Ceramides drive a PKR-dependent integrated stress response. (A) The ISR. (B-C) MV411, OCI-AML3, MOLM13, HL-60, and THP-1 cells were treated with MP-A08 alone (20 μM) or in combination with the GCN2 inhibitor A-92 (5 μM), PKR inhibitors C16 (5 μM) or 2-AP (0.1-10 mM) or PERK inhibitor AMG-44 (5 μM) for 6 hours before immunoblot analysis with the indicated antibodies. (D) MV411 cells were treated with MP-A08 (10 μM) and 2-AP (5 mM) for 16 hours and assessed for cell viability by Annexin V/propidium iodide staining. Data are the mean ± standard error of the mean (SEM) of 3 independent experiments. Statistical significance was assessed by Student t test. (E) Wild-type (WT) or PKR knockout MV411 cells were treated with MP-A08 (15 μM) for 16 hours and assessed for cell viability by Annexin V/propidium iodide staining. Data are the mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student t test. Immunoblot analysis of WT or PKR knockout MV411 cells with the indicated antibodies. (F) PKR-HA was immunoprecipitated from transiently transfected HEK 293T cells, incubated with exogenous C6-ceramide (10 μM) for 30 minutes and subjected to a PKR activity assay, using autophosphorylation as the readout. Data are the mean ± SEM of 4 independent experiments. (G) Lysates from HEK293T cells transfected with pcDNA3/PKR-HA was incubated with ceramide conjugated to agarose beads or control beads overnight at 4°C, washed, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the associated PKR was detected by immunoblot analysis with anti-HA antibodies on an Odyssey imaging system.