Detecting SMAD Pathway Activation Using Dual Adenovirus Transduction

Abstract

Source: Chen, H., et al. Live Cell Imaging of the TGF-β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System. J. Vis. Exp. (2018).

This video demonstrates a method to study SMAD pathway activation in breast cancer cells using dual adenoviral infection. One virus delivers a constitutively expressed GFP gene, while the other carries an RFP reporter under a SMAD-sensitive promoter. Upon TGF-beta stimulation, SMAD proteins are activated, forming a complex that induces RFP expression. Detection of dual signals in live cells confirms pathway activation.

Protocol

1. Dual-adenovirus Transduction in Live Single Cells

1. Seed 1.0 - 1.5 x 10⁵ MDA-MB-231 cells with 500 μL of dulbecco’s modified eagle medium (DMEM) culture media containing 5% fetal bovine serum (FBS) in each well of a 12-well plate (35% cell confluency, 2 wells in total).
2. Infect cells with 75 μL of adenoviral vector Ad-CMV-GFP (titer = 5 x 10⁹ PFU/mL) and 7.5 μL of adenoviral vector Ad-CAGA12-Td-Tom (titer = 5 x 10¹⁰ PFU/mL) to obtain a 2,500 multiplicity of infection (MOI), which can achieve 100% infection positivity.
3. Treat cells with or without 0.25 μL of transforming growth factor-beta (TGF-β) per well (10 mg/mL in stock, 5 ng/μL as the final concentration) immediately after adenovirus infection.
4. Place the plate in the incubator at 37 °C with 10% CO₂ for 24 h.
5. Image live cells with a phase contrast fluorescence microscope. Excite green fluorescent protein (GFP) at 470 nm and Td-Tomato protein at 554 nm.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	ThermoFisher	1881024	Warm in 37 °C waterbath before use
Foetal Bovine Serum	Scientifix Life	FBS500-S	Heat inactivated before use
Recombinant Human TGF-β1	PEPROTECH	100-21	Aliquot in DDW to make final concentration at 10 mg/mL
Phase contrast fluorescence microscopy	OLYMPUS	IX50
MDA-MB-231	ATCC	CRM-HTB-26
Solution			Composition
FBS-DMEM			5% heat inactivated FBS; 10 μg/mL penicillin; 100 μg/mL streptomycin