Journal: Frontiers in immunology

Article Title: Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation.

doi: 10.3389/fimmu.2020.01684

Figure Lengend Snippet: FIGURE 2 | Factor H deficiency increases tubular injury after hemolysis. (A,B) Assessment of acute tubular injury by mRNA renal expression of Kim1 (Havcr1) (A) and NGAL (Lcn2) (B), after PBS or PHZ injection (day 1, 2, and 4) in WT and hepatoFH−/−mice. (C) Examples of NGAL staining (false green color) in tubular cells, by IF on frozen kidneys after PBS or PHZ injection in WT and HepatoFH−/−mice (5x). (D) Renal expression of Heme oxygenase 1 (HO-1, Hmox1) mRNA after PBS or PHZ injection (day 1, 2, and 4) in WT and hepatoFH−/−mice. (E) Heatmap summarizing the impact of hemolysis in renal mRNA expression: assessment of (Continued)

Article Snippet: C3 (Hycult Biotech, HM1065), C5b-9 (Abcam, ab55811), polyclonal anti-serum to human FH (Quidel, A312), goat IgG (Jackson Immunoresearch, 005-000-003), C5aR1 (Hycult Biotech, mAb 20/70), CD31 (Abcam, ab124432), and NGAL polyclonal IgG (R&D system, AF1857) were stained for 2 h at RT and revealed by secondary Donkey anti-Goat AF647 (ThermoScientific, A-21447) or goat anti-rat AF647 (ThermoScientific, A-21434).

Techniques: Expressing, Injection, Staining

Journal: Frontiers in immunology

Article Title: Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation.

doi: 10.3389/fimmu.2020.01684

Figure Lengend Snippet: FIGURE 3 | LPS-induced but not Cisplatin-induced tubular injury is exacerbated by FH deficiency. WT and hepatoFH−/−received intraperitoneal injection of PBS, cisplatin (15 mg/kg) or LPS (LPS-EB from E. coli O111:B4) (2 mg/kg) at Day 0 and were sacrificed at Day 2. Renal mRNA expression in LPS treated mice of NGAL (A), Kim-1 (B), and HO-1 (C) or Cisplatin treated mice of NGAL (Lcn2) (D), Kim-1 (Havcr1) (E) and HO-1 (Hmox1) (F) were measured by RTqPCR. P-values are derived from two way ANOVA with Sidak correction: ***P < 0.0005; ****P < 0.0001. Values are mean +/- SEM. Cis, cisplatin. Data from two independent experiments, experimental group containing between 2 and 5 mice.

Article Snippet: C3 (Hycult Biotech, HM1065), C5b-9 (Abcam, ab55811), polyclonal anti-serum to human FH (Quidel, A312), goat IgG (Jackson Immunoresearch, 005-000-003), C5aR1 (Hycult Biotech, mAb 20/70), CD31 (Abcam, ab124432), and NGAL polyclonal IgG (R&D system, AF1857) were stained for 2 h at RT and revealed by secondary Donkey anti-Goat AF647 (ThermoScientific, A-21447) or goat anti-rat AF647 (ThermoScientific, A-21434).

Techniques: Injection, Expressing, Derivative Assay

Journal: Frontiers in immunology

Article Title: Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation.

doi: 10.3389/fimmu.2020.01684

Figure Lengend Snippet: FIGURE 5 | Purified FH controls kidney injury in hemolytic conditions. (A) Measurement of plasma FH by WB after PBS or PHZ injection (Day 1 or 4 post-injection) in WT and hepatoFH−/−mice. Each lane correspond to 1 individual mouse. (B) Assessment of mRNA renal expression of FH in total kidney tissue. (C) Plasma human FH was assessed by ELISA at day 2 in WT and hepatoFH-/- mice, injected with human FH or PBS. (D) Plasma of hepatoFH−/−mice was resolved by electrophoresis and probed for C3 cleavage to C3b in hepatoFH−/−mice, treated or not with purified human FH. Each lane correspond to one individual mouse. (E) Quantification of the α’/α bands ratio, showing the absence of C3 activation in case of FH administration. (F,G) Blood urea was measured in plasma samples collected from PHZ-injected WT (F) or hepatoFH−/−(G) mice, pre-treated with human FH or PBS, at day 0 (D0) and day 2 (D2). (H–J) Assessment of mRNA renal expression for NGAL (Lcn2) (H), Kim-1 (Havcr1) (I) and HO-1 (Hmox1) (J) at day 2 in PHZ-treated WT and hepatoFH−/−mice pre-treated with human FH or PBS. P-values are (Continued)

