Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: FPN1 is localized to the plasma membrane of K562 erythroblast cells and primary erythroblasts. (A) K562 cells were transiently transfected with plasmids to express FPN1GFP and the plasma membrane marker pDsRed-Monomer F for 24 hours to examine their colocalization. (B) Endogenous FPN1 localized mainly to the plasma membrane of primary erythroblasts. Fetal liver erythroblasts were incubated with rabbit anti-FPN1 and mouse anti-TfR1 Abs and were then labeled with FITC-donkey anti–rabbit and Cy3-donkey anti–mouse Abs. Pictures were taken with the Leica SP5 confocal microscope with 63×/1.4 oil objective lens under room temperature. (C) Immunoprecipitations of cell-surface proteins showed that FPN1 localizes on the plasma membrane. Fetal liver erythroblasts were incubated with or without hepcidin for 24 hours, then the cell-surface proteins were labeled with or without Sulfo-NHS-LC-Biotin. The cell lysates were incubated with streptavidin gel, and the immunoprecipitated proteins were immunoblotted with anti-FPN1 and anti-TfR1 Abs.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Transfection, Marker, Incubation, Labeling, Microscopy, Immunoprecipitation

Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: FPN1 expression is regulated by hepcidin in transfected erythroblast cell lines in a dose- and time-dependent manner as indicated by FACS analysis. (A) Validation of the use of FACS analysis to measure iron-dependent TfR1 expression. K562 cells were treated with 100μM FeNTA or 100μM DFO for 18 hours, and cells were then labeled by APC-labeled TfR1 Abs and checked by FACS analysis. (B) Expression of FPN1 increases TfR1 expression in K562 cells. K562 cells were transiently transfected with pEGFP-N1-FPN1 to express the fusion protein FPN1GFP for 24 hours, and the expression of TfR1 was then measured by FACS. Expression of FPN1 in K562 (C) and MEL (D) cells was down-regulated by hepcidin. Cells were transiently transfected with pEGFP-N1 or pEGFP-N1-FPN1 and incubated with 1 μg/mL hepcidin for 24 hours, and the GFP-positive cells were then measured by FACS. (E) Time-dependent regulation of FPN1 by hepcidin. K562 cells were transfected with pEGFP-N1-FPN1 or pEGFP-N1 vectors for 12 hours, and then incubated with or without 1 μg/mL hepcidin. The GFP-positive cells were measured by FACS at the indicated time points. (F) Dose-dependent regulation of FPN1 by hepcidin. K562 cells transfected with pEGFP-N1-FPN1 or pEGFP-N1 vectors were incubated with hepcidin of indicated concentrations for 24 hours. The GFP-positive cells were measured by FACS. (G) Hepcidin treatment reverses the FPN1-induced increase of TfR1 in K562 cells. K562 cells were transiently transfected with pIRES2EGFP-FPN1 and incubated with or without 1 μg/mL hepcidin for 24 hours. The expression levels of TfR1 were measured by FACS analysis in GFP-positive cells. All of the experiments (A-G) were independently repeated at least twice with triplicate samples assessed at each time point. a.u. indicates arbitrary unit. **P < .01, ***P < .001.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Expressing, Transfection, Labeling, Incubation

Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: FPN1 protein expression levels in transfected erythroblast cell lines are directly regulated by hepcidin in a dose- and time-dependent manner. (A) Hepcidin regulates FPN1 expression and intracellular iron status of K562 cells. K562 cells transiently transfected with pEGFP-N1 or pEGFP-N1-FPN1 plasmids to generated cells that expressed GFP alone, or the FPNGFP fusion proteins, and were incubated with or without 1 μg/mL hepcidin for 24 hours. (B) Time-dependent regulation of FPN1 by hepcidin in K562 cells. K562 cells were transfected with pEGFP-N1-FPN1 for 12 hours, then incubated with 1 μg/mL hepcidin at the indicated time points. (C) Dose-dependent regulation of FPN1 by hepcidin. K562 cells were transfected with pEGFP-N1-FPN1 and incubated with hepcidin at the indicated concentrations for 24 hours. Total cell lysates were separated with 4%-20% Tris-glycine gels and then were transferred to nitrocellulose membranes. The membranes were then incubated with Abs for GFP, FPN1, TfR1, IRP2, IRP1, and α-tubulin, respectively. All of the experiments were repeated at least twice.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Expressing, Transfection, Generated, Incubation

Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: Distinctive expression patterns of the 2 FPN1 transcripts during differentiation of fetal liver erythroblasts. (A) Expression of c-Kit, TER119, and TfR1 in the process of differentiation of erythroblasts. After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts. At different time points after being cultured in differentiation medium, erythroblasts were harvested, and total RNA or protein was prepared for real-time qPCR (B-C) or Western blot (D) analysis. The real-time qPCR results were normalized with actin mRNA. The real-time qPCR experiments were repeated 3 times with triplicate samples each time; the Western blot analyses were repeated at least twice; a.u. indicates arbitrary units.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Expressing, Cell Culture, Incubation, Labeling, Western Blot

Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: Hepcidin regulates endogenous FPN1 expression and intracellular iron levels of fetal liver erythroblasts. (A) Expression of endogenous FPN1 in erythroblasts was decreased after a 24-hour hepcidin treatment. Erythroblasts in proliferation medium were incubated with 1 μg/mL hepcidin for 24 hours, and protein expression was then measured by Western blot analysis for FPN1 and TfR1 in membrane fractions of sample lysates and for L-ferritin in soluble fractions of sample lysates. Ponceau S staining of a specific portion of the gel was used as a loading control. (B) Expression of FPN1 at different time points of incubation with hepcidin during growth in proliferation medium. (C) IRP2 activity was decreased by hepcidin in erythroblasts. Erythroblasts were incubated with 1 μg/mL hepcidin for 24 or 48 hours or with 100μM FeNTA or DFO for 18 hours, and samples were then prepared with or without β-mercaptoethanol to measure IRP activity by electrophoresis mobility shift assay. The density of the bands was analyzed with ImageJ (http://imagej.nih.gov/ij/) and normalized to IRP1 activity after β-mercaptoethanol treatment and statistical comparisons were performed. (D) Western blot analyses showed decreased IRP2 protein levels in samples treated by hepcidin. The cells were treated as described in panel C. (E) Hepcidin increases intracellular iron contents of erythroblasts. Erythroblasts in 1 well of a 6-well plate in proliferation medium were loaded with 55Fe by incubation with 5 μCi 55Fe (0.185 Bq; 0.2 μg) for 2 hours and were then washed twice and incubated with 1 μg/mL hepcidin for 24 or 48 hours, after which time intracellular 55Fe levels were measured with a Liquid Scintillation Counter. (F) Hepcidin increases intracellular iron levels in a dose-dependent manner. Erythroblasts in proliferation medium were loaded with 55Fe as described in panel E and were then incubated with hepcidin of indicated concentrations for 24 hours, after which time intracellular 55Fe levels were measured with Liquid Scintillation Counter. a.u. indicates arbitrary unit. Experiments in panels A through D were repeated ≥ 3 times, and experiments in E and F were repeated twice with triplicate samples for each time point. *P < .05, **P < .01, and ***P < .001.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Expressing, Incubation, Western Blot, Staining, Activity Assay, Electrophoresis, Mobility Shift

Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: Expression of FPN1 in erythroblasts of mouse BM increased in mice maintained on a low-iron diet. (A) Expression of hepcidin mRNA was decreased to barely detectable levels in the livers of mice on the low-iron diet. Total RNA was prepared, and hepcidin expression was measured by real-time qPCR and normalized to actin mRNA levels. a.u. indicates arbitrary unit. (B) FPN1 expression increased in Ter119-positive erythroblasts from the BM of mice maintained on a low-iron diet compared with mice on the control diet. Expression of FPN1 and L-ferritin was measured by Western blot analyses in total cell lysates. A distinctive band in Ponceau S staining was used as a loading control. (C) FPN1 expression increased in the spleens of mice maintained on the low-iron diet. Expression levels of FPN1, TfR1, L-ferritin, and α-tubulin were measured by Western blot analyses. (D) FPN1 expression, unlike TfR1, is not regulated by iron status in cultured primary erythroblasts. Fetal liver erythroblasts were cultured in proliferation medium and incubated with 100μM FeNTA or DFO for 18 hours. Then the membrane fractions were prepared, and the expression levels of FPN1, TfR1, and α-tubulin was measured by Western blot analyses. The experiments in panels A through C were repeated in 3 animals in each group and panel D was repeated twice.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Expressing, Western Blot, Staining, Cell Culture, Incubation

