Fig 1: Predictive value of biomarkers and their corresponding models. ROC curves of endostatin, NGAL, cystatin C and their corresponding models for predicting failure to recover in derivation cohort. a AUCs of endostatin, NGAL and cystatin C alone for prediction. b AUCs of biomarker–clinical risk prediction models for prediction. ROC receiver operating characteristic, AUC area under the ROC, NGAL neutrophil gelatinase-associated lipocalin
Fig 2: An LCN2-neutralizing antibody enhances the ferroptosis-inducing effect of sorafenib on HCC patient-derived xenograft tumors with low LIFR expression and high LCN2 expression.a, b Quantification of immunohistochemical staining (IHC, see images in Supplementary Fig. 9a) of MDA (a) and 4-HNE (b) in tumor tissues from NSG mice bearing PDX line #5. Mice were treated with anti-LCN2 and sorafenib, alone or in combination. n = 7 mice. Statistical significance was determined by a one-way ANOVA (to compare the means among three or more groups) and a two-tailed unpaired t-test (to compare the means between two groups). Error bars are s.e.m. c, d Transmission electron microscopy images of tumor tissues from NSG mice bearing PDX line #5 (c) or #4 (d). Mice were treated with anti-LCN2 and sorafenib, alone or in combination. Blue arrows indicate normal mitochondria. Red arrows indicate shrunken mitochondria with heavily condensed membrane, and pink arrows indicate mitochondria with increased membrane density, but to a lesser extent than those indicated by red arrows. Scale bars, 500 nm. e Model for the role of a LIFR-NF-?B-LCN2 axis in liver tumorigenesis and ferroptosis. Source data are provided as a Source Data file.
Fig 3: LIFR and SHP1 positively regulate ferroptosis while LCN2 negatively regulates ferroptosis.a-d LIFR-knockdown (a, b) or SHP1-knockdown (c, d) HT1080 cells were treated with 10 µM erastin for 0, 5, or 10 h. a, c: staining of 7-aminoactinomycin (7-AAD) and annexin V. b, d: the percentage of annexin V and 7-AAD double-negative population. e The percentage of annexin V and 7-AAD double-negative population in LIFR-knockdown HT1080 cells treated with 0.5 µM RSL3 for 12 h, 50 µM FIN56 for 6 h, or 10 µM FINO2 for 24 h. Supplementary Figure 6e shows representative flow cytometry plots. f The percentage of annexin V and 7-AAD double-negative population in SHP1-knockdown HT1080 cells treated with 0.5 µM RSL3 for 12 h, 50 µM FIN56 for 6 h, or 10 µM FINO2 for 24 h. Supplementary Figure 6f shows representative flow cytometry plots. g The percentage of annexin V and 7-AAD double-negative population in LCN2-knockdown HT1080 cells treated with 10 µM erastin for 0, 4, or 8 h, alone or in combination with liproxstatin-1 (lip-1, 10 µM) or DFO (100 µM). Supplementary Figure 7b shows representative flow cytometry plots. h The percentage of annexin V and 7-AAD double-negative population in LCN2-knockdown HT1080 cells treated with 0.5 µM RSL3 for 10 h, 50 µM FIN56 for 3 h, or 10 µM FINO2 for 12 h, alone or in combination with liproxstatin-1 (lip-1, 10 µM) or DFO (100 µM). Supplementary Fig. 7c shows representative flow cytometry plots. i, j Lipid peroxidation levels in LIFR-knockdown (i) and SHP1-knockdown (j) HT1080 cells treated with 10 µM erastin for 3 h or 0.5 µM RSL3 for 4 h. k Lipid peroxidation levels in LCN2-knockdown HT1080 cells treated with 10 µM erastin for 3 h or 0.5 µM RSL3 for 4 h, alone or in combination with liproxstatin-1 (10 µM) or DFO (100 µM). Lipid peroxidation levels were gauged by C11-BODIPY staining in i–k. Statistical significance in b and d–k was determined by a two-tailed unpaired t-test. Error bars are s.e.m. n = 3 samples per group. Source data are provided as a Source Data file.
