rat kidney fibroblast cell line nrk49f  (ATCC)

Journal: PLoS ONE

Article Title: Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

doi: 10.1371/journal.pone.0084063

Figure Lengend Snippet: (A) Western blot showing expression of fibronectin, α-smooth muscle actin (α-SMA), collagen I and GAPDH in NRK49F cells cultured with L-NAME-treated or control MMEC media in the presence or absence of TGF-β1 stimulation for 24 hrs. (B) Quantification of the relative expression of fibronectin, α-SMA and collagen I compared to GAPDH. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. * p <0.05, ** p <0.01. Experiments were repeated at least three times.

Article Snippet: Rat kidney fibroblast cell line NRK49F cells were purchased from ATCC and grown in a 5% CO 2 atmosphere at 37°C in DMEM containing 10% fetal bovine serum.

Techniques: Western Blot, Expressing, Cell Culture, Control, Comparison

Journal: PLoS ONE

Article Title: Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

doi: 10.1371/journal.pone.0084063

Figure Lengend Snippet: Sub-confluent NRK49F cells were incubated in L-NAME-treated or control MMEC media in the presence or absence of PDGF-BB stimulation for 24 hr. (A) Immunostaining of Ki67 (brown) with nuclear staining (blue) in NRK49F cells. (B) Quantification of the percentage of NRK49F cells positive for Ki67 immunostaining. (C) Quantification of cell proliferation using incorporation of bromodeoxyuridine (Brdu). Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. ** p <0.01, *** p <0.001. Experiments were repeated at least three times.

Article Snippet: Rat kidney fibroblast cell line NRK49F cells were purchased from ATCC and grown in a 5% CO 2 atmosphere at 37°C in DMEM containing 10% fetal bovine serum.

Techniques: Incubation, Control, Immunostaining, Staining, Comparison

Journal: PLoS ONE

Article Title: Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

doi: 10.1371/journal.pone.0084063

Figure Lengend Snippet: (A) Western blot (WB) showing phosphorylation of JNK and p-S208 in the Smad3 linker region in NRK49F cells incubated with L-NAME conditioned MMEC media which is inhibited by RvD1. (B) Immunoprecipitation (IP) of Smad3 followed by WB identified phosphorylation of the Smad3 C-terminal domain (p-C-Smad3) and linker region (p-T179) following culture of NRK49F cells in L-NAME conditioned MMEC media which is inhibited by RvD1. (C) Quantification of phosphorylation of JNK and the different sites on Smad3. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. * p <0.05, *** p <0.001, N.S., not significant. Experiments were repeated at least three times.

Article Snippet: Rat kidney fibroblast cell line NRK49F cells were purchased from ATCC and grown in a 5% CO 2 atmosphere at 37°C in DMEM containing 10% fetal bovine serum.

Techniques: Western Blot, Phospho-proteomics, Incubation, Immunoprecipitation, Comparison

Journal: PLoS ONE

Article Title: Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

doi: 10.1371/journal.pone.0084063

Figure Lengend Snippet: (A) Western blot showing that incubation of NRK49F cells in L-NAME treated MMEC media for 24 hr up-regulates expression of fibronectin and collagen I which is prevented by RvD1. (B) Quantification of fibronecting and collagen I expression relative to GAPDH. *** p <0.001. (C) Quantification of bromodeoxyuridine (Brdu) uptake shows that L-NAME treated MMEC media can increase proliferation of NRK49F cells which is prevented by the addition of RvD1. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *** p <0.001. Experiments were repeated at least three times.

Article Snippet: Rat kidney fibroblast cell line NRK49F cells were purchased from ATCC and grown in a 5% CO 2 atmosphere at 37°C in DMEM containing 10% fetal bovine serum.

Techniques: Western Blot, Incubation, Expressing, Comparison

Journal: PLoS ONE

Article Title: Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

doi: 10.1371/journal.pone.0084063

Figure Lengend Snippet: (A) NRK49F cells were transfected with a reporter plasmid in which luciferase production is driven by the collagen I promoter. Incubation of these cells with L-NAME treated MMEC media for 24 hr up-regulated collagen I promoter activity which was prevented by the addition of RvD1. (B) 293T cells were transfected with Smad3 plasmids containing point mutations of various phosphorylation sites plus a collagen I promoter plasmid. Twenty-four hours later, cells were cultured with or without TGF-β1 and luciferase activity measured 24 hr later. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. * p <0.05, *** p <0.001, N.S., not significant. Experiments were repeated at least three times.

Article Snippet: Rat kidney fibroblast cell line NRK49F cells were purchased from ATCC and grown in a 5% CO 2 atmosphere at 37°C in DMEM containing 10% fetal bovine serum.

Techniques: Transfection, Plasmid Preparation, Luciferase, Incubation, Activity Assay, Phospho-proteomics, Cell Culture, Comparison