Fig 2: Equilibrium distributions of organelle marker proteins and ACSL4 in MCF-7 density gradient fractions. a Equal volume samples from each MCF-7 sucrose gradient fraction were subjected to SDS-PAGE separation and immunoblotted for ACSL4 using two different commercially available antibodies; antibody#1 was supplied by GeneTex and antibody#2 from Invitrogen. The gradient distributions of a panel of organelle marker proteins was similarly determined by immunoblotting. Data presented are representitive of experiments repeated 2–3 times with similar results. b The normalised distributions of anti-ACSL3 and anti-ACSL4 immunoreactivties in the gradient fractions. Western blotting signals were quantified using imageJ software. Data are representitive of experiments repeated 3–4 times with similar results

Fig 3: ACSL4 partially localises to MAM. MAM, cytoplasmic and mitochondrial fractions, were prepared from MCF-7 cells. Equal volumes of samples from each MCF-7 fraction were subjected to SDS-PAGE separation and immunoblotted for ACSL4, calnexin, caveolin, HMGCR and Sigma1R. Each panel represents the results of an experiment repeated twice with similar results

Fig 4: Inhibition of PUFA synthesis prevents ferroptotic cell death in ALCLsa. Immunoblotting of ACSL4 in the indicated Karpas299 cells. Actin is used as a loading control (top). Relative fold change in cell viability of indicated Karpas299 cell lines in the presence or absence of ML162 (20 nM) and Fer-1 (1 uM) for 5 days (bottom) (mean ± SD, n = 3 biologically independent samples). Statistics: two-tailed unpaired t-test.b. Correlation of mRNA levels of ACSL4 with SQLE (left) and ALK (right) in primary ALCLs dataset (n = 22 biologically independent samples).c. Correlation of mRNA levels of ACSL4 with SQLE in CCLE dataset (n = 935 independent cell lines).d. Lipid peroxidation assessed by flow cytometry measuring C11-BODIPY fluorescence of indicated Karpas299 cell lines after an 18 hr treatment with ML162 (200 nM). Representative data from one of three experiments is shown.e. Lipid peroxidation assessed by flow cytometry measuring C11-BODIPY fluorescence of indicated Karpas299 cell lines after an 18 hr treatment in the presence and absence of ML162 (200 nM) and Fer-1 (1 uM).

Fig 5: Immunohistochemistry reveals differential expression of ACSL3 and ACSL4 in sarcomas. Multiple tissues arrays were probed using antibodies specific for either ACSL3 or ACSL4. Representative images are shown for ACSL3 and ACSL4 immunohistochemical staining of matched patient samples from a and b liposarcoma, c and d fibrosarcoma, e and f leiomyosarcoma, and g and h rhabdomyosarcoma cores on multiple tissue arrays. Examples of negative and weak staining are presented in panels a, b and d, and high-intensity staining in the remaining panels The main image in each panel ×10 objective magnification, and each inset is ×100 objective magnification. Cytosplamic staining can be seen in higher-magnification inset images in panels c, and e–h. Note that, while ACSL3 staining was classified as weak overall in liposarcomas, it was nevertheless possible to visualise ACSL3 staining on the perimeter of lipid droplets in these samples a inset