Extended Data Fig. 5. viFSP1 is a potent FSP1 inhibitor in different cell lines.
a. Cell viability of Pfa1 Gpx4-KO cells stably overexpressing hFSP1 mutants treated with viFSP1 for 48 h with indicated cell death inhibitors. Ferrostatin-1 (Fer-1), deferoxamine (DFO) and liproxstatin-1 (Lip-1) were used as ferroptosis inhibitors. b. Cell viability of Pfa1 Gpx4-KO cells stably overexpressing wildtype FSP1 enzyme from different organisms treated with viFSP1 for 48 h. Data represents the mean ± SD of 3 wells of a 96-well plate from one out of 2 independent experiments (See also Supplementary Videos 1-8). c. An initial structure–activity relationship study (SAR) for viFSP1. Pfa1 Gpx4-KO cells stably expressing wildtype FSP1 from different species were treated with the different viFSP1 analogs for 48 h. EC50 values were obtained from 3 wells of a 96-well plate from a single experiment and calculated by GraphPad Prism9. d. Heat maps represent cell viability of 4T1, B16F10, A375, and H460 cells treated with viFSP1 and RSL3 for 48 h. Data represents the mean of 4 wells of a 384-well plate from 3 independent experiments. e. 3D-Heat maps represent the same data as d and are visualized by MuSyC (https://musyc.lolab.xyz)38,39. f. Schematic models for the synergistic effect of 2 given compounds. g. Synergistic potency and efficacy. Data is calculated by MuSyC using the data from d. Data represent the mean ± 95% confidence intervals of three independent experiments. h. Heat map represents cell viability of 786-O and Rat1 cells treated with viFSP1 and RSL3 for 48 h. Data represents the mean of 4 wells of a 384-well plate (786-O) or a single well of a 96-well plate (Rat1) from one out of 3 or 2 independent experiments.