1/5 and proliferation to disturbed flow.</a> Journal of Thrombosis and Haemostasis. 11 (4), 741-755 (2013).</li><li>Zhou, 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=Force-specific+activation+of+Smad1/5+regulates+vascular+endothelial+cell+cycle+progression+in+response+to+disturbed+flow.">Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (20), 7770-7775 (2012).</li><li>van Dijk, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+TGF-&#946;+and+BMP+signaling+in+human+atherosclerosis:+A+histological+evaluation+based+on+Smad+activation.">Visualizing TGF-&#946; and BMP signaling in human atherosclerosis: A histological evaluation based on Smad activation.</a> Histology and Histopathology. 27 (3), 387-396 (2012).</li><li>Derwall, 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=Inhibition+of+bone+morphogenetic+protein+signaling+reduces+vascular+calcification+and+atherosclerosis.">Inhibition of bone morphogenetic protein signaling reduces vascular calcification and atherosclerosis.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 32 (3), 613-622 (2012).</li><li>Fredriksson, 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=Protein+detection+using+proximity-dependent+DNA+ligation+assays.">Protein detection using proximity-dependent DNA ligation assays.</a> Nature Biotechnology. 20 (5), 473-477 (2002).</li><li>S&#246;derberg, O., 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=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nature Methods. 3 (12), 995-1000 (2006).</li><li>Alam, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+Ligation+Assay+(PLA).">Proximity Ligation Assay (PLA).</a> Current Protocols in Immunology. 123 (1), 58 (2018).</li><li>Application Note 03: Growing Cells in &#181;-Channels. ibidi Available from: <a target="_blank" href="https://ibidi.com/img/cms/support/AN/AN03_Growing_cells.pdf">https://ibidi.com/img/cms/support/AN/AN03_Growing_cells.pdf</a> (2012)</li><li>Application Note 13: HUVECs under perfusion. ibidi Available from: <a target="_blank" href="https://ibidi.com/img/cms/support/AN/AN13_HUVECs_under_perfusion.pdf">https://ibidi.com/img/cms/support/AN/AN13_HUVECs_under_perfusion.pdf</a> (2019)</li><li>ibidi. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+Note+31:+Instructions+&#181;-Slide+VI+0.4.">Application Note 31: Instructions &#181;-Slide VI 0.4.</a> ibidi. , (2013).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Reichenbach, 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=Differential+impact+of+fluid+shear+stress+and+YAP/TAZ+on+BMP/TGF-&#946;+induced+osteogenic+target+genes.">Differential impact of fluid shear stress and YAP/TAZ on BMP/TGF-&#946; induced osteogenic target genes.</a> Advanced Biology. 5 (2), 2000051 (2021).</li><li>Hiepen, C., Mendez, P. L., Knaus, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=It+takes+two+to+tango:+Endothelial+TGF&#946;/BMP+signaling+crosstalk+with+mechanobiology.">It takes two to tango: Endothelial TGF&#946;/BMP signaling crosstalk with mechanobiology.</a> Cells. 9 (9), 1965 (2020).</li></ol>

The authors declare no conflict of interest.

