Extended Data Fig. 7: Mechanism of action of viFSP1.
From: Integrated chemical and genetic screens unveil FSP1 mechanisms of ferroptosis regulation
a. Enzyme kinetics in vitro assay. Schematic representation of the FSP1 enzyme activity assay using resazurin and NADH as the substrates. Representative reduction kinetics of resazurin, bar plots of the Km and relative Vmax values, Lineweaver-Burk plot, and Dixon plot in the presence of different concentrations of either NADH or resazurin with viFSP1 at indicated concentrations. Reduction kinetics, Lineweaver-Burk plot, and Dixon plot represent a single well of a 96-well plate from one out of 2 independent experiments. Bar plots represent the mean from 2 independent experiments. b. Representative resazurin reduction assay in the presence of FSP1 mutants with indicated concentrations of viFSP1. Data represents a single well of a 96-well plate from one out of 3 independent experiments. c. Cell viability of Pfa1 Gpx4-KO cells stably overexpressing wildtype mouse FSP1 or its mutants treated with viFSP1 for 24 h. Data represents the mean ± SD of 3 wells of a 96-well plate from one out of 2 independent experiments. d. Immunoblot analysis of GPX4, FSP1 (HA), and VCP expression in Pfa1 cells stably overexpressing wildtype FSP1 or its mutants from a single experiment. e. Alignment of different FSP1 orthologues. f. Comparison between predicted human (blue) and mouse (magenta) FSP1 structures in the respective viFSP1 binding pocket. Amino acid differences are highlighted in magenta/blue.
