Fig. 3: Ferroptotic cells release metabolites of active anabolism.

A Primary mouse lung fibroblasts (PMLFs) were treated with RSL3 [1 µM] or erastin [1 µM] ± Fer-1 [1 µM] for 15 h. ATP release was measured using the RealTime-Glo™ Extracellular ATP Assay and a luminescence plate reader. Relative Luciferase Units; RLU. B Cells and treatments as in A were stained with Draq7 [100 nM]. Cells were imaged every 2 h for near-infrared (NIR) count as a measure of dead cells using the IncuCyte live cell imaging system. % Cell death was normalized to confluency. C Parental Pfa1 or Pfa1 MEFs with stable FSP1 overexpression [9] (mFSP1) were treated with RSL3 [1 µM] ± Fer-1 [1 µM], and ATP release was quantified using the RealTime-Glo™ Extracellular ATP Assay and a luminescence plate reader. Relative Luciferase Units; RLU. D Cells and treatments as in (C) were stained with Draq7 [100 nM]. Cells were imaged every 2 h for near-infrared (NIR) count as a measure of dead cells using the IncuCyte live cell imaging system. % Cell death was normalized to confluency. E Heatmap of pentose phosphate pathway metabolites in cell pellets 30 h after 4OHT [1 µM] stimulation of Pfa1 cells ± Fer-1 [1 µM]. Log2 fold change is shown. F Heatmap of pyrimidine synthesis metabolites in cell pellets 30 h after 4OHT [1 µM] stimulation of Pfa1 cells ± Fer-1 [1 µM]. Log2 fold change is shown. G Heatmap of all metabolites detected within supernatants of all 4 experimental conditions, 48 h after stimulation. Log2 fold distance is shown. H Heatmap of significantly different (p = 0.05) metabolites within supernatants of Pfa1 cells ± 4OHT [1 µM] for 48 h. Log2 fold distance is shown. I KEGG pathway enrichment of metabolites listed in (H) in ferroptotic supernatants (48 h + 4OHT [1 µM]) is plotted by −log¹⁰ p-value using MetaboAnalyst 6.0 [49, 50]. Data information: A–D Graphs show data of means ± SEM of 3 independent biological replicates. E–H Heatmaps show 5 independent biological replicates for metabolomics measurement. 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.