Integrated Stress Response Assay

The integrated stress response is a cellular signaling pathway initiated by a variety of intrinsic or extrinsic cellular stresses, including endoplasmic reticulum stress, hypoxia, amino acid deprivation, glucose deprivation, viral infection, and oxidants.   Different types of stress activate different kinases that all act upon the translation initiation factor subunit eIF2α.  This phosphorylation reduces translation initiation and the synthesis of most cellular proteins.  However, translation of the mRNA for the ATF4 transcription factor is increased under these circumstances.  ATF4 translocates to the nucleus where it upregulates genes that promote cellular homeostasis or, alternatively, apoptosis.

The Integrated Stress Response Assay is a high content assay in which cells grown in microplates are treated with test article for a desired duration and then fixed and immunofluorescently labeled for the ATF4 transcription factor. Cells are then imaged, nuclei are segmented from the images, and ATF4 staining intensity is quantified per nucleus. ATF4 induction is evident within 2 hours of a treatment and is maintained for up to 24 hours. The relationship between stress response and  ATF4 is depicted below.

The integrated stress response.