Fig. 3. Ferroptosis is characterized by impaired electrophile detoxification and associated increased cellular electrophile levels. (A–E) RSL3-induced ferroptosis is marked by decreased electrophile adduct export and associated increased electrophile levels. (A) Representative 20× widefield AcroB fluorescence and corresponding DIC (inset) images of HT-1080 cells following 0–60 minute incubations with 1 μM RSL3. (B) Average AcroB CTCF and fluorescence background curves representing n = 15 FOV (control), n = 12 FOV (15 min) and n = 16 FOV (30 min, 60 min) obtained from 20× images. Arrow indicates 60 minute timepoint depicted in (A). (C) 100× imaging of HT-1080 cells following 60 minute treatment with 1 μM RSL3 shows large cytoplasmic fluorescence followed by loss of cell fluorescence in the emission channel and loss of pseudo three-dimensional relief shading in the DIC channel during cell membrane rupture. Fluorescence and corresponding DIC images presented. Scale bar is 12 μm. (D) Aldehyde probe Na-FA fluorescence levels (5 μM, 30 minute incubation) in HT-1080 cells after 0–60 minute incubations with 1 μM RSL3. Average of n = 8 FOV for all conditions. (E) AcroB CTCF or fluorescence background vs. Na-FA CTCF following RSL3 treatment. Values used are those calculated 30 minutes following dye treatment as shown in (B) and (D). (F–J) BSO-induced ferroptosis is marked by decreased electrophile conjugation and associated increased electrophile levels. (F) Representative 20× widefield AcroB fluorescence and corresponding DIC (inset) images of HT-1080 cells following 0–24 hour incubations with 500 μM BSO. (G) Average AcroB CTCF and fluorescence background curves representing n = 12 FOV (control), n = 16 FOV (16 h), n = 11 FOV (24 h) obtained from 20× images. Arrow indicates 60 minute timepoint depicted in (F). (H) ElectrophileQ plot constructed using values recorded 20 min after AcroB application for indicated conditions presented in (B), (G) and Fig. S3B and S4.† (I) Aldehyde probe Na-FA fluorescence levels (5 μM, 30 minute incubation) in HT-1080 cells after 0–24 h incubations with 500 μM BSO. Average of n = 16 FOV for all conditions. (J) AcroB CTCF or fluorescence background vs. Na-FA CTCF following BSO treatment. Values used are those calculated 30 minutes following dye treatment as shown in (G) or (I). AcroB concentration = 100 nM. λ_ex = 488 nm (0.1 mW for 20×, 0.05 mW for 100×). 20× imaging: images acquired over 60 minutes following AcroB addition, LUT range 0–4000, scale bar is 64 μm. All values presented as mean ± SEM.