Journal: Journal of the American Society of Nephrology

Article Title: Proteinuria Impairs Podocyte Regeneration by Sequestering Retinoic Acid

doi: 10.1681/asn.2012090950

Figure Lengend Snippet: Figure 1. Transferrin and IgG overload affects human RPC survival, while albumin overload impairs RPC differentiation into podocytes. (A) Effect of HSA, transferrin, or IgG treatment on the proliferative capacity of RPC, as assessed by [3H]-thymidine incorporation (n=4). (B) Flow cytometry analysis of RPC viability after treatment with HSA, transferrin, or IgG, as assessed by annexin-V and propidium iodide costaining. One representative of four experiments is shown (control: 90.82%60.69% live cells; HSA: 90.35%61.44%, transferrin: 51.68%64.83%, IgG: 72.48%65.28%; P=0.75 con- trol versus HSA, P=0.01 control versus transferrin-treated and control versus IgG- treated cells by Mann-Whitney test, n=4). (C) NPHS1 mRNA levels in RPCs cultured in control medium or VRAD medium alone, or supplemented with HSA, transferrin, or IgG (n=6). (D) Nephrin protein expression assessed by FACS analysis in RPCs cultured in control medium or VRAD medium alone or supplemented with HSA, transferrin, or IgG. Representative histograms obtained in one of three independent experiments are shown. Percentages of nephrin-positive cells are calculated over their respective isotype control. All data are means 6 SEM, **P,0.01 by ANOVA with Bonferroni post hoc analysis (A) and by Mann-Whitney test (C).

Article Snippet: For the determination of uri- nary transferrin and IgG, a mouse transferrin ELISA kit (Bethyl Lab- oratories Inc., Montgomery, TX) andmouse IgG ELISA kit (Cusabio, Wuhan, Hubei, P.R.

Techniques: Flow Cytometry, Control, MANN-WHITNEY, Cell Culture, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Proteinuria Impairs Podocyte Regeneration by Sequestering Retinoic Acid

doi: 10.1681/asn.2012090950

Figure Lengend Snippet: Figure 3. Evaluation of urinary protein levels and podocyte markers in mice with genetically tagged podocytes validate AN as a model to study podocyte loss and regeneration. (A) Evaluation of albumin, transferrin, and IgG levels in the urine of three different mice strains (n=15 each) after induction of AN (day 11). (B) Three-dimensional reconstruction of glomerulus of healthy and Adriamycin-treated NPHS2.Cre;mT/mG transgenic mouse (green = GFP). (C) Representative electron micrographs of glomerular podocytes in AN show

Article Snippet: For the determination of uri- nary transferrin and IgG, a mouse transferrin ELISA kit (Bethyl Lab- oratories Inc., Montgomery, TX) andmouse IgG ELISA kit (Cusabio, Wuhan, Hubei, P.R.

Techniques: Transgenic Assay

Journal: Journal of Nanobiotechnology

Article Title: Macrophage-derived extracellular vesicles represent a promising endogenous iron-chelating therapy for iron overload and cardiac injury in myocardial infarction

doi: 10.1186/s12951-024-02800-1

Figure Lengend Snippet: Iron overload and cell ferroptosis contributes to MI-induced cardiac injury. a Quantitative analysis of serum iron level in sham and MI mice. b Quantitative analysis of cardiac iron level in sham and MI mice. c , d Representative western blotting images and quantitative analysis of transferrin and FTH1 expression in sham and MI mice. e Quantitative analysis of MDA level in sham and MI mice. f Quantitative analysis of GSH in sham and MI mice. g , h Representative western blotting images and quantitative analysis of GPX4 expression in sham and MI mice. i , j Representative immunohistochemical images and quantitative analysis of 4-HNE expression in sham and MI mice. Scale bar = 30 μm. N = 6 each group. Data are expressed as Mean ± SEM

Article Snippet: The measurement of transferrin level was performed with Transferrin ELISA kit (Elabscience, E-EL-M1184c) following manufacturer’s instructions.

Techniques: Western Blot, Expressing, Immunohistochemical staining

Journal: Journal of Nanobiotechnology

Article Title: Macrophage-derived extracellular vesicles represent a promising endogenous iron-chelating therapy for iron overload and cardiac injury in myocardial infarction

doi: 10.1186/s12951-024-02800-1

Figure Lengend Snippet: Macrophage-derived EVs attenuated myocardial injury by preventing iron overload in post-MI heart. a Diagram of MI construction and EVs injection protocol. b Quantitative analysis of serum iron level in mice. c Quantitative analysis of cardiac iron level in mice. d , e Representative western blotting images and quantitative analysis of transferrin and FTH1 expression in hearts of mice. f Quantitative analysis of MDA level in hearts of mice. g Representative DHE staining images of hearts of mice. Scale bar = 50 μm. h , i Representative western blotting images and quantitative analysis of GPX4 expression in hearts of mice. j Representative immunohistochemical images of 4-HNE expression in hearts of mice. Scale bar = 50 μm. k , l Representative TTC staining images and quantitative analysis of infarcted area of hearts. m Representative M-mode echocardiography images. n , o Quantitative analysis of LVEF and LVFS. p , q Quantitative analysis of serum CKMB and LDH1 in mice. N = 6 each group. Data are expressed as Mean ± SEM

Article Snippet: The measurement of transferrin level was performed with Transferrin ELISA kit (Elabscience, E-EL-M1184c) following manufacturer’s instructions.

