Fig. 7

Ellipticine inhibits ferroptosis by downregulation of TFRC and iron homeostasis. A Ellipticine toxicity assay in HT1080 cells. Data represent mean ± s.d. from three biological repeats. (B) Protection of RSL3-induced cell death by the indicated concentrations of ellipticine. The best protection is achieved at 0.5 µM. Data represent mean ± s.d. from three repeats and p-value is determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Ellipticine (0.5 µM) protects neomycin/ Fe³⁺ stimulated ferroptosis sensitivity to RSL3. Assays were performed as described in Fig. 3A and data represent mean ± s.d. from three biological repeats. (D) Ellipticine protects RSL3-induced (2 µM) ferroptosis, but not hydrogen peroxide-induced (100 µM) necroptosis or staurosporine-induced (2 µM) apoptosis in HT1080 cells. Data represent mean ± s.d. from three independent repeats and p-value is determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test. ns, no significance; ***, p < 0.001. (E) In vitro DPPH assay for ROS scavenging activity. Data represent mean ± s.d. from three repeats and p-value is determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance; **, p < 0.01; ***, p < 0.001. (F) Western blot for the indicated ferroptosis related proteins. The concentration used is 0.5 µM (for ellipticine) or 5 µM (for all other ferroptosis inhibiting compounds). GAPDH is the loading control. Assays were repeated twice, and representative blots are presented. (G) Ellipticine dose-dependently downregulates TFRC in either absence or presence of RSL3 in HT1080 cells. Representative western blots and quantitative results are presented. Data represent mean ± s.d. of 4 blots and p-value is determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (H) qRT-PCR analysis for TFRC and FTH1 gene expression in HT1080 cells. β-actin is the internal control. Data represent mean ± s.d. of three independent repeats and p-value is determined by student’s t-test. ns, no significance; *, p < 0.05. (I) Cell iron content assay in HT1080 cells. Data represent mean ± s.d. of three independent assays and p-value is determined by student’s t-test. *, p < 0.05. (J) qRT-PCR analysis for zebrafish tfr1a, tfr1b and fth1a gene expression. β-actin is the internal control. Data represent mean ± s.d. of three independent repeats and p-value is determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance; *, p < 0.05; **, p < 0.01. (K) Western blot for transferrin receptor expression in zebrafish total embryonic lysate. β-Actin is the loading control. A representative blot and quantitative results are presented. Data represent mean ± s.d. of 3 blots and p-value is determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. *, p < 0.05; **, p < 0.01