The PLA based protocol described here offers an efficient way to determine close proximity of two proteins (e.g., their direct interaction) in ECs exposed to shear stress with quantitative and spatial resolution. By using flow slides with multiple parallel channels, several different protein interactions can be examined at the same time in cells under identical mechanical conditions. In contrast, custom-build flow chamber systems often make use of a single channel that is built around a glass coverslip, which would allow only a single PLA experiment without the necessary controls per slide and pump. Although this protocol focuses on the detection of SMAD interactions, it can be adapted to detect any other protein interactions. However, analysis of results must be done carefully as two proteins may also reside in close proximity without forming complexes. If definite statements on interactions of proteins are desired, PLA experiments should be complemented with additional methods such as co-immunoprecipitations. Additionally, PLA cannot be used to detect the protein-protein interaction in live cells since samples need to be fixed for subsequent antibody binding and DNA amplification steps.
For successful detection of protein interactions by PLA, the most critical step is to choose a combination of primary antibodies that fulfil several criteria: (1) The antibodies detecting the individual protein-partners must be generated in different species (e.g., mouse or rabbit) as the secondary antibodies are species-specific; ideally, antibodies were already successfully tested by conventional immunofluorescence microscopy; (2) the distance between epitope bound antibodies should be &#60;40 nm23; (3) as the affinities for antibody-epitope binding might differ, the final concentration of used antibodies has to be adjusted for each experimental setting, as shown here (Figure 2A, lower panel versus Figure 2C, lower panel). Therefore, if very few but specific PLA events are detected, it may be worth increasing the amount of antibody used. However, this must be carefully titrated to avoid oversaturation and unspecific binding events. Also, antibody concentration used in control samples must match the concentration used in the PLA samples.
As for any other biochemical assay, suitable controls are indispensable in PLA. Unspecific binding of the used antibodies might, for example, lead to PLA signals originating from just one primary antibody. Therefore, essential antibody controls for PLA experiments should include addition of only one of the two primary antibodies but both PLA Probes (Plus and Minus). Furthermore, controls with no antibodies added can be used to determine unspecific binding of the PLA probes to the sample. In general, those technical controls should yield no to a very few PLA signals. If several signals are observed, concentration of the primary antibody used, and its specificity should be reconsidered. In addition, it is useful to include a positive biological control, if possible. In the protocol described above, this could be stimulation with a BMP ligand that is known to induce phosphorylation of SMADs and, therefore, trimeric complex formation with SMAD4.
For PLA experiments, it is normally recommended to use cells that are 50-70% confluent, since this simplifies the penetration of reagents. However, when performing experiments with ECs, we would strictly argue against this, except if semi-confluency is part of the experimental set-up. In vivo ECs form monolayers with tight cell-to-cell junctions, which are essential for EC homeostasis and mechano-transduction29. Therefore, experiments on non-confluent ECs could give rise to false results. Furthermore, non-confluent cells are more prone to detach from flow channel slides if high levels of shear stress are used during the experiment. We advise to seed cells (2-2.5 x 106 cells/ mL, see protocol step 1.2) two to three days in advance of experimental start as EC monolayers need time to form and develop mature junctions. Therefore, we would not recommend seeding a higher number of cells (&#62;2.5 x 106 cells/mL) in flow channels just one day before the experiment to achieve a confluent monolayer.
Although various liquid mounting media containing DAPI exist it is worth to include a separate DAPI staining step and mount the cells with liquid mounting medium without DAPI, at least if polymer-based flow slides are used. This prevents heavy background signals during image acquisition. Images should be acquired from positions in the channel center rather than the edges since shear stress is inhomogeneous at the channel walls. We recommend to take at least 5-10 images per biological replicate and condition of random areas along the center region. 3 or more biological replicates are normally used to gain statistical relevance. For image analysis we advise to use the ImageJ/FIJI macro function. In the protocol above, we mentioned an ImageJ macro that is suitable to count cytosolic and nuclear PLA events based on DAPI staining. Users should be aware that parameters like particle size need to be adjusted depending on nuclear size or bigger/smaller PLA dots. The macro saves the threshold PLA images and masks that should be compared to raw images to evaluate specificity of PLA signal detection.
In conclusion, the method presented here allows rapid and efficient spatial and quantitative analysis of transcription factor complex formation at atheroprotective and atheroprone SS conditions. It will allow scientists to further decipher the impact of SMAD complex formation in atherogenesis and vascular disease in general. It can, furthermore, be adapted to investigate the consequences of proximity of different proteins in those vascular pathologies.