Article Snippet: C3 (Hycult Biotech, HM1065), C5b-9 (Abcam, ab55811), polyclonal anti-serum to human FH (Quidel, A312), goat IgG (Jackson Immunoresearch, 005-000-003), C5aR1 (Hycult Biotech, mAb 20/70), CD31 (Abcam, ab124432), and NGAL polyclonal IgG (R&D system, AF1857) were stained for 2 h at RT and revealed by secondary Donkey anti-Goat AF647 (ThermoScientific, A-21447) or goat anti-rat AF647 (ThermoScientific, A-21434).

Techniques: Clinical Proteomics, Injection, Expressing, Enzyme-linked Immunosorbent Assay, Electrophoresis, Activation Assay

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: (A) LCN2 in human sera from patients with normal pancreas controls (n=10), chronic pancreatitis (n=10), or PDAC (n=10) as well as plasma from patients with metastatic PDAC (n=69), as measured by ELISA *p<0.05; **p<0.01 (B) mRNA expression levels of lipocalin family member molecules in normal (n=10), pancreatitis (n=16) and PDAC (n=14) tissue samples. ***p<0.001 (C) Lcn2 in serum from mice with and without KRasG12D expression before and after being fed a HFD for 7 weeks as measured by ELISA (n=8). *p<0.05 (D) Pancreas mRNA levels of Lcn2 in mice fed a CD vs a HFD for 7 weeks (n=3). *p<0.05 (E) Representative immunohistochemical staining of Lcn2 from mice expressing KRasG12D fed a CD or a HFD for 7 weeks. (F) Lcn2 in fat lysates from mice with and without KRasG12D expression in mice fed a CD vs a HFD for 7 weeks (n=3–5). *p<0.05 and p=0.0512 when compared to CRE in CD. See also supplemental figure 1, 2, and 9

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemical staining, Staining

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: (A) Body weight over time after long-term consumption of CD or a HFD from mice with or without Lcn2 expression. *p<0.05 (B) Representative images from mice expressing pancreas specific KRasG12D with or without Lcn2 expression fed a HFD >50 days, visceral adipose tissue, traced with white line. (C) Representative images of H & E, Picrosirius Red (collagen stain) and immunohistochemical staining for alpha-SMA, CD45 and F4/80, from mouse pancreas with endogenous Lcn2 (top panel) or Lcn2 null (bottom panel) expressing pancreas specific KRasG12D. (D) Pathologist scoring of pancreas inflammation, fibrosis, and PanIN lesions from mice expressing pancreas specific KRasG12D, with (n=10) and without (n=10) Lcn2 expression fed a HFD for 7 weeks. *p<0.05, **p<0.01 See also Table 1 and supplemental figure 3.

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: Timeline changes in mouse body weight The weight of Lcn2 +/+ and Lcn2 −/− animals fed either a CD or HFD was recorded over time. The mean weight of each group was calculated and CD weights were compared with HFD weights for each group to determine differences in weight gain over time. * Lcn2 +/+ CD vs LCN2 −/− CD: p=0.0203 (240 days), p=0.0157 (270 days), ° Lcn2 +/+ HFD vs LCN2 −/− HFD: p <0.001, Lcn2 +/+ CD vs LCN2 −/− HFD: p> 0.05, All values are expressed as median and standard deviation (SD)

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Standard Deviation

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: (A) Survival curves of mice fed a long-term CD (KRasG12D/CRE, n=48; Lcn2−/−/KRasG12D/CRE, n=21) vs a HFD (KRasG12D/CRE, n=32; Lcn2−/−/KRasG12D/CRE, n=26) expressing pancreas specific KRasG12D, with endogenous Lcn2 (shown in black) or without whole body expression of Lcn2 (shown in gray). Corresponding p-values are shown in table 2. (B) Representative mouse pancreas H & E staining at median survival age. (C) Representative Picrosirius Red (collagen stain) and immunohistochemical staining for F4/80, CD45 and alpha-SMA of mouse pancreas from A were euthanized at around the medium survival age. Animals fed a CD are shown in the top two rows and animals fed a HFD are shown in the bottom two rows.

