Detecting SMAD Pathway Activation Using Dual Adenovirus Transduction

Take cultures of breast cancer cells.

Co-infect with two types of adenoviruses. One carries a green fluorescent protein or GFP gene under a constitutively active promoter.

The other contains a reporter gene encoding red fluorescent protein or RFP, under a SMAD-responsive promoter.

To the test culture, add TGF-beta and incubate.

The viruses bind to the receptor to enter the host cell, deliver the genes into the nucleus, and enable GFP expression.

In test cells, TGF-beta binds with its receptor and activates the SMAD pathway by phosphorylating proteins SMAD2 and SMAD3.

These proteins form a complex with SMAD4.

This complex binds to the promoter of the reporter gene, enabling RFP expression in addition to GFP.

In the control, the SMAD pathway remains inactive, allowing cells to express only GFP.

Capture fluorescent images of live cells.

The presence of both signals confirms SMAD pathway activation using dual adenovirus transduction.

On the day of adenovirus infection, seed the wells of a 12-well plate with approximately 1.25 times 10 to the fifth MDA-MB-231 cells in 500 microliters of medium for a confluency of 35%. Infect cells in all wells with 75 microliters of adenovirus-CMV-GFP and 7.5 microliters of adenovirus CAGA12-Td-Tomato to obtain a 2,500 MOI which can achieve 100%infection positivity. Then add TGF-beta to the treatment well immediately after adenovirus infection.

Incubate at 37 degrees Celsius with 10%carbon dioxide for 24 hours. The next day, image live cells with a phase-contrast fluorescence microscope.