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

Paul-Lennard Mendez; Leon Obendorf; Petra Knaus

doi:10.3791/62608

JoVE Journal > Biochemistry

Biochimie

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

Published: September 14, 2021

doi:

10.3791/62608

Paul-Lennard Mendez^1,2, Leon Obendorf¹, Petra Knaus¹

¹Institute for Chemistry and Biochemistry,Freie Universität Berlin, ²International Max-Planck Research School for Biology and Computation

Summary

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.

Abstract

Transforming Growth Factor β (TGFβ)/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 TGFβ/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGFβ/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 TGFβ/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGFβ 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 TGFβ/BMP signaling in ECs, both quantitatively and spatially.

Introduction

Proteins of the transforming growth factors beta (TGFβ) superfamily are pleiotropic cytokines with a variety of members, including TGFβs, 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 TGFβs and Activins mainly induce phosphorylation of SMAD2/3, BMPs induce SMAD1/5/8 phosphorylation. However, there are accumulating evidences that BMPs and TGFβs/Activins also activate R-SMADs of the respective other sub-family, in a process termed as 'lateral signaling'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 TGFβ/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 TGFβ/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.

Figure 1: Schematic representation of the protocol described. (A) Cells seeded in 6-channel slides are exposed to shear stress with a pneumatic pump system. (B) Fixed cells are used for PLA experiment or for control conditions. (C) Images of PLA experiments are acquired with a fluorescence microscope and are analyzed using ImageJ analysis software. Please click here to view a larger version of this figure.

Protocol

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.

Coat 6-channel slide with 0.1% porcine skin gelatin in PBS for 30 min at 37 °C.
Seed HUVECs in pre-coated 6-channel slides at a density of 2.5 x 10⁶ cells per mL in 30 µL of M199 full medium.
NOTE: For further information on how to seed cells in the flow slide, see reference²⁴.
Let cells adhere for 1 h at 37 °C in a humidified incubator.
Add 60 µL of pre-warmed M199 full medium to each of the reservoirs.
Culture for 2 days, with a gentle medium exchange once a day, at 37 °C in a humidified incubator.
1. Aspirate the reservoirs completely, add 120 µL of pre-warmed M199 full medium in one of the reservoirs, and aspirate from the other side.
2. Add 60 µL of pre-warmed M199 full medium to both reservoirs.
Assemble and start the flow set-up as detailed in the reference²⁵.
1. 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.
2. Fill the reservoirs with an appropriate amount of pre-warmed M199 full medium (minimum 10 mL).
3. 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.
4. 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.
5. For the exposure of cells to high levels of shear stress (>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.

2. Fixation

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.
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.
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 °C) with three times the channel volume (90 µL). 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.
Fix the cells by adding 90 µL 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.
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.
Quench the PFA-fixation by adding 90 µL 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.
Wash as described in step 2.5.
NOTE: At this point, the samples may be stored at 4 °C overnight, or the protocol can be immediately continued with blocking and primary antibody incubation (see step 3).

3. Blocking and primary antibody incubation

To permeabilize the cells, add 90 µL 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.
Wash as described in step 2.5.
Add 90 µL 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 °C in a humidified chamber.
1. 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).
Prepare primary antibodies (1:100) in PLA antibody diluent. Prepare 30 µL 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.
Before the application of primary antibodies, aspirate the blocking solution from the reservoirs and, also, carefully from the channel. Pipette 30 µL 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.
Incubate samples with the primary antibodies overnight in humidified chambers at 4 °C.
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.

4. PLA probe incubation

NOTE: For all steps in section 4.1-7.3, the washing buffers A and B are stored at 4 °C and need to be warmed to RT prior to the use.

Dilute PLA-probes (+)-mouse and (-)-rabbit to 1:5 in PLA antibody diluent (or 1% BSA) solution. Prepare 30 µL per channel.
Wash samples 2x for 5 min using 90 µL 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.
Aspirate the wash buffer A carefully and add 30 µL of PLA probe solution (prepared in step 4.1), similar to the addition of primary antibodies in step 3.5.
Incubate samples for 1 h at 37 °C in a humidified chamber.

5. Ligation

Wash samples 2x for 5 min using 90 µL of the wash buffer A at RT, as described in 4.2.
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 µL per channel.
Aspirate the wash buffer A completely and add the ligation solution as described in 3.5.
Incubate samples for 30 min at 37 °C in a humidified chamber.

6. Amplification

Wash samples 2x for 2 min using 90 µL of wash buffer A at RT, as described in 4.2.
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 µL per channel.
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 °C in a humidified chamber.

7. Mounting

Wash samples 2x for 10 min using 90 µL 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.
Dilute the wash buffer B in deionized water (1:10) and wash 1x with 90 µL of 0.1x buffer B solution as described in 4.2.
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 °C in a humidified environment until imaging.

8. Image acquisition

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.

9. Image analysis and quantification using ImageJ/FIJI

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.

Representative Results

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.

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) Confocal images of single antibody controls for PLA in A. (C) 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 µm and 10 µm 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. Please click here to view a larger version of this figure.

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).

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 µM (10 µM for zoom in). Statistical significance was calculated with two-sided t-Test. ns – non-significant, *P<0.05, **P<0.01, ***P<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. Please click here to view a larger version of this figure.

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.

Figure 4: Example for failed PLA experiment. Confocal images, scale bar: 20 µm and 10 µm 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. Please click here to view a larger version of this figure.

Discussion

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 <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 (>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.

Divulgations

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

References

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

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Mendez, P., Obendorf, L., Knaus, P. Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay. J. Vis. Exp. (175), e62608, doi:10.3791/62608 (2021).