Kinase Assay: High-content microscopy-based secondary screen; U2OS cells carrying the ATF4-dGFP-IRES-Cherry reporter are plated in 96 well plates and treated with 100 nM Thapsigargin and 10μM of the cherry-picked compounds for 8 hr. Cells are stained with Hoechst 33,258 and are visualized using an automated microscope. Data acquisition and image analyses are performed with the INCell Developer Toolbox Software, version 1.9. Compounds that block induction of the ATF4-dGFP reporter, do not block the accumulation of mCherry downstream of the IRES, and are deemed non-toxic as determined by cell number measured by counting nuclei, are repurchased for further analyses.

Cell Assay: ISRIB blocks production of endogenous ATF4, whereas XBP1 mRNA splicing and XBP1s production persisted. ISRIB prevents cells from re-establishing ER homeostasis by blocking signaling through the PERK branch of the UPR, and decreases the viability of cells that are subjected to ER-stress.