Proteins of the transforming growth factors beta (TGF&#946;) superfamily are pleiotropic cytokines with a variety of members, including TGF&#946;s, bone morphogenetic proteins (BMPs), and Activins1,2. Ligand binding induces the formation of receptor oligomers leading to the phosphorylation and, thereby, activation of cytosolic regulatory SMAD (R-SMAD). Depending on the sub-family of ligands, different R-SMADs are activated1,2. While TGF&#946;s and Activins mainly induce phosphorylation of SMAD2/3, BMPs induce SMAD1/5/8 phosphorylation. However, there are accumulating evidences that BMPs and TGF&#946;s/Activins also activate R-SMADs of the respective other sub-family, in a process termed as &#39;lateral signaling&#39;3,4,5,6,7,8 and that there are mixed SMAD complexes consisting of both, SMAD1/5 and SMAD2/3, members3,9. Two activated R-SMADs subsequently form trimeric complexes with the common mediator SMAD4. These transcription factor complexes are then able to translocate into the nucleus and regulate the transcription of target genes. SMADs can interact with a variety of different transcriptional co-activators and co-repressors, leading to the diversification of the possibilities to regulate target genes10. Deregulation of SMAD signaling has severe implications in a variety of diseases. In line with this, unbalanced TGF&#946;/BMP signaling may lead to severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, or atherosclerosis3,11,12,13,14.
Endothelial cells (ECs) form the innermost layer of blood vessels and are, therefore, exposed to shear stress (SS), a frictional force exerted by the viscous flow of the blood. Interestingly, ECs residing at the parts of the vasculature, which are exposed to high levels of uniform, laminar SS, are kept in a homeostatic and quiescent state. In contrast, ECs that experience low, non-uniform SS, e.g., at bifurcations or the lesser curvature of the aortic arch, are proliferative and activate inflammatory pathways15. In turn, sites of dysfunctional ECs are prone to develop atherosclerosis. Interestingly, ECs in these atheroprone areas display aberrantly high levels of activated SMAD2/3 and SMAD1/516,17,18. In this context, enhanced TGF&#946;/BMP signaling was found to be an early event in the development of atherosclerotic lesions19 and interference with BMP signaling was found to markedly reduce vascular inflammation, atheroma formation, and associated calcification20.
Proximity Ligation Assay (PLA) is a biochemical technique to study protein-protein interactions in situ21,22. It relies on the specificity of antibodies of different species that can bind target proteins of interest, allowing highly specific detection of endogenous protein interactions at a single-cell level. Here, primary antibodies have to bind to their target epitope at a distance of less than 40 nm to allow for the detection23. Therefore, PLA is greatly beneficial over traditional co-immunoprecipitation approaches, where several million cells are needed to detect endogenous protein interactions. In PLA, species-specific secondary antibodies, covalently linked to DNA fragments (termed Plus and Minus probes), bind the primary antibodies and if the proteins of interest interact, Plus and Minus probes come in close proximity. The DNA gets ligated in the following step and the rolling circle amplification of the circular DNA is made possible. During amplification, fluorescently labeled complementary oligonucleotides bind to the synthesized DNA, allowing these protein interactions to be visualized by conventional fluorescence microscopy.
The protocol described here enables scientists to quantitatively compare the number of active SMAD transcription complexes at atheroprotective and atheroprone SS conditions in vitro using PLA. SS is generated via a programmable pneumatic pump system that is able to generate laminar unidirectional flow of defined levels and allows stepwise increases of flow rates. This method allows for the detection of interactions between SMAD1/5 or SMAD2/3 with SMAD4, as well as mixed-R-SMAD complexes. It can easily be expanded to analyze interactions of SMADs with transcriptional co-regulators or to transcription factor complexes other than SMADs. Figure 1 shows the major steps of the protocol presented below.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62608/62608fig01.jpg" /> 
