Fig. 1: Characterization of proteomes released from ferroptotic cells.

A Experimental setup: Pfa1 MEFs were treated with ±4OHT [1 µM] to achieve near 100% cell death and further processed for (1) proteomics (Fig. 1, 72 h), (2) lipidomics (Fig. 2, 72 h), and (3) metabolomics (Fig. 3, 48 h). B Volcano plot of proteins enriched in GPX4 WT (−4OHT) and KO (+4OHT) conditions is shown. T-test difference and corresponding −log₁₀ T-test p-value are plotted for the individual hits. p-value < 0.05 cutoff was used to identify enriched proteins. Representative hits relevant to innate immune system pathway enrichment are highlighted within GPX4 KO supernatants. C Proteins significantly enriched in ferroptotic secretomes were analyzed for protein-protein interaction networks using STRING. Reactome pathways enriched within ferroptotic supernatants that are relevant to immune activation are plotted. −log₁₀ of the False discovery rate (FDR) is plotted. D Venn diagram of the total detected secretome (red, 353 proteins in sum), newly translated secretome (blue, 589 proteins in sum), and overlapping proteins (231). E Parental Pfa1 or Pfa1 MEFs with stable FSP1 overexpression [9] were stained with Draq7 [100 nM] and treated with 4OHT [1 µM] for 72 h. Cells were imaged for near-infrared (NIR) count as a measure of dead cells using the IncuCyte live cell imaging system. % Cell death was normalized to confluency. F Supernatants from cells and treatments as in (F) were subjected to LDH quantification using a colorimetric assay. G Cells as in (F) were treated with 4OHT [1 µM] ± Ferrostatin-1 [1 µM] for 72 h. MIF was quantified in supernatants using ELISA. H Pfa1 MEFs were treated as in (H). qPCR-mediated quantification of MIF cDNA is shown. I GPX4 control or GPX4-deficient SCLC cell lines [35] were kept in the presence of Fer-1 [1 µM]. Supernatants were collected 16 h after Ferrostatin-1 withdrawal, and MIF was quantified using ELISA. J Primary mouse lung fibroblasts (PMLFs) were treated with RSL3 [1 µM] ± Fer-1 [1 µM] for 24 h. Supernatants were collected and subjected to MIF ELISAs. K Pfa1 MEFs treated ±4OHT [1 µM] ± Fer-1 [1 µM] for 36 h were subjected to Western Blot analysis of the indicated proteins. Representative blots of three independent repeats are shown. L Supernatant from cells, as in (K), was subjected to MIF ELISAs. All schemes were created with BioRender.com. Data information: E–J, L Graphs show data of means ± SEM of 3 independent biological replicates. One- or two-way ANOVA was used to calculate p-values. ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Source data are available online for this figure.