Journal: Blood

Article Title: Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

doi: 10.1182/blood-2011-01-330241

Figure Lengend Snippet: Expression levels of FPN1 in erythroblasts of mouse BM and in mouse spleen were decreased by hepcidin injection. (A) Expression levels of FPN1 and TfR1 in Ter119-positive erythroblasts or the Ter119-negative cells from BM were compared by Western blot analyses after injection of either hepcidin or PBS for 24 hours. A band from Ponceau S staining of the gel was used as a loading control. (B) Hepcidin injections decreased FPN1 expression in mouse spleen. Expression levels of FPN1, TfR1, L-ferritin, and α-tubulin were measured by Western blot analyses. The experiments were performed in mice maintained on a low-iron diet as described in “Methods.” Experiments in panels A and B were repeated in 3 animals in each group.

Article Snippet: After being cultured in either proliferation or differentiation medium for 48 hours, erythroblasts were incubated with PE-labeled anti–c-Kit, PE Cy7-labeled anti-TER119, or FITC-labeled anti-TfR1 Abs (BD PharMingen), followed by FACS analysis. (B) mRNA expression of hemoglobin adult α-chain (HBA) and TfR1 during the differentiation of erythroblasts. (C) mRNA expression of FPN1A, FPN1B, and FPN1 during the differentiation of erythroblasts. (D) Protein expression of FPN1, TfR1, and α-tubulin during the differentiation of erythroblasts.

Techniques: Expressing, Injection, Western Blot, Staining

Journal: Drug Design, Development and Therapy

Article Title: Puerarin Ameliorates the Ferroptosis in Diabetic Liver Injure Through the JAK2/STAT3 Pathway Inhibition Based on Network Pharmacology and Experimental Validation

doi: 10.2147/DDDT.S487496

Figure Lengend Snippet: Effects of Puerarin on JAK2/STAT3 pathway and ferroptosis in mice with DLI. ( A-C ) p-JAK2 and p-STAT3 protein expression and the Western blot bands. ( D-J ) GPX4, ACSL4, PTGS2, SLC7A11, FTH1, and TFR1 protein expression and the Western blot bands. Data represent means ± SD (n = 5). Statistical analysis tested by one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control; ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Anti- NF-κB p65 (YT3108, 1:2000), anti-GPX4 (YN3047, 1:2000), anti-SLC7A11 (YT8130, 1:2000), anti-TFR1 (YT5374, 1:2000), anti-FTH1 (YT1692, 1:1000), and anti-ACSL4 (YT8070, 1:2000) were purchased from Immunoway (California, USA).

Techniques: Expressing, Western Blot, Control

Journal: Drug Design, Development and Therapy

Article Title: Puerarin Ameliorates the Ferroptosis in Diabetic Liver Injure Through the JAK2/STAT3 Pathway Inhibition Based on Network Pharmacology and Experimental Validation

doi: 10.2147/DDDT.S487496

Figure Lengend Snippet: Effects of Puerarin and AG490 on JAK2/STAT3 pathway and ferroptosis in HG-induced AML12 cells. ( A-C ) p-JAK2 and p-STAT3 protein expression and the Western blot bands. ( D-J ) GPX4, ACSL4, PTGS2, SLC7A11, FTH1, and TFR1 protein expression and the Western blot bands. Data represent means ± SD (n = 5). Statistical analysis tested by one-way ANOVA test. ** P < 0.01, *** P < 0.001 vs control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Anti- NF-κB p65 (YT3108, 1:2000), anti-GPX4 (YN3047, 1:2000), anti-SLC7A11 (YT8130, 1:2000), anti-TFR1 (YT5374, 1:2000), anti-FTH1 (YT1692, 1:1000), and anti-ACSL4 (YT8070, 1:2000) were purchased from Immunoway (California, USA).

Techniques: Expressing, Western Blot, Control