Fig 2: The Nrf2 KO cell line was resistant to the ferroptosis inducer RSL3. (a) LDH release levels in control and Nrf2 KO cells treated with different concentrations of RSL3 for 24 h. (b) Image showing control and Nrf2 KO cells after 1 μM RSL3 treatment for 24 h in a light field. (c) LDH release levels in B. (d) Western blots of Nrf2, CTR1, TfR1, p53, and GPX4 protein expression in control and Nrf2 KO cells treated with 1 μM RSL3 at different time points. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 100 μm.

Fig 3: Copper depletion impacts invasion and metastasis.a Patient survival with CTR1 or SLC31A1 expression using METABRIC dataset. The graph depicts survival for patients with low expression (first quartile) and high expression (fourth quartile) of the SLC31A1 gene (n = 425 patients/group). Significance was calculated using Log-rank (Mantel–Cox) test. b Distribution of SLC31A1 expression with tumor stage. Significance was calculated using one-way ANOVA with Tukey post-test for comparing multiple groups. N = 433 for Stage 0-I patients, n = 702 for Stage II patients, n = 111 for Stage III–IV patients. p-value (0-I vs. II) < 0.0001, p-value (0-I vs. III–IV) = 0.0011. c Distribution of SLC31A1 mRNA expression in breast cancer subtypes. Significance was calculated using one-way ANOVA with Tukey post-test for comparing multiple groups. n = 290 for ER−/PR− patients, n = 603 for ER+/HER2− High proliferative patients, n = 619 for ER+/HER2− Low proliferative patients, n = 188 for HER2+ patients. p-value < 0.0001. d Event-free survival (EFS) for Stage 4 NED breast cancer patients from TM clinical (Chan et al. 2017). 1 cycle = 4 weeks. e (Top panel) Development of EO771.ML1 model. (Lower panel) Timeline for metastasis assay with or without TM using EO771.ML1 model. f Ceruloplasmin levels in control vs. TM-treated mice in ML1 model, 2 weeks after TM administration (n = 6/group). Analysis was performed by unpaired two-sided t-test. Center lines of box plots denote median values, top whiskers denote maxima and bottom whiskers minima. p-value = 0.0224. g Primary tumor growth in control and TM cohorts in ML1 model, measured with calipers (n = 10 in the control group, n = 9 in TM group). Analysis was performed by unpaired two-sided t-test. p-value = 0.8365. h BLI measurements of lungs in ML1 model showing metastatic burden (n = 4/group). Analysis was performed by unpaired two-sided t-test. p-value = 0.0015. i Measurement of intracellular copper (Cu) in LM2 cells using graphite furnace atomic absorption spectrometry (y-axis refers to Cu content measured at 324.75 nm wavelength). Significance was calculated using one-way ANOVA with Tukey post-test for comparing multiple groups. p-value (0 vs. 0.1 nM) = 0.0003, p-value (rest) < 0.0001. j MTT assay for viability with increasing TM concentrations, 72 h after treatment of LM2 cells (n = 4/group). Significance was calculated using One-way ANOVA with Tukey post-test for comparing multiple groups. p-value (0 vs. 1 nM) = 0.0162, p-value (0.2 vs. 1 nM) = 0.0041. k Matrigel coated transwell assay showing invasion of LM2 cells treated with TM (0.5 µM) for a total of 72 h (48-h pretreatment, followed by 24 h treatment on Matrigel-coated transwells) and rescue with addition of copper (0.5 µM CuCl2) for the last 24 h in the transwell (along with TM for that cohort). n = 3 replicates/group (7 fields of view/sample). Significance was calculated using one-way ANOVA with Tukey post-test for comparing multiple groups. p-value (Control vs. TM + Cu) = 0.0115. Representative data of at least two independent experiments are depicted. Results are expressed as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 4: IL-17 induces cellular copper uptake through induction of STEAP4.a Western blots analysis for copper trafficking-associated proteins from lysates of mouse colon epithelial organoids treated with different inflammatory cytokines for 8 h. Three independent experiments were done and representative blots were shown. Untr untreated. b IL-17 primed or unprimed mouse colon organoids were stained for STEAP4 (St4), E-cadherin (E-cad) and Ctr1 and integrin α. Scale bar, 100 µm. c SOD enzyme activity recovery from TTM inhibition in wild-type (WT) mouse organoids with or without 12 h of IL-17 priming. d SOD enzyme activity recovery in Steap4 wild-type (WT) and knockout (KO) organoids. e, f Fluorometric detection of intracellular copper in primary mouse colon organoids (e) or Ls174t cells (f). CF4 copper Fluor-4 probe, Ctrl CF4 control probe. Scale bar,100 µm. n = 5 biologically independent cell cultures. g Fluorometric detection of intracellular copper in Steap4 WT and Ctr1 knockdown cells. Cells were treated with or without IL-17 for 12 h and copper were supplemented into the medium for 6 h in all the groups. Scale bar,100 µm. n = 5 biologically independent cell cultures. h Atomic absorption spectroscopy measurement of copper content in Steap4 WT and KO Ls174t cells after copper addition. Cells were primed with or without IL-17 for 12 h and then 10 µM Cu(II) were supplemented into the medium of the indicated group for 6 h. n = 5 biologically independent cell cultures. i Copper content in cytosolic and mitochondrial fraction in Steap4 WT and KO cells, respectively. Cells were treated the same as described in panel (h). n = 5 biologically independent cell cultures. Results were normalized to cell numbers. All data show mean ± SEM. Two-tailed Student’s t test was performed for panel e (**P = 0.0057; ***P = 0.0004; ****P < 0.0001), f (***P = 0.0004; ****P < 0.0001), g (*P = 0.015; **P = 0.0027; ***P = 0.0004), h (*P = 0.0383; **P = 0.0022; ***P = 0.0009), i (***P = 0.0003; ****P < 0.0001; n.s., not significant). Data represent three independent experiments with similar results.

Fig 5: Nrf2 KO affects ferroptosis signaling pathway-induced cisplatin resistance. (a) Western blots of Nrf2, TfR1, and GPX4 protein expressions in control and Nrf2 KO cells with or without 25 μM cisplatin treatment for 24h. (b) RT -PCR verification of TfR1 mRNA expression levels in control and Nrf2 KO cells with or without 25 μM cisplatin treatment for 24h. (c) RT -PCR verification of GPX4 mRNA expression levels in control and Nrf2 KO cells with or without 25 μM cisplatin treatment for 24h. (d) FerroOrange indicated the intracellular Fe2+ content after treatment with 25 μM cisplatin for 24h. (e) Relative fluorescence intensity of FerroOrange for the cells in (d). (f) Western blots of Nrf2, CTR1, TfR1, p53, and GPX4 protein expressions in control and Nrf2 KO cells treated with 25 μM cisplatin for different lengths of time. ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001. Scale bar, 20 μm.