Fig 4: Loss of LIFR activates NF-?B signaling through SHP1, leading to upregulation of LCN2.a HEK293T cells were transfected with HA-FLAG-SHP1 and SFB-tagged GFP or LIFR. LIFR-SFB protein was pulled down with S-protein beads, followed by immunoblotting with antibodies against FLAG and HA. b HEK293T cells were transfected with MYC-SHP2 and SFB-tagged GFP or LIFR. LIFR-SFB protein was pulled down with S-protein beads, followed by immunoblotting with antibodies against FLAG and MYC. c HEK293T SFB-GFP and SFB-LIFR stable cell lines were infected with the scrambled (Scr) or sh-SHP1 lentivirus, followed by transfection with a K63-specific mutant of His-Xpress-ubiquitin (Ub). 48 h later, cells were subjected to pulldown with nickel beads and immunoblotting with antibodies against TRAF6 and Xpress. d Control and LIFR-overexpressing PLC/PRF/5 cells were transduced with SHP1 shRNA and immunoblotted with the indicated antibodies. e Control (Scr) and LIFR-knockdown HEK293T cells were transfected with FLAG-TRAF6. 48 h later, cells were immunoprecipitated with a FLAG-specific antibody and immunoblotted with antibodies against LIFR, SHP1, and FLAG. f, g qPCR of LCN2, LIFR, and RELA in HEK293T (f) and PLC/PRF/5 (g) cells transduced with LIFR shRNA alone or in combination with p65 shRNA. n = 3 technical replicates. h qPCR of Lcn2, Lifr, and RelA in control and Lifr-knockout PHM cells transduced with the scrambled shRNA (Scr) or p65 shRNA. n = 3 technical replicates. i, j Immunohistochemical staining (i) and quantification (j) of Lcn2 in livers from Lifrfl/fl (F/F) and Lifrfl/fl;Alb-Cre (LKO) mice, 57 days after hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase, myrAKT, RasV12, and shRNA (sh-p65, sh-Lcn2, or scrambled). n = 10, 8, 10, and 12 mice from left to right. Scale bars, 200 µm. k, l Liver weight (k) and liver-to-body weight ratio (l) of the mice described in i and j. n = 10, 8, 10, and 12 mice from left to right. m H&E staining of livers described in i and j. Scale bars, 300 µm. Statistical significance in f-h and j-l was determined by a two-tailed unpaired t-test. Error bars are s.e.m. Source data are provided as a Source Data file.
Fig 5: LCN2 mediates ferroptosis resistance and is a therapeutic target for enhancing sorafenib efficacy.a qPCR of Lifr and Lcn2 in Lifr-knockout PHM cells transduced with the scrambled (Scr) or Lcn2 shRNA. n = 3 samples. b Lifr-knockout PHM cells were transduced with Lcn2 shRNA and treated with 10 µM erastin or 20 µM sorafenib. Cell viability was determined by a CCK8 assay. n = 5 wells. c Fe2+ levels in liver tissues of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice in the absence or presence of hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase, myrAKT, and RasV12. n = 4 mice. d Fe2+ levels in control and Lifr-knockout PHM cells transduced with the scrambled (Scr) or Lcn2 shRNA. n = 3 wells. e Malondialdehyde (MDA) levels in control and Lifr-knockout PHM cells transduced with the scrambled (Scr) or Lcn2 shRNA. n = 5 wells. f Glutathione (GSH) levels in control and Lifr-knockout PHM cells transduced with the scrambled (Scr) or Lcn2 shRNA. n = 3 wells. g Immunoblotting of Lifr, Slc7a11, Fsp1, Gpx4, and Gapdh in control and Lifr-knockout PHM cells transduced with the scrambled (Scr) or Lcn2 shRNA. h Immunoblotting of LCN2, LIFR, p-p65, p65, and GAPDH in tumors generated from four PDX lines of HCC. i ELISA of lipocalin 2 in the serum collected from NSG mice bearing PDX line #5. Mice were treated with anti-LCN2 and sorafenib, alone or in combination. n = 7 mice. j, k Growth curves of tumors in NSG mice bearing PDX line #4 (j) or #5 (k). When tumors grew to 50–150 mm3, mice were treated with 100 µg anti-LCN2 and 30 mg kg-1 sorafenib, alone or in combination. The treatments were given 6 days a week for 4 weeks. Statistical significance was determined by a two-way ANOVA. n = 6 mice in j and n = 7 mice in k. l Endpoint tumor images of the mice described in j and k. Statistical significance in b–f and i was determined by a two-tailed unpaired t-test. Error bars are s.e.m. Source data are provided as a Source Data file.
Supplier Page from Abcam for Human Lipocalin-2 ELISA Kit (NGAL)