This study investigates the role of CaeB, an anti-apoptotic bacterial protein, in modulating apoptosis in human cells. By utilizing a tetracycline-inducible gene expression system, the effects of CaeB on pro-apoptotic signaling are examined.
Take test human cells expressing CaeB, an anti-apoptotic bacterial protein.
Use cells lacking CaeB as the control.
Introduce a transfection reagent complexed with a tetracycline-inducible gene expression system containing both regulator and response plasmids.
The regulator plasmid encodes a transcriptional activator and silencer, while the response plasmid encodes a pro-apoptotic protein controlled by TRE.
Incubate the cells to facilitate plasmid uptake and regulatory protein expression.
Without tetracycline, the activator remains inactive, and the silencer represses TRE-driven transcription.
Add a tetracycline analog and incubate.
The analog enables activator–TRE binding, initiating pro-apoptotic protein expression.
In control cells, this triggers apoptotic signaling, activating an apoptotic marker.
In CaeB-expressing cells, CaeB inhibits apoptotic signaling, reducing the marker activation.
A reduced marker activation in CaeB-expressing cells suggests that CaeB inhibits apoptosis.
Begin this procedure by seeding HEK293 cells stably expressing GFP or GFP-CaeB in a 12 well plate at a density of 100,000 cells per well.
Next, prepare DNA and polyethylenimine transfection reagent separately in 75 microliters of Opti-MEM medium, and incubate for five minutes at room temperature. For complex formation, incubate both mixtures together for 15 minutes at room temperature. After that, add the polyethylenimine DNA solution dropwise to the cells and incubate at 37 degrees Celsius in 5% carbon dioxide.
Five hours after transfection induce expression of the pro apoptotic proteins by adding one microgram per milliliter of doxycycline to the culture medium. Then, incubate the cells for 18 hours at 37 degrees Celsius in 5% carbon dioxide.
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