Figure 1: Schematic representation of the protocol described. (A)&#160;Cells seeded in 6-channel slides are exposed to shear stress with a pneumatic pump system. (B)&#160;Fixed cells are used for PLA experiment or for control conditions. (C)&#160;Images of PLA experiments are acquired with a fluorescence microscope and are analyzed using ImageJ analysis software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62608/62608fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&amp;micro;-Slide VI 0.4</td><td>ibidi</td><td>80606</td><td>6-channel slide</td></tr><tr><td>Ammonium Chloride</td><td>Carl Roth</td><td>K298.1</td><td>Quenching</td></tr><tr><td>Bovine Serum Albumin</td><td>Carl Roth</td><td>8076.4</td><td>Blocking</td></tr><tr><td>DAPI</td><td>Sigma Aldrich/ Merck</td><td>D9542</td><td>Stain DNA/Nuclei</td></tr><tr><td>DPBS</td><td>PAN Biotech</td><td>P04-53500</td><td>PBS</td></tr><tr><td>Duolink In Situ Detection Reagents Green</td><td>Sigma Aldrich/ Merck</td><td>DUO92014</td><td>PLA kit containing Ligase, ligation buffer, polymerase and amplification buffer (with green labeled oligonucleotides)</td></tr><tr><td>Duolink In Situ PLA Probe Anti-Mouse MINUS</td><td>Sigma Aldrich/ Merck</td><td>DUO92004</td><td>MINUS probe</td></tr><tr><td>Duolink In Situ PLA Probe Anti-Rabbit PLUS</td><td>Sigma Aldrich/ Merck</td><td>DUO92002</td><td>PLUS probe</td></tr><tr><td>Duolink In Situ Wash Buffers, Fluorescence</td><td>Sigma Aldrich/ Merck</td><td>DUO82049</td><td>PLA wash buffers A and B</td></tr><tr><td>Endothelial Cell Growth Supplement</td><td>Corning</td><td></td><td>supplement for medium (ECGS)</td></tr><tr><td>Fetal calf Serum</td><td></td><td></td><td>supplement for medium</td></tr><tr><td>FIJI</td><td></td><td></td><td>Image Analysis software</td></tr><tr><td>Formaldehyde solution 4% buffered</td><td>KLINIPATH/VWR</td><td>VWRK4186.BO1</td><td>PFA</td></tr><tr><td>Full medium</td><td></td><td></td><td>M199 basal medium +20 % FCS +1 % P/S + 2 nM L-Glu +&amp;nbsp; 25 &amp;micro;g/mL Hep +&amp;nbsp;&amp;nbsp; 50 &amp;micro;g/mL ECGS</td></tr><tr><td>Gelatin from porcine skin, Type A</td><td>Sigma Aldrich</td><td>G2500</td><td>Use 0.1% in PBS for coating of flow channels</td></tr><tr><td>GraphPad Prism v.7</td><td>GarphPad</td><td></td><td>Statistical Program used for the Plots and statistical calculations</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma Aldrich</td><td>H4784-250MG</td><td>supplement for medium (Hep)</td></tr><tr><td>HUVECs</td><td></td><td></td><td></td></tr><tr><td>ibidi Mounting Medium</td><td>ibidi</td><td>50001</td><td>Liquid mounting medium</td></tr><tr><td>ibidi Pump System</td><td>ibidi</td><td>10902</td><td>pneumatic pump</td></tr><tr><td>Leica TCS SP8</td><td>Leica</td><td></td><td>confocal microscope</td></tr><tr><td>L-Glutamin 200mM</td><td>PAN Biotech</td><td>P04-80100</td><td>supplement for medium (L-Glu)</td></tr><tr><td>Medium 199</td><td>Sigma Aldrich</td><td>M2154</td><td>Base medium</td></tr><tr><td>mouse anti- SMAD1 Antibody</td><td>Abcam</td><td>ab53745</td><td>Suited for PLA</td></tr><tr><td>mouse anti- SMAD2/3 Antibody</td><td>BD Bioscience</td><td>610843</td><td>Not suited for PLA in combination with CST 9515</td></tr><tr><td>mousee anti- SMAD4 Antibody</td><td>Sanata Cruz Biotechnology</td><td>sc-7966</td><td>Suited for PLA</td></tr><tr><td>Penicillin 10.000U/ml /Streptomycin 10mg/ml</td><td>PAN Biotech</td><td>P06-07100</td><td>supplement for medium (P/S)</td></tr><tr><td>Perfusion Set WHITE</td><td>ibidi</td><td>10963</td><td>Tubings used for 1 dyn/cm2</td></tr><tr><td>Perfusion Set YELLOW and GREEN</td><td>ibidi</td><td>10964</td><td>Tubings used for 30 dyn/cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>rabbit anti- phospho SMAD1/5 Antibody</td><td>Cell Signaling Technologies</td><td>9516</td><td>Suited for PLA</td></tr><tr><td>rabbit anti- SMAD2/3 XP Antibody</td><td>Cell Signaling Technologies</td><td>8685</td><td>Suited for PLA</td></tr><tr><td>rabbit anti- SMAD4 Antibody</td><td>Cell Signaling Technologies</td><td>9515</td><td>Not suited for PLA in combination with BD 610843</td></tr><tr><td>Serial Connector for &amp;micro;-Slides</td><td>ibidi</td><td>10830</td><td>serial connection tubes</td></tr><tr><td>Triton X-100</td><td>Carl Roth</td><td>6683.1</td><td>Permeabilization</td></tr></tbody></table>

