Ceramides drive a PERK-independent ISR. (A-C) MV411 cells were treated with vehicle control (0.1% dimethyl sulfoxide [DMSO]; blue bars) or 20 μM MP-A08 (red bars) for 6 hours and analyzed by liquid chromatography-mass spectrometry. (A) Quantitation of individual ceramide (Cer) and dihydroceramide (dhCer) species. (B) Quantitation of sphingosine (Sph) and dihydrosphingosine (dhSph) species. (C) Quantitation of S1P and dihydrosphingosine 1-phosphate (dhS1P). All data are presented as picomoles per million cells; mean ± standard deviation of 4 independent experiments. Statistical significance was assessed by Student t test. *P < .05; **P < .01; ***P < .001. (D) MV411 cells were treated with MP-A08 (20 μM), PF-543 (1 μM), and SK1-I (10 μM) for 6 hours; lysed; and subjected to immunoblot analysis. (E) MV411 cells were treated with ceranib-2 for 16 hours and assessed for cell viability by Annexin V/propidium iodide staining. Data are the mean ± standard deviation of 2 independent experiments. MV411 cells were treated with ceranib-2 (10 μM) for 6 hours and subjected to immunoblot analysis with the indicated antibodies. (F) MV411 cells were treated with MP-A08 (20 μM), C2-ceramide (10 μM), C6-ceramide (10 μM), C2-dhCeramide (10 μM), and C6-dhCeramide (10 μM) (all introduced from 2.5 mM stock solutions in dimethyl sulfoxide) for 6 hours; lysed; and subjected to immunoblot analysis with the indicated antibodies. (G) HAP1 wild-type and PERK^−/− cells were treated with MP-A08 (20 μM) for 6 hours, lysed, and subjected to immunoblot analysis with the indicated antibodies. (H-I) MV411 cells were stably transduced with a doxycycline-inducible shRNA targeting PERK. Cells were treated with 1 μg/mL doxycycline for 48 hours and MP-A08 (20 μM) for 6 hours before (H) quantitative PCR analysis of PERK messenger RNA levels and (I) immunoblot analysis with the indicated antibodies. Data are the mean ± standard error of the mean of 3 independent experiments. Statistical significance was assessed by Student t test. **P < .01.