Fig 2: SLC39A7 transporter function is required to relieve ER stress, restore TNFR1 surface expression and induce cell death. a KBM7 FADD- SLC39A7- cells lentivirally transduced with either V5-tagged SLC39A7 or GFP were lysed and immunoblotted with the indicated antibodies, KBM7 FADD- sgRen cells serve as control. Immunoblots shown are representative of two independent experiments, asterisk (*) indicates non-specific band. b Flow cytometry analysis for TNFR1 surface expression in KBM7 FADD- SLC39A7- cells reconstituted with either V5-tagged SLC39A7 or GFP. KBM7 FADD- sgRen and empty KBM7 FADD- SLC39A7- cells serve as positive and negative control, respectively. Data shown are representative of two independent experiments. c Cell viability in KBM7 FADD- SLC39A7- cells reconstituted with either V5-tagged SLC39A7 or GFP, KBM7 FADD- sgRen and empty KBM7 FADD- SLC39A7- serve as controls. Cells were treated overnight (16 h) with TNFa (10 ng/ml), SMAC mimetic (0.5 µM), or a combination thereof. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. d Cell viability in KBM7 FADD- SLC39A7- cells stably reconstituted with GFP or the indicated V5-tagged SLC39A7 constructs. Cells were treated as indicated for 24 h and cell viability was assessed as in c. Data represent mean value ± s.d. of two independent experiments performed in triplicates. e KBM7 FADD- SLC39A7- or KBM7 FADD- cells stably reconstituted with the specified constructs were lysed and subjected to immunoblotting with the indicated antibodies. Asterisk (*) indicates non-specific band

Fig 3: Haploid genetic screens in KBM7 FADD- cells identify genes required for necroptosis. a–c Circos plots of haploid genetic screens in KBM7 FADD- cells with necroptosis induction by 10 µM SMAC mimetic birinapant (a) 100 ng/ml TNFa (b) and 1 µM SMAC mimetic and 100 ng/ml TNFa combined (c). Each dot represents a mutagenized gene identified in the resistant cell population, dot size corresponds to the number of independent insertions identified for each gene and distance from center indicates the significance of enrichment compared to an unselected control data set. Hits with an adjusted p-value < 10?10 are labeled. d Bubble plot depicting the top hits over all three screens ranked according to adjusted p-value in the TNFa screen. Bubble size corresponds to the number of independent insertions identified and color gradient reflects the significance of enrichment. e, f Multi-color competition assay (MCA) of KBM7 FADD- SpCas9 cells transduced with a GFP marker (GFP+) and sgRNAs targeting either SLC39A7 or RIPK1 (e), SP1 or TNFR2 (f), or Renilla luciferase (sgRen) as control, against cells transduced with sgRen and an mCherry marker (mCherry+). The cell populations were mixed at 1:1 ratio, treated with SMAC mimetic (1 µM) or TNFa (10 ng/ml), and analyzed after 14 days by flow cytometry. Data represent mean value ± s.d. of two independent experiments performed in duplicates, n.d. (not determined) indicates wells with no outgrowth

Fig 4: ZIP7 protein level is significantly increased in TamR and FasRL cells. (A) Immunoblotting of anti-hormone (tamoxifen or Faslodex®) resistant cell lines probed with total ZIP7 antibody and compared to MCF-7 cells. Statistical analysis was performed on densitometric data normalised to ß-actin showing the mean values of n = 4 ± SEM with *** = p < 0.001 and * = p < 0.05 versus responsive MCF-7 cells. (B) KM plotter (; www.kmplot.com) derived survival plot showing patients with higher SLC39A7 mRNA expression had a shortened relapse free survival in both the tamoxifen treated ER+ (left graph; n = 740) and whole ER+ breast cancer patient cohorts treated why any endocrine treatment or chemotherapy (right graph, n = 2061).

Fig 5: Validate the essential role of ZIP7 in ferroptosis by genetic and chemical means.A, B, C ZIP7 knockdown conferred resistance to cystine-deprivation or erastin mediated ferroptosis in MDA-MB-231 cells. A MDA-MB-231 cells were transfected with two independent siRNAs targeting ZIP7 for 48 h and treated with the cystine-deficient medium. B The knockdown efficiency of both ZIP7 siRNA in MDA-MB-231 was validated by the lower levels of ZIP7 protein (arrow) using Western blots. C MDA-MB-231 cells were transfected with indicated siRNA for 72 h and treated with 5 µM erastin for an additional 22 h before measuring the viability with CellTiter-Glo®. D, E, F ZIP7 knockdown conferred resistance to erastin-mediated ferroptosis in a zinc-dependent manner. D RCC4 cells were treated with non-targeting (NC) or individual ZIP7 siRNA for 48 h, followed by treatment of 100 µM ZnCl2, 2.5 µM erastin, or combination for an additional 24 h. E MDA-MB-231 cells were treated with siNC and ZIP7-targeting siRNAs for 50 h, followed by treatment of 100 µM ZnCl2, 10 µM erastin, or combination for 18 h. F The knockdown efficiency of ZIP7 siRNA in MDA-MB-231 was validated by Western blots (arrow for ZIP7). G, H ZIP7 inhibitor protected against erastin-mediated ferroptosis. MDAMB231 or RCC4 cells were pretreated with ZIP7 inhibitor (NVS-ZP7-4 (48 h for 231 and 24 h for RCC4) followed by the addition of erastin for an additional 24 h. The cell viability was measured by CellTiter-Glo®. The data were represented as mean ± SD (n = 3; **p < 0.01; ****p < 0.0001; two-way ANOVA).