Gimap4 accelerates stress-induced death in T cells. Sorted T cells from Gimap ko mice and heterozygous littermates were incubated for 1 day in serumfree medium. Data are representative of at least 3 independent experiments using 3 mice in each experiment. Error bars indicate standard deviation. (A) Live, apoptotic, or dead cells were distinguished by fluorochrome–annexin V and PI labeling. The apoptotic population was always 2- to 4-fold higher in Gimap4-deficient T cells. Student t test revealed significant differences; asterisk represents P value of less than .005. (B) Sorted T cells were incubated in the presence or absence of the effector-caspase inhibitor DEVD (100 μM) and subsequently stained with fluorochrome–annexin V and PI. (C) After 1 day of serum deprivation, live and apoptotic T cells were sorted based on their annexin V and PI staining pattern and stained for intracellular active caspase-3. (D) For the mitochondrial membrane potential (ΔΨ) cells were stained with the voltage-dependent membrane permeable dye TMRE. (E) Cells were stained with fluorochrome-labeled cytochrome c antibody. (F) Equal amounts of proteins from lysates from purified live and dying T lymphocytes after 1 day of serum starvation were used for 2D analysis.