Fig. 1

TGF-β1/SMAD3-transactived HLF promotes TNBC ferroptosis resistance, proliferation, metastasis and chemoresistance through GGT1 signaling. a Real-time PCR analysis of HLF in 40 pairs of TNBC samples and their corresponding peritumoral normal samples. b The overall survival time after surgery of the patients in TNBC patients was compared between the “HLF low, n = 40” and “HLF high, n = 40” groups. c MB-231/HCC1937 cells were transfected with siSMAD3 followed by western blot analysis. d MB-231 transfected with siSMAD3 or siNC was subjected to dual immunofluorescence staining. Representative images are shown. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 20 μm. e TAMs were transfected with siTGF-β or siNC. MB-231/HCC1937 cells were cocultured with TAMs siTGF-β or TAMs siNC followed by western blot analysis. f MB-231 cells subjected to ChIP assay with anti-SMAD3 or anti-IgG antibody. g Venn diagram representing the overlap between HLF targets, identified by ChIP-seq, and RNA-seq. h Real-time PCR analysis of the mRNA expression of HLF and GGT1 in shHLF or control TNBC cells. i Western blot analysis of the protein expression of HLF and GGT1 in shHLF or control TNBC cells. j MB-231 cells subjected to ChIP assay with anti-Flag or anti-IgG antibody. k The ratio of reductive GSH to oxidative GSH was measured by the GSH/GSSG quantification kit in the indicated cells. l Substantially lower GPX4-specific activity was detected in the indicated cells using PCOOH as a substrate. m Total iron in the indicated cells was analyzed using Iron Assay Kit. n Oxidative stress in the indicated cells was assessed by the levels of lipid ROS. o MB-231 shHLF or control cells were subcutaneously injected into nude mice (n = 6) for xenograft assay. Tumor average weight in each group was shown. p Lung H&E staining of nude mice inoculated MB-231 shHLF and control cells via tail vein for 12 weeks. The number of lung metastatic foci in each group (n = 7) were also calculated, *p < 0.05. q Cell survival curves of shHLF and control TNBC cells treated with cisplatin with a dose escalation from 0 to 100 μg/ml. Data are presented as mean ± SD (n = 4) from one of three independent experiments. r Representative xenograft images at 6 weeks after the injection of indicated TNBC cells into nude mice. The weight of xenografts in each group (n = 6) was calculated. s Representative microscopic images of pulmonary metastatic lesions at 12 weeks after the injection of indicated TNBC cells into the tail vein of nude mice. The number of lung metastatic tumors in each group (n = 6) was calculated. t Kaplan–Meier analysis of overall survival of TNBC patients with high or low HLF and GGT1. p < 0.001. All results are presented as the mean ± SD, and statistical significance was assessed using a two-tailed Student’s t test. *p < 0.05