Fig 1: Myeloid cell-specific lipin-1 deficiency reduced circulating levels of lipocalin-2 (Lcn2) and serum amyloid A1 (Saa1) in mice after ethanol feeding.Wild-type (WT) or mLipin-1KO (KO) mice were pair-fed either a control diet or an ethanol-containing diet for 10 days followed by single gavage of ethanol. (A) Relative liver mRNA levels of Lcn2 and Saa1. (B) Representative Western analysis of liver Lcn2 and Saa1. (C) Relative liver protein levels of Lcn2 and Saa1. (D) Relative serum Lcn2 levels. (E) Relative serum Saa1 levels. Results are expressed as means ± SEM (n = 4–6 mice). Means without a common letter differ, P < 0.05.
Fig 2: LIFR negatively regulates NF-?B signaling and LCN2 in the liver.a Volcano plot of genes upregulated (red) or downregulated (blue) in Lifrfl/fl;Alb-Cre mice (n = 3) relative to Lifrfl/fl mice (n = 2). Statistical analysis of RNA-seq data was performed using Cuffdiff and P values are false discovery rate (FDR)-adjusted. b Cytokine arrays of the conditioned medium of Lifr-knockout PHM cells. Boxed: the top five upregulated (red) and downregulated (blue) cytokines. c qPCR of Lcn2 in Lifr-knockout PHM cells. n = 3 samples. d ELISA of lipocalin 2 in the conditioned medium of Lifr-knockout PHM cells. n = 5, 4, and 5 wells from left to right. e ELISA of lipocalin 2 in the serum of 3-month-old Lifrfl/fl and Lifrfl/fl;Alb-Cre mice. n = 4 mice. f Pathway analysis of Lifr-knockout PHM cells with or without LIFR add-back. g, h Immunoblotting of p-p65, p65, and LIFR in LIFR-knockdown (g) and LIFR-overexpressing (h) Mahlavu cells. i, j Pathway analysis of livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice that received hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase and oncogenes (i: ß-catenin + YAP; j: myrAKT + RasV12). k, l qPCR of Lcn2 in livers described in i and j, respectively. n = 3 samples per mouse; n = 2 mice per group. m, n Immunohistochemical staining (m) and quantification (n) of Lcn2 in livers described in i and j, respectively. n = 6 mice. o Immunoblotting of Lifr, p-p65, p65, p-Stat3, Stat3, and Gapdh in livers of Lifrfl/fl and Lifrfl/fl;Cre-ERT2 mice, 28 days after hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase, myrAKT, and RasV12. From day 7, all mice received 5-day tamoxifen treatment. p, q Immunohistochemical staining (p) and quantification (q) of Lcn2 in livers of the mice that received control or LIFR-expressing adenovirus 3 days and 17 days after hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase, myrAKT, and RasV12. Scale bars, 200 µm. n = 4 mice. Statistical significance in c–e, n, and q 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 3: 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 4: Cerebrovascular changes associated with S. epidermidis infection in the neonatal hippocampus(A) IPA regulatory network analysis identified an increase in lcn2 (red) and as a molecule involved in neuroinflammatory responses. Orange dashed lines predict activation, blue dashed lines predict inhibition and gray dashed lines indicate non-predicted responses.(B–I) Representative image of CD31+ immunostained capillaries (brown color). CD31 capillary length was quantified in PND5 mice (24 h after S. epidermidis infection) in MDG (C) and CA1.SR (G) hippocampal subregions. CD31 capillary diameter in MDG (D) and CA1.SR (H). AQP4 positive capillary length in MDG (E) and CA1SR (I) was quantified in PND5 mice (24 h after S. epidermidis infection). AQP4/CD31 length ratios in MDG (F) and CA1SR.(L). Data are presented as median, 10–90th percentile. Statistical comparison between the S. epidermidis and saline groups was performed using the non-parametric Mann-Whitney test. **p < 0.01; ***p < 0.001 (see also Figure S1).
Fig 5: 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.
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