The CellSensor™ T-REx™ FOXO3 DBE-bla HeLa cell line was engineered by lentiviral transduction of HeLa cervical cancer cells with a FOXO3 response element driving beta-lactamase expression (DBE-bla), along with a tetracycline repressor and tetracycline (or the tetracycline analog doxycycline)-inducible FOXO3 constructs. Addition of doxycycline to these cells allows for FOXO3 transcription factor expression and subsequent beta-lactamase expression, which in turn can be down-regulated by insulin-mediated activation of the PI3K/AKT signaling pathway which leads to phosphorylation and inactivation of FOXO3 and concomitant suppression of beta-lactamase expression. This cell line has been tested for robust assay performance by assessing a variety of assay parameters, including: cell plating number, DMSO tolerance, stimulation time, and substrate loading time. Assay validation was performed using serial dilutions of insulin to antagonize doxycycline- induced FOXO3 activity (Figure 1). Additional testing data using RNAi and alternate stimuli are also available.