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: Lcn2 depletion results in increased survival The median survival of KRas G12D /CRE and Lcn2 −/− / KRas G12D /CRE animals fed either a CD or HFD long term was calculated and assessed for significant changes in survival. (Mantel-Cox test)

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques:

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: (A–B) Bioluminescent signal over time, indicating tumor growth from a syngeneic orthotopic model of PDAC using luciferase-labeled mouse PDAC cells (derived from a PDAC genetic mouse model; KRasG12D/p53−/−/pdx1-CRE) (KPC-LUC) implanted in Lcn2+/+ (n=20) and Lcn2−/− mice (n=17).***p<0.001 (C) LCN2 expression as measured by ELISA in serum from mice with or without Lcn2 expression and with (n=7) or without (n=4) tumor cell implantation in the pancreas after 3 weeks. (D) Survival curve of Lcn2+/+ and Lcn2−/− mice from A after tumor cell implantation. ****p<0.0001 See also supplemental figure 4 and 5.

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Luciferase, Labeling, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: (A) Representative mouse pancreas with endogenous Lcn2 expression (top panel) and Lcn2 null (bottom panel) after tumor cell implantation (H & E staining), Picrosirius Red (collagen stain) and immunohistochemical staining for F4/80, CD45, alpha-SMA and Ki67. See also supplemental figure 6 for staining of a representative Lcn2 null mice implanted with KPC-LUC cells which formed a tumor. (B) Serum levels of putative PDAC serum biomarkers in animals with (n=6) or without (n=6) endogenous Lcn2 expression after tumor cell implantation as measured by densitometry analysis of a cytokine array assay. **p<0.01, ***p<0.001, ****p<0.0001 (C) mRNA expression of pro-inflammatory and ECM molecules in the pancreas of animals with (n=6) or without (n=6) endogenous Lcn2 expression after tumor cell implantation. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Cancer research

Article Title: Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment

doi: 10.1158/0008-5472.CAN-16-1986

Figure Lengend Snippet: (A) mRNA expression of LCN2 and its two receptors SLC22A17 and LRP2 in normal pancreas cells (HPDE and HPNE), PDAC cells (BxPC3, HPAC, CAPAN1, CAPAN2, MIAPACA2, MPANC96) and two immortalized PSC#1 and PSC#2. (B) LCN2 expression as measured by ELISA in supernatants from various primary pancreas PSCs (n=10) (cultured to reach 70% confluency, at passage 3–4) and western blot (insert) for LCN2 of BXPC3, PSC#1 and PSC#1 treated with recombinant human LCN2 cell lysates. Recombinant human LCN2 (rhLCN2) was used as positive control. (C) Levels of pro-inflammatory molecules secreted into the media from PSC#1 after 48hrs of treatment with 500ng/ml of rhLCN2 as measured by densitometry analysis of a cytokine array assay. *p<0.05, ***p<0.001. (D) 100 ng/mL of rhLCN2 was ectopically added to PSC#1 expressing shRNA vector control or shRNA SLC22A17 and incubated for 24 and 48 hrs with 100 ng/mL of rhLCN2. The resulting effects on the mRNA expression of IL-6, MCP-1, IL-8, IL-1B, MMP-1, MMP-3, MMP-9, C5a, C5aR1, and alpha SMA were examined. rhLCN2 induced a significant upregulation of all molecules after rhLCN2 treatment. This effect is diminished upon downregulation of SLC22A17 expression. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (student t-test). See also supplemental figure 7 and 8.

