Figure 4. IL4i1 metabolites form an anti-ferroptotic hierarchy.

(A) Simplified schema of ferroptosis control showing the points of chemical perturbation by erastin and RSL3. (B) Quantification of cell death of HeLa cells treated with the ferroposis inducer erastin in the presence of 200 µM PP, 4HPP, or I3P by live cell imaging using CellTox straining. Ferrostatin-1 (Fer-1) was added as a control to block erastin-induced death and has an equivalent suppressing effect as I3P. Right panel, representative CellTox staining images from an experiment similar to (B). (C) As in B, using RSL3 to induce ferroptosis. B,C: n = 3 biological replicates. The graphs are representative for three independent experiments. (D) I3P blocks lipid peroxidation induced by erastin and RSL3 determined by flow cytometry using C11-BODIPY. n = 3 biological replicates; data were analyzed by one-way ANOVA with Tukey's multiple comparisons test; ****p<0.0001. All error bars represent standard deviation. (E) Flow cytometry analysis of the anti-ferroptotic activity of I3P (200 µM) using Fer-1 as positive control. Murine NIH3T3 cells were treated with erastin, RSL3, FINO2, or ferroptocide to induce ferroptosis in the absence or presence of Fer-1or I3P and death quantified by 7-AAD and Annexin-V staining. See 'Materials and methods' for details of reagent concentrations and timing. The plots are representative for two independent experiments.

Figure 4—figure supplement 1. IL4i1 metabolites form an anti-ferroptotic hierarchy.

(A) HeLa cells were concurrently treated with erastin in the presence of increasing doses of PP, 4HPP, or I3P and death quantified over time using Incucyte imaging of CellTox straining. Ferrostatin-1 was added as a control. (B) As in (A), but using RSL3 to induce ferroptosis. (C) As in (A), but with 24 hr pre-treatment with the keto-acids before inducing ferroptosis with erastin. (D) As in (C), using RSL3 to induce ferroptosis. (E) HeLa cells were concurrently treated with erastin or RSL3 in the presence of 200 µM L-Trp, 200 µM I3P, or control DMEM medium. All experiments: n = 3 biological replicates; the graphs are representative for three independent experiments. All error bars represent standard deviation.

Figure 4—figure supplement 2. IL4i1 metabolites form an anti-ferroptotic hierarchy.

(A) HT1080 cells were concurrently treated with erastin (left) and RSL3 (right) in the presence of increasing doses of I3P and death quantified over time using Incucyte imaging of CellTox straining. Ferrostatin-1 was added as a control. (B) As in (A), comparing 200 µM of PP, 4HPP, and I3P. (C) As in (B), but with 24 hr of pre-treatment with the keto-acids before inducing ferroptosis. A–C: n = 3 biological replicates. The graphs are representative for two independent experiments. All error bars represent standard deviation. (D) Flow cytometry analysis of the anti-ferroptotic activity of I3P (200 µM) using Fer-1 as positive control. HT1080 cells were treated with erastin, RSL3, FINO2, or ferroptocide to induce ferroptosis in the absence or presence of ferrostatin or I3P and death quantified by 7-AAD and Annexin-V staining. The plots are representative for two independent experiments.