Ernest Heimsath1, Antony Wood2, Lisa Arvidson2, and AACR 2023
Reginaldo Prioli2

A Multiscale Approach to Quantitatively Evaluate
the SMAD Signaling Pathway

Growth factor treatment
Before exposure with defined concentrations of growth
factors, cells seeded in culture plates (12-well for
PathScan sandwich ELISA and 96-well for 2D cultures)
were subjected to an overnight, 18-hour serumstarvation period using the same basal culture medium
as listed above, lacking FBS (A-DMEM) or BulletKit
supplements (EBM). Growth factor treatment wasconducted by adding hTGF-β1, hBMP-2 (part numbers
75362 and 4697, respectively; Cell Signaling
Technology, Danvers, MA, USA), or rhBMP-6 (partnumber 507-BP; R&D Systems, Minneapolis, MN, USA)
diluted in serum-free media to adherent cells at theindicated concentrations and allowed to incubate for 1hour, followed by 4% paraformaldehyde fixation andimmunostaining.
Results and Discussion

Cellular assay: nuclear translocation
Cell Signaling Technology antibodies:• Phospho-SMAD2 (S465/467) (E8R3R), part number #56532• Phospho-SMAD1 (S463/465)/SMAD5 (S463/465)/SMAD9 (S465/467)(D3B10), part number 55605

CI-huFIB + BMP-20 n g/mL

1 0 0 ng/mL

100µma-tubulin; pSMAD1/5/9NoHUVEC + BMP-60 n g/mL

100 ng/mLF-actin; pSMAD1/5/9

100 ng/mLF-actin; pSMAD2/3

SMAD4

4
3210PECAM1t-2enm
MPt
B
a+Tre200 µm

654Maximum intensity projection of a 187slice Z-stack spanning 185 µm.
Green: SMAD4; red: PECAM1; gray: DRAQ7.

320etmea
Trnt+BM

6P-A549 lung adenocarcinoma spheroids serve as a 3D model for EMT when
cultured in the presence of TGF-β10 n g/mL TGF-β1

109876543210No

1.5
1.4tmeaTrten+TG

β1F-100 ng/mL TGF-β1

415

3

10EC500.034 ng/mL

2
1

100

10

1

0.
101
0.

0.

00
00
01
0.
00
00
10.
00
01
0.
00
10

[TGF-β1] (ng/mL)

1.1
1.0
0.9

0.01

0.1

1

10100

1000

A549 lung adenocarcinoma spheroids were embedded in acollagen matrix and cultured for 5 days in the presence ofincreasing concentrations of TGF-β1. Green: AF488-Phalloidin;
red: DRAQ7. Scale bar = 200 µm.

65huFIB: BMP-24HUVEC: BMP-63A549: TGF-β1Conclusion

2101001000

51020

The nuclear translocation assay can be scaled to a highthroughput 96-well microplate array

160.
125

Fold Change

70.
00
00
10.
00
01
0.
00
10.
01

pSMAD2/3
Fold Change

30

Total SMAD2 (Abs450)

Attribution 4.0 International (CC BY 4.0)

1.2

[TGF-β1] (ng/mL)

0Cells. 2014, 3 (4), 981–993. doi: 10.3390/cells3040981

1.30.80.001

SMAD2/3 phosphorylation in CI-huFIBs

5

Enlarged region of thebox on the left panel

1NoA549 + TGF-β10 n g/mL

Cell Signaling Technology antibodies:• SMAD4 (D3R4N), part number #24483

CST reagents can be used for antibody-based detection of SMAD4expression during in vitro angiogenesis development

Biochemical analysis: high-throughput ELISA assay
Cell Signaling Technology PathScan sandwich ELISA kits:• Phospho-SMAD2 (S465/467)/SMAD3 (S423/425), part number #12001• Total SMAD2/3, part number #12000

3D assay: spheroid sprouting angiogenesis, and EMT model

Spheroid Area
Fold Change

Canonical TGF-β/SMAD and BMP/SMAD signaling pathway

Cell lines
Immortalized human fibroblasts (CI-huFIB, part number
INS-CI-1010) were a kind gift from InSCREENex GmbH
(Braunschweig, Germany). Human Umbilical Vein
Endothelial Cells (HUVECs) were purchased from Lonza
Bioscience (part number C2519A; Basel, Switzerland).
A549 lung carcinoma cells were purchased from ATCC
(part number CCL-185; Manassas, VA, USA). CI-huFIBs
and A549s cells were cultured in Advanced DMEM (partnumber 12491; Gibco Thermo Fisher Scientific;Waltham, MA, USA) containing 10% FBS and 1x
penicillin/streptomycin/glutamine.
HUVECsweremaintained in Endothelial Cell Grown Medium-2 BulletKit(EGM-2; part numbers CC-3162; Lonza).

Results and Discussion

Nuclear Fluorescent Signal
Fold Change

Angiogenesis and cell proliferation are fundamental
biological processes, and their pathologic states
represent two hallmarks of cancer. Central to controllingboth processes is the TGF-β/BMP signaling pathway, forwhich the SMAD proteins serve as major downstream
effector molecules. Upon ligand binding, TGF-β or BMP
type I/II receptor heterotetramers phosphorylate
receptor-regulated SMADs (R-SMADs). TGF-β-receptor
binding leads to SMAD2/3 phosphorylation, whereas
BMP-receptor binding induces phosphorylation ofSMADs 1/5/8. Phosphorylated SMADs (pSMADs) thenform a complex with the co-SMAD SMAD4, at which
point they undergo nuclear translocation, where they
operate as transcriptional modulators of target genes.
The magnitude of SMAD phosphorylation and functional
output (e.g., stimulated angiogenesis or attenuated cellproliferation) depends on both the ligand concentrationand the duration of exposure. In this study, wequantitatively evaluate the phosphorylation states of RSMADs, as well as their downstream biological effects
at three different biological scales: 1) biochemical; 2)cellular; and 3) tissue/3D models. At the biochemicallevel, the temporal and dose-dependent phosphorylationof R-SMADs in response to growth factor stimulation
can be quantified using a quantitative ELISA approach.
Growth factor stimulation can be quantified at thecellular level by high-content imaging of SMAD
phosphorylation and nuclear translocation, while
simultaneously monitoring cell proliferation rates.
Lastly, high-resolution confocal imaging can be
employed to observe developmental consequences ofphospho-SMAD signaling using a HUVEC-based 3D
angiogenesis model system.

Results and Discussion

Nuclear Fluorescent Signal
Fold Change

Experimental

Nuclear Fluorescent Signal
Fold Change

Introduction

Poster # 30
Abstract # 3656

1 Agilent Technologies, Inc., 2 Cell Signaling Technology[Growth Factor] (ng/mL)

The high-quality reagents from Cell Signaling
Technology combined with the multifunctional
capabilities of the Agilent BioTek Cytation C10 confocal
imaging reader enable quantitative evaluation of theTGF-β/BMP pathway activation at different biologicalscales.
For Research Use Only. Not for use in diagnostic
procedures. RA45026.4146412037