Article Snippet: LCN2 ELISA Human and mouse LCN2 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) were used to measure LCN2 levels in human serum and plasma as well as mouse serum and fat lysates as per manufacturer instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Recombinant, Positive Control, shRNA, Plasmid Preparation, Control, Incubation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: LCN2 expression is elevated in CD4+ T cells and the kidneys of patients with LN. (A) Screening of the top 20 upregulated genes in PBMCs from patients with SLE (SLE; n=3) and healthy controls (C; n=3). (B) Real-time PCR validation of gene expressions in PBMCs from patients with SLE (n=6) and healthy control (HCs; n=6). (C) Expression of LCN2 in naive CD4+ T cells derived from patients with SLE and LN (n=14), patients without LN (non LN, n=16), and HCs (n=20). (D) Immunochemical staining of LCN2 in the kidneys from patients with class 2 LN (n=5), class 3 LN (n=5), class 4 LN (n=8), class 4+5 (n=3), and class 5 (n=4), respectively. Quantification of LCN2-positive area is shown in the right panel. (E) The data plots show the correlation analysis of LCN2-positive area in kidneys with the active index, chronic index, and interstitial inflammation in patients with LN (n=25). P values are determined by Mann–Whitney U test in (B), one-way ANOVA with Tukey multiple comparisons test in (C), and one-way ANOVA with Dunnett multiple comparisons test in (D). Both correlation coefficient r and P values are calculated by the Spearman r test in (E). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Original magnification, ×100. Ctrl, control.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Control, Derivative Assay, Staining, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: LCN2 expression is increased in T cells, macrophages, neutrophils, and renal TECs in LN kidneys. Representative photos of immunofluorescence staining of LCN2 and different cell type markers in renal biopsy sections of controls (Ctrl), patients with LN, and those with DN. (A) LCN2+ cells (green) and CD3 (red) are stained. (B) LCN2+ cells (green) and CD68 (red) are stained. (C) LCN2+ cells (green) and CD15 (red) are stained. (D) LCN2+ cells (green) and E-cadherin (red) are stained. Colocalization is visualized by the yellow merge of red and green signals. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). (E) Quantification of LCN2 immunofluorescence intensity in the experiments of (A–D) (n=6). One-way ANOVA with Dunnett multiple comparisons test is used. Data are shown as mean±SEM. *P<0.05, **P<0.01. Original magnification, ×100. Mφ, macrophages; Neu, neutrophils; T, T cells; TEC, tubular epithelial cells.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: LCN2 accelerates the development of LN. (A) Albuminuria is expressed as micrograms of albumin per milligram of urinary creatinine during the course of treatments in LCN2- (n=6) or PBS- (n=7) treated mice. (B) Spleen/body weight ratio of both groups. (C) Immunofluorescence staining for mouse IgG and C3. Original magnification, ×400. (D) Representative photographs of kidney sections stained with hematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Original magnification, ×100. Quantification of glomerular, tubulointerstitial, and perivascular pathology. (E) The mRNA expression of IFN-γ, IL-6, TNFα, IL-1β, and MCP-1 in the kidneys. (F) Representative photographs of CD68 and Gr-1–stained kidney sections from LCN2- or PBS-treated mice. Original magnification, ×400. Quantification of positive cells per high-power field (HPF) is shown in the right panels. (G) Representative flow cytometric analysis of Th1 cells in spleens and lymph nodes from mice treated with LCN2 or PBS. The percentage and number of Th1 cells are shown in the right two panels, respectively. Data are representative of two independent experiments. P values are determined by two-tailed unpaired t test in (A–D, F, and G) and by Mann–Whitney U test in (E). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Immunofluorescence, Staining, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: LCN2 deficiency prevents LN development by suppressing Th1 cells. (A) Percentage survival, showing animals euthanized due to pristane treatment (n=6 for WT or LCN2−/− mice, n=20 for WT+pristane or LCN2−/−+pristane mice). (B) The spleen/body weight ratio from PBS-treated WT (n=6) and LCN2−/− mice (n=6) and pristane-treated WT (n=14) and LCN2−/− mice (n=20). (C) Albuminuria and (D) serum creatinine levels from each treatment group. (E) Representative photographs of kidney sections stained with hematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Original magnification, ×100. Quantification of glomerular, tubulointerstitial, and perivascular pathology. (F) Photos and graphs of IgG and C3 deposition in glomeruli. Original magnification, ×400. (G and H) Representative flow cytometric analysis of Th1 cells in (G) splenic and (H) renal (n=4 for each group) CD4+ T cells. The percentage and number of Th1 cells are shown in the right two panels, respectively. Data are representative of three independent experiments. P values are determined by one-way ANOVA with Dunnett multiple comparisons test in (B–D, G, and H) and by two-tailed unpaired t test in (E and F). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Staining, Two Tailed Test

