Extended Data Fig. 5: Subcellular localization and effect on ferroptosis of myristic acid and cholesterol, overexpression and distribution of ACSL4.
From: Identification of essential sites of lipid peroxidation in ferroptosis
a. Structure and SRS image of myristic acid-d27 (20 µM) in HT-1080 cells. b. Structure and SRS image of cholesterol-d6 (20 µM) in HT-1080 cells. c. Fluorescence and confocal imaging of cholesterol-d6 to evaluate its subcellular localization. d. Solution Raman spectra of the FAs and cholesterol used in these experiments. e. Dose-response curves of HT-1080 cells pretreated with either MA-d27 or cholesterol-d6 (20 µM) followed by varying concentrations of RSL3. Data are represented as mean ± SEM, n=3. f. Western blot of HT-1080 cells overexpressing GFP (control) or GFP-ACSL4. ACSL4 antibody was used, with actin as a control. g. Immunofluorescence staining of HT-1080 cells labeled for ACSL4 (anti-ACSL4 antibody), ER (anti-calnexin antibody), and nucleus (DAPI). Individual channels and overlay is shown. h. Western blotting of mitochondrial and ER HT-1080 fractions stained for ACSL4 PDI (ER), and Cytochrome C (mitochondria), indicating presence in both fractions, representative of two experiments.
