(A) Reaction mechanism of L-amino acid oxidases (LAAOs). (B) Construct design to express venom LAAOs in mammalian cells. The N. naja LAAO variants contain the human VEGF signal sequence and a …
Source data for the graphs in Figure 1.
(A) Multiple sequence alignment of Calloselasma rhodostoma, N. naja, LAAO, and Mus musculus and Homo sapiens IL4i1. Sites displayed in green represent residues used to generate enzyme-dead point …
(A) Human or mouse IL4i1 fail to induce cell death of HeLa cells at equivalent concentrations to Naja naja LAAO (all enzymes added at 2.5 µg/ml) (n = 3 biological replicates). (B) Representative …
Source data for the graphs in Figure 2.
(A) Recombinant murine IL4i1 and the enzyme-dead variant are glycosylated in their secreted forms. (B) WT and mutant murine IL4i1 activity, quantified by H2O2 production with 1 mM of L-Phe as …
(A–C) THP-1 human monocytic cells were trated for 24 hr with PP, 4HPP, or I3P (200 µM) and RNAseq analysis was used to quantify gene expression changes compared to untreated controls. (D) Top hits …
(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 …
Source data for the graphs in Figure 4.
(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. …
(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. …
(A) Cell-free scavenging activity of 200 µM PP, 4HPP, I3P, ascorbic acid (Asc), and Fer-1 determined by changes in the absorbance at 517 nm of the stable radical DPPH relative to H2O control. n = 4 …
Source data ffor the graphs in Figure 5.
(A) Absorption (grey line) and emission (light blue line) spectra of I3P in neutral aqueous solution. (B) Flow cytometric evaluation of I3P uptake (200 µM, 24 hr) by HeLa cells. Data acquired using …
(A) I3P and 4HPP induce the expression of SLC7A11 and HO-1 at protein level. HeLa cells were treated with erastin in the presence of 200 µM of PP, 4HPP, and I3P for the indicated time and SLC7A11 or …
(A) Heat map of differentially expressed genes in HeLa cells after 24 hr incubation with IL4i1 (WT or K351A inactive mutant [Figure 2—figure supplement 1])-conditioned DMEM, 200 µM I3P, or untreated …
Source data for the graphs in Figure 6.
(A) Quantification of RSL3-induced ferroptosis in HeLa cells in the presence of IL4i1 conditioned DMEM. WT but not K351A mutant IL4i1 conditioned medium (1 µg/ml) suppressed ferroptosis (left). The …
(B) ROS quantification in HeLa cells treated for 4 hr with WT and mutant LAAO by flow cytometry using the ROS probe H2DCFDA. 200 µM H2O2 was used as a positive control. n=3 biological replicates, …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Chemical compound, drug | Doxycycline | Sigma | D9891 | 1 µg/ml |
Chemical compound, drug | PNGaseF | New England Biolabs | P0704 | |
Chemical compound, drug | Erastin | Selleckchem | S7242 | |
Chemical compound, drug | (1S, 3R)-RSL3 | Selleckchem | S8155 | |
Chemical compound, drug | Catalase | Sigma | C1345 | |
Chemical compound, drug | Zinc (II) protoporphyrin IX | Sigma | 691550 | |
Chemical compound, drug | Ketoconazole | Acros Organics | 455470010 | |
Chemical compound, drug | Ferrostatin-1 | Sigma | SML0583 | 2 µM |
Chemical compound, drug | Liproxstatin-1 | Selleckchem | S7699 | 1 µM |
Chemical compound, drug | Indole-3-pyruvic acid | Sigma | I7017 | |
Chemical compound, drug | Sodium phenylpyruvate | Alfa Aesar | H56767.06 | |
Chemical compound, drug | 4-Hydroxyphenylpyruvic acid | Sigma | 114286 | 200 µM |
Chemical compound, drug | 2,2-Diphenyl-1-pikryl-hydrazyl | Sigma | D9132 | |
Chemical compound, drug | Horse radish peroxidase | Sigma | P8375 | |
Peptide recombinant protein | Recombinant human IL4i1 | R and D systems | 5684-AO-020 | |
Sequence-based reagent | HO-1 siRNA | Invitrogen | 4390824 (S6673) | 50 nM |
Sequence- based reagent | Scrambled siRNA | Invitrogen | 4390843 | 50 nM |
Antibody | Anti-Slc7a11 (rabbit monoclonal) | Cell Signaling Technology | 12691 | 1:1000 |
Antibody | Anti-HO-1 (rabbit polyclonal) | Enzo | ADI-SPA-895 | 1:1000 |
Commercial assay, kit | CellTox Green | Promega | G8731 | 1:4000 |
Commercial assay, kit | Amplex UltraRed | Thermo Fisher Scientific | A36006 |
I3P RNAseq GO terms.