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: Administration of anti-LCN2 antibodies ameliorates the pathologic phenotype of lupus mice. (A) Percentage survival of mice treated with anti-LCN2 (n=8) or control antibodies (n=10). (B) Albuminuria during the course of treatments in mice treated with anti-LCN2 or control antibodies. (C) Spleen/body weight ratio in mice treated with anti-LCN2 (n=8) or control antibodies (n=8). (D) Representative photographs of kidney sections stained with hematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Original magnification, ×100. Quantification of glomerular, tubulointerstitial, and perivascular pathology. (E) Photos and graphs of IgG and C3 deposition in glomeruli. Original magnification, ×400. (F) Representative photographs of CD68- and Gr-1–stained kidney sections. Original magnification, ×400. Quantification of positive cells per high-power field (HPF) is shown in the right two panels. (G) The mRNA expression of IFN-γ, IL-6, TNFα, IL-1β, and MCP-1 in the kidneys. (H) Representative flow cytometric analysis of Th1 cells in the spleens and lymph nodes from mice treated with anti-LCN2 or control antibodies. The percentage and number of Th1 cells are shown in the right two panels, respectively. Data are representative of two independent experiments. P values are determined by two-tailed unpaired t test in (B–E), the top scatter diagram in (F), IFN-γ, IL-6, and MCP-1 in (G), and the percentage of Th1 cells in (H); and by Mann–Whitney U test in the lower scatter diagram in (F), TNFα and IL-1β in (G), and the number of Th1 cells in (H). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Control, Staining, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: LCN2 deficiency suppresses STAT4-mediated Th1 cell differentiation in vitro. (A) Representative CD4 and IFN-γ staining profile in isolated T cells from WT and LCN2−/− mice with/without recombinant LCN2 (rhLCN2) under Th1 cell–polarizing conditions. The percentage of Th1 cells is shown in the right panel. n=5 per group. (B) Protein levels of IFN-γ (n=5) in cultured supernatants of CD4+ T cells from WT and LCN2−/− mice with/without rhLCN2 under Th1 cell–polarizing conditions for 5 days. (C) Protein level of 24p3R in splenic naive CD4+ T cells (n=2). (D) Western blot analysis of the time course of phosphorylated-STAT4 (p-STAT4) expression in anti-CD3/CD28 antibody–treated CD4+ T cells from WT and LCN2−/− mice with/without rhLCN2 after exposure to IL-12. p-STAT4 expression is normalized to STAT4. Time course of p-STAT4 expression is shown in the right panel. Data are representative of three independent experiments. (E) mRNA expression for IL12Rβ2, T-bet, and IFN-γ (n=5) of CD4+ T cells from WT and LCN2−/− mice under Th1 cell–polarizing conditions. mRNA levels were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) Representative CD4 and IFN-γ staining profile in isolated T cells from MRL/lpr mice adding anti-LCN2 or control antibodies under Th1 cell–polarizing conditions. The percentage of Th1 cells is shown in the right panel. n=5 per group. P values are determined by one-way ANOVA with Dunnett multiple comparisons in (A, B, and D), and by Mann–Whitney U test in (E and F). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Cell Differentiation, In Vitro, Staining, Isolation, Recombinant, Cell Culture, Western Blot, Expressing, Control, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

doi: 10.1681/ASN.2019090937

Figure Lengend Snippet: Schematic diagram showing the contribution of LCN2 to LN. LCN2 promotes Th1 cell differentiation via the IL-12/STAT4 pathway, leading to the exacerbation of LN. During pathologic exacerbation of LN, the kidney (TECs and infiltrating leukocytes) can also serve as the source of increased LCN2, forming a positive feedback loop. Mφ, macrophages; Neu, neutrophils; TEC, tubular epithelial cells; T, T cells; P, phosphorylated.

Article Snippet: The 16-week-old MRL/ lpr mice received weekly i.p. injections of 100 μ g of a blocking monoclonal LCN2 antibody (clone 228418; R&D Systems, Minneapolis, MN) or purified rat IgG2a isotype control antibody (R&D Systems) for 4 weeks.

Techniques: Cell Differentiation

Journal: Cells

Article Title: Human Liver Organoids as an Experimental Tool to Investigate Lipocalin-2 in Hepatic Inflammation

doi: 10.3390/cells15030216

Figure Lengend Snippet: LCN2 expression in human liver organoids. ( A ) Comparison of protein expression of various liver-specific proteins between male (m) and female (f) liver tissue and organoid lysates. For liver tissue, 80 µg of protein lysate was used, while for organoids, 40 µg of protein lysates were applied for Western blotting. ( B ) mRNA expression of exemplary genes over a cultivation period of 6 passages is shown. All genes are stably expressed during long-term cultivation of organoids. ( C ) LCN2 protein expression and ( D ) mRNA expression of LCN2 increase after stimulation of organoids for 24 h with certain inflammatory cytokines. The concentrations of cytokines used are 2.5 ng/mL IL-1β, 10 ng/mL TNF-α, 10 ng/mL IL-6, and 10 µg/mL LPS. HSP90, Cyclophilin A and β-Actin protein expression in ( A , C ) are used as controls. Data are shown as mean ± SD (n ≥ 6). Please note that signals labeled in grey resulted from previous probing. Multiple comparisons of data were performed to unstimulated (ctrl) samples. Statistical significances are highlighted with asterisks, **** p < 0.0001.

Article Snippet: The primary antibody against LCN2 (AF1757, R&D Systems) was diluted 1:100 in blocking buffer and incubated overnight at 4 °C.

Techniques: Expressing, Comparison, Western Blot, Stable Transfection, Labeling

Journal: Cells

Article Title: Human Liver Organoids as an Experimental Tool to Investigate Lipocalin-2 in Hepatic Inflammation

doi: 10.3390/cells15030216

Figure Lengend Snippet: Immunofluorescence staining of LCN2 under inflammatory conditions. LCN2 expression (green) was visualized after 24 h of stimulation. Nuclei were counterstained with DAPI (blue). Organoids were stimulated with cytokines at the following concentrations: 2.5 ng/mL IL-1β, 10 ng/mL TNF-α, 10 ng/mL IL-6, and 10 µg/mL LPS. Fluorescence images were captured using a Nikon Eclipse 80i microscope at a magnification of 200×. Representative images are displayed with a scale bar indicating 100 µm.

Article Snippet: The primary antibody against LCN2 (AF1757, R&D Systems) was diluted 1:100 in blocking buffer and incubated overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Microscopy

Journal: Cells

Article Title: Human Liver Organoids as an Experimental Tool to Investigate Lipocalin-2 in Hepatic Inflammation

doi: 10.3390/cells15030216

Figure Lengend Snippet: Downstream signaling pathway activation and inhibition. ( A ) Stimulation of liver organoids with LCN2 induces inflammatory cytokines for 15 min and 1 h to detect activated downstream pathways through phosphorylation or activation of selected proteins. Representative membranes are shown (n = 6). HSP90 protein expression was used as an internal control. ( B ) NF-κB inhibitor QNZ, JNK inhibitor JNK-IN-8 and p38 inhibitor SB203580 were utilized to inhibit the signaling pathways before organoids were stimulated with 2.5 ng/mL IL-1β for 24 h. Protein expression of downstream targets and LCN2 was analyzed after 24 h. Representative Western blot images are shown (n = 6). ( C ) LCN2 mRNA expression was measured after 24 h of stimulation with IL-1β and treatment with the respective inhibitors. Data is presented as mean ± SD. Multiple comparisons of data were made to IL-1β-treated samples. Statistical significances (n = 6) are indicated with asterisks * p < 0.05, **** p < 0.0001.

Article Snippet: The primary antibody against LCN2 (AF1757, R&D Systems) was diluted 1:100 in blocking buffer and incubated overnight at 4 °C.

Techniques: Activation Assay, Inhibition, Phospho-proteomics, Expressing, Control, Protein-Protein interactions, Western Blot