Fig. 2: icFSP1 indirectly inhibits FSP1 by inducing condensate formation.
a, Schematic representation of the FSP1 enzyme activity assay using resazurin as the substrate. b, Representative dose–response curves for the effect of iFSP1 and icFSP1 on hFSP1 activity using recombinant purified hFSP1 protein. Data represent the mean ± s.d. of 3 wells of a 96-well plate from one of three independent experiments. c, Representative time-lapse fluorescence images acquired immediately after treatment of wild-type Gpx4 Pfa1 cells stably overexpressing hFSP1–EGFP–Strep with 2.5 µM icFSP1. Scale bars, 10 μm. Representative results from one of three independent experiments. See also Supplementary Video 1. d, Number of condensates per cell quantified from time-lapse images at different time points (0, 60, 120, 180 and 240 min) after treatment obtained from one of two independent experiments. Dots represent each cell and n corresponds to cell number (n = 129, 124, 130, 130 and 134 (left to right)). P values were calculated by one-way ANOVA followed by Dunnett’s multiple-comparison test. e, Representative time-lapse fluorescence images before and after treatment of Gpx4-knockout Pfa1 cells stably overexpressing hFSP1–mTagBFP with 10 µM icFSP1 in FluoroBrite DMEM containing propidium iodide (PI; 0.2 µg ml–1). Cells were prestained with 5 µM Liperfluo for 1 h. Scale bars, 10 μm. Representative results from three independent experiments. Differential interference contrast (DIC) is displayed with refractive index (RI). See also Supplementary Video 2.