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.

We have previously used PLA to detect interactions of different SMAD proteins3 and analyzed shear stress induced changes in SMAD phosphorylation28.
Here, both methods were combined with the protocol described above. HUVECs were subjected to shear stress of 1 dyn/cm2 and 30 dyn/cm2 and analyzed for interactions of SMAD transcription factors. We show that, when compared to the high shear stress (30 dyn/cm2), the low shear stress (1 dyn/cm2) leads to a significant increase in SMAD1-SMAD2/3 interactions, the so called mixed-SMAD complexes in both, the cytosol, and nuclei of analyzed cells (Figure 2A, lower panel). PLA events are visible as distinct spots in both samples, and one can distinguish between cytosolic and nuclear events with reference to DAPI staining (Figure 2A, upper panel). In contrast, antibody controls, where only one of both primary antibodies but still both PLA probes were incubated, showed negligible numbers of PLA signals (Figure 2B). Thus, it can be concluded that the experiment was successful.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62608/62608fig02.jpg" /> 
Figure 2: SMAD2/3-SMAD1 PLA comparing different antibody concentrations. (A) Confocal fluorescence images and quantification of SMAD2/3-SMAD1-PLA in HUVECs exposed to 24 h of indicated SS levels. Antibodies dilution ratio: 1:100. (B)&#160;Confocal images of single antibody controls for PLA in A. (C)&#160;Confocal fluorescence images and quantification of SMAD2/3-SMAD1-PLA in HUVECs exposed to 24 h of indicated SS levels. Antibodies dilution ratio: 1:50. Scale bars for A-C: 20 &#181;m and 10 &#181;m in zoom in. Dyne equals dyn/cm2. Antibodies used were rabbit anti-SMAD2/3 and mouse anti-SMAD1 (see Table of Materials). For each condition 5 random areas along the center of the flow channel were used for image acquisition. N=1 biological replicate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62608/62608fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To show how different concentrations of antibodies change PLA results, the same experiment was performed with a 1:50 instead of a 1:100 dilution of antibodies. Under these conditions, twice the amount of antibody result in more than four-fold higher PLA signals per cell (compare Figure 2A and Figure 2C). The difference in signals between 1 dyn/cm2 and 30 dyn/cm2 decreases when a higher antibody concentration is used and statistical significance is lost for the total and cytosolic PLA events (Figure 2A, lower panel; Figure 2C, lower panel). This might be due to the signal coalescence and problems of distinguishing PLA events. If such accumulation of signals occurs, antibody concentrations should be decreased.
Furthermore, we showed that self-made buffers for blocking and antibody dilution can be used as an alternative for commercial buffers included in in situ PLA kits. PLA events per cell were compared for SMAD1-SMAD2/3 (Figure 3A versus Figure 3D), SMAD2/3-SMAD4 (Figure 3B versus Figure 3E) and pSMAD1/5-SMAD4 (Figure 3C versus Figure 3F) complexes under 1 dyn/cm2 and 30 dyn/cm2 using either commercial solutions (Figure 3A-C) or self-made BSA/PBS based solutions (Figure 3D-F). Quantifications for both commercial and self-made diluent/ blocking solutions show the same trend of PLA signals in cytosolic and nuclear areas. However, the total number of PLA events per cell is higher when using commercial solutions (Figure 3A-C, lower panels versus Figure 3D-F, lower panels).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62608/62608fig03.jpg" /> 
Figure 3: PLA experiment comparing commercial and self-made antibody buffers. Confocal images (upper part of each panel) and quantification (lower part of each panel) of different SMAD-SMAD PLAs in HUVECs exposed to 24 hours of indicated shear stress levels. (A-C) Commercial buffers (see Table of Materials). (D-F) Self-made buffers (3% BSA in PBS for blocking, 1 % BSA in PBS for antibody dilution). All primary antibodies were diluted 1:100. Scale bar, 20 &#181;M (10 &#181;M for zoom in). Statistical significance was calculated with two-sided t-Test. ns - non-significant, *P&#60;0.05, **P&#60;0.01, ***P&#60;0.001. Values are depicted as mean + SEM. Dyne equals dyn/cm2. Antibodies used were rabbit anti-SMAD2/3, mouse anti-SMAD1, rabbit anti-phospho SMAD1/5 and mouse anti-SMAD4 (see Table of Materials). For each condition 5 random areas along the center of the flow channel were used for image acquisition. N=1 biological replicate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62608/62608fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We also included an example for a failed PLA experiment. Here, a combination of SMAD2/3-SMAD4 antibodies was used where the SMAD4 antibody was not suited for performing immunofluorescence experiments. When compared to single antibody controls, no increase in spots in either the 1 dyn/cm2 or 30 dyn/cm2 samples was observed (Figure 4A versus Figure 4B). As the formation of SMAD2/3-SMAD4 complexes is induced by shear stress (see Figure 3B,E), it can be concluded that this PLA experiment was unsuccessful. This highlights the importance of choosing the correct antibody combinations to detect PLA events, as orientation and distance of bound antibodies might be crucial for successful annealing of the oligonucleotide probes.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62608/62608fig04.jpg" /> 
Figure 4: Example for failed PLA experiment. Confocal images, scale bar: 20 &#181;m and 10 &#181;m in zoom in. (A) SMAD2/3-SMAD4 PLA. (B) Single antibody controls. Antibodies used were mouse anti-SMAD2/3 and rabbit anti-SMAD4 (see Table of Materials). Dyne equals dyn/cm2. For each condition 5 random areas along the center of the flow channel were used for image acquisition. N=1 biological replicate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62608/62608fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization and Quantification of TGFÎ²/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay at JoVE.com

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

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 ...

visualization-quantification-tgfbmpsmad-signaling-under-different

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.3K Views.  Freie Universität Berlin. 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.

Video: Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

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

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Bioengineering

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.

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 ...

Cancer Research

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 ...

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Engineering

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Biology

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Summary

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A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition