Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 1. SMILE inhibits hepcidin expression by IL-6 signal. (a) Effect of SMILE on IL-6-induced hepcidin promoter activity. HepG2 cells were transfected with expression vectors encoding hepcidin promoters (hHpeicidn-luc; 100 ng or mHepcidin-luc; 200 ng) and pcDNA3-FLAG-hSMILE (3, 300 ng; 5, 500 ng) and then treated with IL-6 (20 ng/mL) for 12 h. (b) Effect of SMILE on JAK2-mediated hepcidin promoter activity. HepG2 cells were transfected with expression vectors encoding hepcidin promoters, JAK2 (+, 200 ng with hHepcidin-luc or 400 ng with mHepcidin-luc) and pcDNA3-FLAG-hSMILE (1, 100 ng; 3, 300 ng; 5, 500 ng). (c) Effect of SMILE on STAT3-induced hepcidin promoter activity. HepG2 cells were transfected with expression vectors encoding hepcidin promoters, STAT3-c (+, 200 ng with hHepcidin-luc or 500 ng with mHepcidin-luc) and pcDNA3-FLAG-hSMILE (1, 100 ng; 3, 300 ng; 5, 500 ng). (d–e) Inhibitory effect of SMILE on hepcidin mRNA expression. HepG2 cells (d) and AML12 cells (e) were infected with Ad-GFP (50, 100 MOI) or Ad-SMILE (30, 50, 100 MOI) for 36 h and then treated with IL-6 (20 ng/mL) for 12 h. All experiments were performed in duplicate or triplicate and repeated at least three times. The values are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Expressing, Activity Assay, Transfection, Infection, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 2. SMILE suppresses IL-6-induced hepcidin secretion. (a–f) Huh7 and AML12 cells were transfected with vector encoding SMILE (5 µg) and treated with IL-6 for 12 h after 2 h of serum starvation. Secreted hepcidin was measured using ELISA from culture medium. Ferroportin (FPN) expression was analyzed using western blot. Hepcidin secretion (a), FPN expression (b) and graphical representation (c) in Huh7 cells. Hepcidin secretion (d), FPN expression (e) and graphical representation (f) in AML12 cells. All gels for western blot analysis in (b,e) were run under the same experimental conditions including equal amounts (100 µg) of protein. The independent experiments were repeated at least three times. The values are presented as means ± SD. ns; not significant. * P < 0.05, ** P < 0.01 using two-tailed Student’s t-test.

Article Snippet: Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 3. SMILE interacts with and represses STAT3 binding to hepcidin promoter. (a) Subcellular co-localization of SMILE and STAT3. HepG2 cells were co-transfected with vectors encoding GFP- SMILE and FLAG STAT3 and treated with IL-6 for 12 h. Scale bar shows 10 µm. (b) Relative fluorescence of SMILE (left) and FoxO1 (right) between nucleus and cytoplasm. (c) Co-immunoprecipitation analysis showing interaction between SMILE and STAT3. HepG2 cells were transfected with vectors encoding HA-SMILE and FLAG-STAT3 and treated with IL-6 for 12 h. Gels for western blot analysis were run under the same experimental conditions. (d) ChIP assay showing inhibitory effects of SMILE on STAT3 binding activity to hepcidin promoter. HepG2 cells were transfected with vectors encoding hepcidin promoter, HA-SMILE and FLAG-STAT3 and then treated with IL-6 for 12 h. Soluble chromatin was immunoprecipitated using anti-FLAG antibody. ChIP signals were measured using Q-PCR. STAT3-RE, STAT3-response element. The values are presented as means ± SD. ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 6. Effects of SMILE knockdown on inhibiting hepcidin expression by EGCG. (a) EGCG effect on IL-6-induced hepcidin expression in mouse primary hepatocytes treated with IL-6 (20 ng/mL) and EGCG (100 µM) for 12 h. (b) Q-PCR analysis showing efficiency of SMILE knockdown. HepG2 cells were transfected with si-Con and si-SMILE for 48 h. (c) Western blot analysis showing efficiency of SMILE knockdown. HepG2 cells were transfected with si-Con and si-SMILE for 66 h. (d) Transient transfection analysis showing the effect of SMILE knockdown on EGCG-reduced hepcidin promoter activity. HepG2 cells were transfected with Hepcidin-luc (200 ng) and si-SMILE and then treated with IL-6 (20 ng/mL) and EGCG (100 µM) for 12 h. (e) Q-PCR analysis showing SMILE knockdown effect on EGCG-mediated inhibition of hepcidin expression. HepG2 cells were transfected with si-SMILE and then treated with IL-6 (20 ng/mL) and EGCG (100 µM) for 12 h. The independent experiments were repeated at least twice. The values are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Activity Assay, Inhibition, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 7. EGCG effects on LPS-induced iron metabolism in mice. (a–h) C57BL/6J mice were intraperitoneally injected with LPS (1 mg/kg, n = 7) for 12 h after 2 h of the PBS (n = 5) and EGCG (100 mg/kg, n = 5, EGCG-LPS: n = 6) injection. Serum iron levels (a). Hepcidin mRNA levels in liver (b). Serum hepcidin levels (c). IL-6, FoxO1 and SMILE mRNA levels in liver (d–f). Western blot analysis showing FoxO1 and SMILE expression (g). Graphical representation of FoxO1 (right) and SMILE (left) (h). mRNA levels were measured using Q-PCR. Gels for western blot analysis were run under the same experimental conditions. The values are presented as means ± SEM in (a–f) and ±SD in h. ns; not significant (Con vs. EGCG). * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student’s t-test.

Article Snippet: Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Injection, Western Blot, Expressing, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

doi: 10.3390/antiox9060514

Figure Lengend Snippet: Figure 8. A model for SMILE function in IL-6-mediated hepcidin expression. SMILE expression is increased by EGCG-mediated induction of FoxO1 expression. In addition, SMILE expression inhibits STAT3 binding to hepcidin promoter and subsequently contributes to suppression of hepcidin production and secretion triggered by the IL-6 signal in hepatocytes.

Article Snippet: Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Expressing, Binding Assay

Journal: Experimental and therapeutic medicine

Article Title: Reduced hepcidin expression enhances iron overload in patients with HbE/β-thalassemia: Α comparative cross-sectional study.

doi: 10.3892/etm.2021.10838

Figure Lengend Snippet: Figure 1. Serum ferritin concentrations. Results are presented as the median ferritin concentrations and show a significant increased ferritin level in HbE/β‑thalassemia patients and their parents (P<0.001) compared with healthy controls. HbE/β‑thalassemia, hemoglobin E/β‑thalassemia. Figure 3. Gene expression analysis of HEPC. Results display a significant downregulation of HEPC gene expression in HbE/β‑thalassemia patients and their parents (P=0.001) compared with healthy controls. However, there was no significant difference in HEPC gene expression in HbE/β‑thalassemia patients (P=0.208) compared to their parents. HEPC, hepcidin; HbE/β‑thalassemia, hemoglobin E/β‑thalassemia.

Article Snippet: Serum HEPC level was determined using Human HEPC ELISA Kit (cat. no. E‐EL‐H0077; Elabscience Biotechnology Co., Ltd.) according to the manufacturer's protocol.

Techniques: Gene Expression

Journal: Experimental and therapeutic medicine

Article Title: Reduced hepcidin expression enhances iron overload in patients with HbE/β-thalassemia: Α comparative cross-sectional study.

doi: 10.3892/etm.2021.10838

Figure Lengend Snippet: Figure 2. Serum hepcidin concentrations. Results show a significantly lower HEPC concentration in HbE/β‑thalassemia patients (P=0.001) compared with their parents and healthy controls. Results also demonstrate a significant decrease in HEPC concentration in parents (P=0.001) compared with healthy controls. HbE/β‑thalassemia, hemoglobin E/β‑thalassemia.

Article Snippet: Serum HEPC level was determined using Human HEPC ELISA Kit (cat. no. E‐EL‐H0077; Elabscience Biotechnology Co., Ltd.) according to the manufacturer's protocol.

Techniques: Concentration Assay

Journal: Journal of the Formosan Medical Association = Taiwan yi zhi

Article Title: Real-world experience with Ropeginterferon-alpha 2b (Besremi) in Philadelphia-negative myeloproliferative neoplasms.

doi: 10.1016/j.jfma.2020.08.021

Figure Lengend Snippet: Figure 4 Effects of Ropeg on cytokine profiles. (A) Relative change in the percentages of levels of five cytokines over time in one particular patient. The plasma cytokine levels were measured with multiplex ELISA-based Q-plex Human Cyokine HS Screen Array (Quansys Biosciences), which contained more than a dozen of cytokines. Only cytokines with significantly altered levels after treatment are shown here. (B) The absolute values of plasma hepcidin levels over time in six PV patients. Human Hepcidin ELISA Kit (Cusabio Technology) was used for the quantification.

Article Snippet: Human Hepcidin ELISA Kit (Cusabio Technology) was used for the quantification.

Techniques: Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Medicine

Article Title: The Role of Iron and Erythropoietin in the Association of Fibroblast Growth Factor 23 with Anemia in Chronic Kidney Disease in Humans

doi: 10.3390/jcm9082640

Figure Lengend Snippet: Demography and biomarkers of 225 study participants according to the stage of CKD.

Article Snippet: For c-terminal FGF23 (cFGF23), the Immutopics Human FGF-23 (C-Term) ELISA (Quidel, San Diego, CA, USA), for Klotho the Soluble alpha-Klotho ELISA (IBL, Imuno-Biological Laboratories Co., Ltd., Fujioka, Japan), for Hepcidin the Human Hepcidin Quantikine ELISA (R&D Systems Inc., Minneapolis, MN, USA) were used according to the manufacturers’ instructions.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: The Role of Iron and Erythropoietin in the Association of Fibroblast Growth Factor 23 with Anemia in Chronic Kidney Disease in Humans

doi: 10.3390/jcm9082640

Figure Lengend Snippet: Dependence of hemoglobin on biomarkers of mineral metabolism, iron metabolism, EPO concentrations, and inflammation biomarkers.

Article Snippet: For c-terminal FGF23 (cFGF23), the Immutopics Human FGF-23 (C-Term) ELISA (Quidel, San Diego, CA, USA), for Klotho the Soluble alpha-Klotho ELISA (IBL, Imuno-Biological Laboratories Co., Ltd., Fujioka, Japan), for Hepcidin the Human Hepcidin Quantikine ELISA (R&D Systems Inc., Minneapolis, MN, USA) were used according to the manufacturers’ instructions.

Techniques: Biomarker Discovery

Journal: Journal of Clinical Medicine

Article Title: The Role of Iron and Erythropoietin in the Association of Fibroblast Growth Factor 23 with Anemia in Chronic Kidney Disease in Humans

doi: 10.3390/jcm9082640

Figure Lengend Snippet: Influence of cFGF23 and iFGF23 on biomarkers of iron metabolism, EPO concentrations and inflammation biomarkers.

Article Snippet: For c-terminal FGF23 (cFGF23), the Immutopics Human FGF-23 (C-Term) ELISA (Quidel, San Diego, CA, USA), for Klotho the Soluble alpha-Klotho ELISA (IBL, Imuno-Biological Laboratories Co., Ltd., Fujioka, Japan), for Hepcidin the Human Hepcidin Quantikine ELISA (R&D Systems Inc., Minneapolis, MN, USA) were used according to the manufacturers’ instructions.

Techniques:

Journal: PLoS ONE

Article Title: Evidence Suggesting a Role of Iron in a Mouse Model of Nephrogenic Systemic Fibrosis

doi: 10.1371/journal.pone.0136563

Figure Lengend Snippet: Primary antibodies used in this study. IHC—immunohistochemistry, WB—western blot.

Article Snippet: Anti-hepcidin , Goat (polyclonal) , 1:1,000 WB , Novus Biologicals , NBP1-59337.

Techniques:

Journal: bioRxiv

Article Title: Live-cell invasive phenotyping uncovers the ALK2/BMP6 iron homeostasis pathway as a therapeutic vulnerability in LKB1-mutant lung cancer

doi: 10.1101/2023.06.14.544941

Figure Lengend Snippet: A) Schematic depicting pre-clinical trial workflow. B) Brightfield images of tumors isolated from vehicle and LDN214117 treated mice. C) Mean tumor volume from mice treated with vehicle ( 6 mice/group) and LDN214117 (7 mice/group). D) Graph depicting mean tumor weight from vehicle and LDN214117 treated mice. E) Western blot to assess BMP6 and hepcidin expression level in tumors from vehicle and LDN214117-treated mice. F) Representative images of immunohistochemistry for BMP6 and Hepcidin on tumor sections from mice treated with vehicle or LDN214117. F) Model depicting mechanism of altered iron homeostasis in LKB1-mutant tumor cells.

Article Snippet: Hepcidin Antimicrobial Peptide antibody (NBP1-59337) was from Novus Biologicals.

Techniques: Isolation, Western Blot, Expressing, Immunohistochemistry, Mutagenesis

Journal: PLoS ONE

Article Title: Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication

doi: 10.1371/journal.pone.0240949

Figure Lengend Snippet: A) transcriptional expression levels of SLC40A1 , and (B) HAMP genes in THP-1 macrophages treated overnight with 200U/ml IFN-γ measured by qRT-PCR and compared to untreated controls. C) Ferroportin protein expression assessed in THP-1 macrophages treated as in A. D) Hepcidin secretion levels in THP-1 macrophages treated as in B (magnification = 63X). E) Transcriptional expression levels of the FTH gene in THP-1 macrophages treated as in A and B. **p <0.01, ***p <0.001. All data were from three independent experiments.

Article Snippet: Hepcidin levels in culture supernatants were determined using human hepcidin DuoSet ELISA Kit (R&D Systems, MN, USA), per manufacture’s recommendations.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication

doi: 10.1371/journal.pone.0240949

Figure Lengend Snippet: A) Hepcidin secretion from THP-1 macrophages after infection with M . tuberculosis (24 hours), M . bovis BCG (24 hours), L monocytogenes (eight hours) or S . enterica (16 hours) bacilli. B) Ferroportin levels in THP- 1 macrophages eight hours post-infection with L . monocytogenes (magnification = 40X). C) Ferroportin expression in hepcidin silenced THP-1 macrophages eight hours post infection with L . monocytogenes . Hepcidin gene silencing in THP-1 cells was achieved by lentiviral based shRNA transduction and Scramble short hairpin RNAs (ShScram) were used as a negative control (magnification = 40X). ***p <0.001. All data were from three independent experiments.

Article Snippet: Hepcidin levels in culture supernatants were determined using human hepcidin DuoSet ELISA Kit (R&D Systems, MN, USA), per manufacture’s recommendations.

Techniques: Infection, Expressing, shRNA, Transduction, Negative Control

Journal: PLoS ONE

Article Title: Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication

doi: 10.1371/journal.pone.0240949

Figure Lengend Snippet: A) Ferroportin expression in IFN-γ-activated (200U/ml) macrophages eight hours post infection with L . monocytogenes , (B) 48 hours post infection with M . bovis BCG, or (C) 16 hours post infection with S. enterica (magnification = 40X). D) Hepcidin secretion in the culture supernatants of IFN-γ-activated macrophages eight hours post infection with L . monocytogenes , (E) 24 hours post infection with M . bovis BCG, or (F) 16 hours post infection with S. enterica . **p <0.01, ***p <0.001. All data were from three independent experiments.

Article Snippet: Hepcidin levels in culture supernatants were determined using human hepcidin DuoSet ELISA Kit (R&D Systems, MN, USA), per manufacture’s recommendations.

Techniques: Expressing, Infection