Techniques: Derivative Assay, Injection, Western Blot, Expressing, Staining, Immunohistochemical staining

Journal: Journal of Nanobiotechnology

Article Title: Macrophage-derived extracellular vesicles represent a promising endogenous iron-chelating therapy for iron overload and cardiac injury in myocardial infarction

doi: 10.1186/s12951-024-02800-1

Figure Lengend Snippet: TfR was responsible for the iron-chelating capacity of macrophages-derived EVs. a Quantitative analysis of iron level in iron solution after incubation with PBS or EVs. b Representative western blotting images of TfR expression in macrophages-derived EVs. c Representative immunogold TEM images of macrophages-derived EVs incubated with IgG or anti-TfR. Scale bar = 100 nm. d Schematic diagram of the experiment protocol to determine the origin of TfR on EVs. e Quantitative analysis of fluorescence intensity of culture medium. f , g Representative flow cytometer results and quantitative analysis of fluorescence intensity of EVs released by macrophages transfected with empty-EGFP or TfR-EGFP plasmid. h Schematic diagram of the experiment protocol to determine whether TfR on EVs could bind with transferrin. i Representative flow cytometer results of EVs released by macrophages transfected with TfR-EGFP plasmid after incubation with cell transfected with transferrin-mCherry or empty-mCherry plasmid. j-m Quantitative analysis of transferrin level of myocardium lysate, serum, cardiomyocytes lysate, and culture medium of cardiomyocytes after incubation with macrophages-derived EVs. N = 6 each group. Data are expressed as Mean ± SEM

Article Snippet: The measurement of transferrin level was performed with Transferrin ELISA kit (Elabscience, E-EL-M1184c) following manufacturer’s instructions.

Techniques: Derivative Assay, Incubation, Western Blot, Expressing, Fluorescence, Flow Cytometry, Transfection, Plasmid Preparation

Journal: Journal of Nanobiotechnology

Article Title: Macrophage-derived extracellular vesicles represent a promising endogenous iron-chelating therapy for iron overload and cardiac injury in myocardial infarction

doi: 10.1186/s12951-024-02800-1

Figure Lengend Snippet: EVs released by macrophages with TfR deficiency exhibited reduced cardiac protective effect against MI. a Representative western blotting images of TfR expression in macrophages transfected with siTfR and EVs released by such macrophages. b Quantitative analysis of serum iron level of mice. c Quantitative analysis of cardiac iron level of mice. d , e Representative western blotting images and quantitative analysis of transferrin and FTH1 expression in hearts of mice. f Quantitative analysis of MDA level of mice. g Representative DHE staining images of hearts of mice. Scale bar = 50 μm. h , i Representative western blotting images and quantitative analysis of GPX4 expression in hearts of mice. j Representative immunohistochemical images of 4-HNE expression in hearts of mice. Scale bar = 50 μm. k , l Representative TTC staining images and quantitative analysis of infarcted area of hearts. m Representative M-mode echocardiography images. n , o Quantitative analysis of LVEF and LVFS. p , q Quantitative analysis of serum CKMB and LDH1 in mice. N = 6 each group. Data are expressed as Mean ± SEM

Article Snippet: The measurement of transferrin level was performed with Transferrin ELISA kit (Elabscience, E-EL-M1184c) following manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transfection, Staining, Immunohistochemical staining

Journal: Journal of Nanobiotechnology

Article Title: Macrophage-derived extracellular vesicles represent a promising endogenous iron-chelating therapy for iron overload and cardiac injury in myocardial infarction

doi: 10.1186/s12951-024-02800-1

Figure Lengend Snippet: Schematic figure illustrating the mechanism underlying the therapeutic effect of macrophage-derived EVs on MI. Myocardial iron overload, cardiomyocytes ferroptosis, and cardiac function impairment were detected in the hearts post MI, which contributed to the poor prognosis of MI. This work identified that macrophage-derived EVs exhibited protective effect on reducing myocardial iron overload, alleviating cardiomyocytes ferroptosis, and improving cardiac function post MI. Mechanistically, TfR on macrophage-derived EVs was the key player responsible for binding with transferrin and removing protein-bound iron. These iron-chelating EVs were ultimately captured and processed by macrophages in the liver

Article Snippet: The measurement of transferrin level was performed with Transferrin ELISA kit (Elabscience, E-EL-M1184c) following manufacturer’s instructions.

Techniques: Derivative Assay, Binding Assay