12S-HETE exposure induces senescence in AECII. Primary AECII were enriched from 8–10-week-old C57/Bl6NcR mice and cultured in chamber slides. AECII cultures received 17.5 Gy irradiation or treated with 150 nM 12S-HETE for 3 days. Panel A: Senescence was assessed using SA-β-galactosidase activity assay and followed by immunocytochemical localization of pro-surfactant C. The percentage of senescent AECII was scored. Columns are the mean and bars represent SD, *P < 0.05 relative to vehicle. Panel B: Protein lysates were prepared from enriched primary AECII cultures and subjected to Western blotting. Panel C: Enriched primary AECII cultures were treated with 150 nM 12S-HETE or irradiated at 17.5 Gy. NOX inhibitor, 150 nM GKT137831 or vehicle (DMSO) was added 1 h prior to irradiation or 12S-HETE administration. After 3 days, β-galactosidase activity was determined followed by immunofluorescence for pro-surfactant C. The percentage of AECII with SA-β-gal activity was scored. Columns are the mean and bars represent SD, *P < 0.05 to untreated vehicle by ANOVA with Tukey’s correction; **P < 0.05 to the corresponding treated control (vehicle, 12S-HETE, 17.5 Gy) without NOX4 inhibitor by ANOVA with Tukey’s correction.