Fig. 1: Identification of 2-BP as a potent contributor to ferroptosis.

a Volcano plot showing ferroptotic cell death changes in A375 cells treated with ~3200 metabolism-focused small molecules for 24 h, followed by 24 h RSL3 treatment. b Cell viability analysis in A375 cells pre-treated with 2-BP for 24 h, followed by 48 h treatment with GPX4 inhibitors (RSL3, ML162, ML210) and SLC7A11 inhibitor (erastin). c Cell viability analysis of A375 cells pre-treated with 2-BP for 24 h, followed by 48 h cystine starvation-induced ferroptosis, with or without Fer-1 (2 μM). Phase contrast and fluorescence images (d) and quantitative analysis of SYTOX Green-stained dead cells (e) in A375 cells pre-treated with 2-BP for 24 h, followed by 6 h RSL3 (4 μM) treatment, with or without Fer-1 (2 μM). Scale bar, 100 μm. f Lipid peroxidation analysis in A375 cells pre-treated with 2-BP for 24 h, followed by RSL3 (4 μM) treatment for 6 h, with or without Fer-1 (2 μM). g PTGS2 mRNA level detection in A375 cells pre-treated with 2-BP for 24 h, then treated with RSL3 (4 μM) for 6 h, with or without Fer-1 (2 μM). h Heatmap of cell viability in A375 cells pretreated with 2-BP for 24 h, followed by 24 h RSL3 (1 μM) treatment with various inhibitors, including z-VAD-fmk (20 μM), necrostatin-1 (2 μM), Bafilomycin A1 (10 nM), and Fer-1 (1 μM). i ROS quantification in A375 cells pretreated with 2-BP for 24 h, followed by 6 h RSL3 treatment using CM-H2DCFDA probe. Xenograft experiment with subcutaneously inoculated A375 cells in nude mice treated with IKE (20 mg/kg), 2-BP (20 mg/kg), a combination of IKE (20 mg/kg) and 2-BP (20 mg/kg), and a combination of IKE (20 mg/kg), 2-BP (20 mg/kg), and Lip-1 (10 mg/kg) (i.p., once every two days) for two weeks. Measurements include dissected tumors (j), growth curve (k), and tumor weight (l). n = 6. m, n Orthotopic xenograft tumors from RKO cells transplanted into the cecum of nude mice, treated with 2-BP (20 mg/kg) and a combination of 2-BP and Lip-1 (i.p., once every three days). Tumor volume indicated by fluorescence intensity. n = 8. Scale bar, 2 cm. Data are presented as mean ± SD. Statistical analyses: two-way ANOVA with Tukey’s multiple comparisons test for (c, e–g, i); one-way ANOVA with Tukey’s multiple comparisons test for (a, b, k, l, n). Sample sizes: (a–c, e–i) n = 3 independent experiments; (j–l) n = 6 individual mice; (m, n) n = 8 individual mice. Image in d shows representative results from three independent experiments. ns not significant.