Extended Data Fig. 8: Targeting of FSP1 by icFSP1 as potential anti-cancer therapy using mouse cells.
a. Pharmacokinetic (PK) parameters of icFSP1 and iFSP1. Plasma concentration was measured after single i.p. administration (10 mg/kg). Data represents mean ± SD from 4 mice of one experiment. b. Summary of microsomal stability analysis of icFSP1 and iFSP1. c. Body weight of tumor-baring mice during the treatment of mice with icFSP1 (50 mg/kg i.p. twice a day, n = 7) and vehicle (n = 6) as a control. These mice are the same as in Fig. 4f. Data represents mean ± SD from one out of 2 independent experiments. d. At the end of the in vivo pharmacological studies, tumors were dissected, cryosectioned and stained with anti-HA to visualize hFSP1 and with anti-4-HNE to visualize the lipid peroxidation breakdown product. Representative confocal microscopy images are shown from one out of 2 independent experiments. Arrowheads indicate FSP1 condensates. Scale bars, 20 µm or 10 µm. e. Immunoblot analysis of HA (FSP1) and VCP expression of B16F10 Gpx4 KO/Fsp1 KO cells stably overexpressing FSP1-WT or the Q319K mutant. Representative results from one out of 2 independent experiments. f. Cell viability was measured after treating B16F10 Gpx4 KO/Fsp1 KO cells stably overexpressing FSP1-WT or Q319K mutants with icFSP1 for 48 h. Data represent mean ± SD of 3 wells from one out of 2 independent experiments. g. icFSP1 inhibits tumor growth of hFSP1 WT but not of FSP1-Q319K expressing cells in vivo. B16F10 Gpx4 KO/Fsp1 KO cells stably expressing hFSP1 WT or Q319K were subcutaneously implanted into C57BL/6J mice (n = 33, in total). Treatment with vehicle (n = 10 for WT and 8 for Q319K) or icFSP1 (50 mg/kg i.p. twice a day, n = 8 for WT and 7 for Q319K) was started from day 6 after randomization. Data represents the mean ± SEM from one out of 2 experiments. P values were calculated by two-way ANOVA followed by Tukey’s multiple comparison test. h. At the end of the in vivo pharmacological studies, tumors were dissected, cryosectioned and stained with anti-HA to visualize hFSP1 and with anti-4-HNE to visualize the lipid peroxidation breakdown product. Representative confocal microscopy images are shown from one out of 2 independent experiments. Arrowheads indicate FSP1 condensates. i. Immunoblot analysis of FSP1 and actin expression of H460 WT and FSP1 KO cells with doxycycline (Dox)-inducible FSP1-WT or the Q319K mutant after Dox induction with indicated concentrations for 48 h. The left part of the blot is already shown in Extended Data Fig. 4f. Representative results from one out of 2 independent experiments. j. Confocal microscopy images after treating H460 FSP1 KO cells with doxycycline-inducible FSP1-WT or the Q319K mutant with 1 µg/mL of Dox for 48 h, followed by treatment with and without 10 µM icFSP1 for 240 min. Scale bars, 10 μm. Representative images from one out of 2 independent experiments.