CSF activates the ISR. (A-C) Immunoblots showing the effects of CSF on eIF2α levels. NALM-6 (A), REH (B), and Jurkat (C) leukemia cell lines were cultured in regular media or CSF for 24 hours. Cells treated with brefeldin A 2.5 μg/mL in regular media served as a positive control. Protein lysates were then collected for immunoblotting with phospho-eIF2α (p-eIF2α), total eIF2α, or β-actin antibodies. Representative western blots are shown. The p-eIF2α antibody reproducibly detected a higher molecular weight protein of unclear etiology in leukemia cells in CSF, which is denoted by “?” (D) Protein synthesis in leukemia cells was assessed using O-propargyl-puromycin after 48 hours of culture in either regular media or CSF. Error bars represent the mean ± SD of 3 technical replicates. ∗∗∗P < .001 and ∗∗∗∗P < .0001 by t test. (E) Methotrexate dose-response curves for A549 lung carcinoma cells in regular media (Dulbecco modified Eagle medium + 10% FBS), regular media plus tunicamycin 2.5 μg/mL, or CSF. A549 cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. (F-H) Methotrexate dose-response curves for NALM-6 (F), REH (G), and Jurkat (H) leukemia cells in the absence or presence of doxycycline 7.5 μM. Leukemia cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. LogIC50 and bottom values with confidence intervals were calculated from the dose-response curves and are shown in the supplemental Table.