Journal: eLife

Article Title: The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack

doi: 10.7554/eLife.41301

Figure Lengend Snippet:

Article Snippet: Cell line ( Rattus norvegicus ) , Normal rat kidney (NRK) fibroblast cell , ATCC , ATCC: CRL-1570; RRID: CVCL_2144 , .

Techniques: Recombinant, Plasmid Preparation, Antibody Labeling, Software

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Knockdown of Cldn2 in PTECs enhances fibroblast activation and proliferation. ( A ) Representative immunostaining images showing the nuclear localization of proliferating cell nuclear antigen (PCNA) in activated fibroblasts. Cells were immune-stained for α-SMA (red) and PCNA (green) and counterstained with DAPI (blue). White arrows indicate positive staining. Magnification ×400, scale bar =100µm. ( B ) Quantitative analysis of PCNA + /α-SMA + cells showed knockdown of Cldn2 in PTECs markedly ameliorated the activation and proliferation in fibroblasts by co-culturing with PTECs in NG for 48 hours. (C and D) Representative images and quantitative analysis show knockdown of Cldn2 in PTECs promotes fibroblast proliferation, as demonstrated by EdU incorporation. (E and F) Western blot revealed the expression levels of α-SMA and collagen I in NRK49F cells in 5.5mM D-glucose (NG) medium± co-culture with NRK52E cells ±si- Cldn2 for 48 hours. Representative blots and quantitative analysis of α-SMA and collagen I are shown above. Data are the mean ± SEM (n=3). ** P <0.01, vs NRK52E-siNC+NRK49F+NG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Knockdown, Activation Assay, Immunostaining, Staining, Western Blot, Expressing, Co-Culture Assay

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Overexpression of Cldn2 reverses high glucose-induced fibroblast activation and proliferation. ( A and B ) Immunocytochemistry revealed the nuclear localization of proliferating cell nuclear antigen (PCNA) in activated-fibroblasts (NRK49Fcells) in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ± co-culture with NRK52E ± pcDNA3.1- Cldn2 . Cells were immune-stained for α-SMA (red) and PCNA (green) and counterstained with DAPI (blue). White Arrows indicate positive staining. Magnification ×400, scale bar =100µm. ( C and D ) Representative images and quantitative analysis show HG stimulated fibroblast proliferation, and co-culture with PTECs exacerbated this process. And overexpressed Cldn2 in PTECs inhibited HG-induced fibroblast proliferation. ( E and F ) Western blot revealed the expression levels of α-SMA and collagen I in NRK49F cells in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ± co-culture with NRK52E cells ± pcDNA3.1- Cldn2 for 48 hours. Representative blots and quantitative analysis of α-SMA and collagen I are shown above. Data are expressed as mean data ± SEM (n = 3), ** P <0.01, vs NRK49F+NG; ## P <0.01, vs NRK49F+HG; && P <0.01, vs NRK52E-pcDNA3.1-NC+NRK49F+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Over Expression, Activation Assay, Immunocytochemistry, Co-Culture Assay, Staining, Western Blot, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Claudin-2 regulates CTGF in PTECs. ( A and B ) Western blot and quantitative analysis showed that the CTGF protein expression increased time-dependent when NRK52E cells were exposed to HG for 24 hours, 48 hours, and 72 hours, respectively. Values represent mean ± SEM, n = 3, * P < 0.05, ** P < 0.01, vs NG; # P < 0.05, ## P < 0.01 vs HG (24 h), & P <0.05 vs HG (48 h). ( C ) Concentrations of CTGF in the culture supernatants of PTECs after interference. Experiments were performed in triplicate. ** P <0.01, vs NRK52E-si-NC +NG; ## P <0.01, vs NRK52E-pcDNA3.1-NC+HG. ( D – F ) NRK52E cells were transduced with control (si-NC) or si- Cldn2 followed by co-culture with NRK49F cells in 5.5mM D-glucose (NG) medium for 48 hours. Western blot revealed the CTGF and Claudin-2 expression levels in NRK52E cells in 5.5mM D-glucose (NG) medium ± co-culture with NRK49F cells ± si- Cldn2. Cldn2 siRNA knockdown efficiency was confirmed by Western blot analyses. Representative blots and quantitative analysis of Claudin-2 and CTGF are shown above. Data are expressed as the mean ± S.E.M. ** P <0.01, vs NRK52E-si-NC+NRK49F+NG. (G and H and I) Western blot revealed the CTGF and Claudin-2 expression levels in NRK52E cells in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ±co-culture with NRK49F cells ± pcDNA3.1- Cldn2 for 48 hours. The overexpression efficiency of pcDNA3.1- Cldn2 plasmid was confirmed by Western blot analyses. Representative blots and quantitative analysis of Claudin-2 and CTGF are shown above. ** P <0.01, vs NRK49F+NG; ## P <0.01, vs NRK49F+HG; && P <0.01, vs NRK52E-pcDNA3.1-NC+NRK49F+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Western Blot, Expressing, Transduction, Control, Co-Culture Assay, Knockdown, Over Expression, Plasmid Preparation

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Claudin-2 deficiency induced tubular epithelial CTGF is involved in fibroblast activation and proliferation. ( A and B ) Immunocytochemistry revealed the nuclear localization of proliferating cell nuclear antigen (PCNA) in activated-fibroblasts in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ± co-culture with NRK52E cells ± si- Cldn2 ± pcDNA3.1- Cldn2 ± si- Ctgf ± pcDNA3.1- Ctgf . Cells were immune-stained for α-SMA (red) and PCNA (green) and counterstained with DAPI (blue). White arrows indicate positive staining. Magnification ×400, scale bar =100µm. ( C and D ) Representative image and quantitative analysis show the effect of interfering with Claudin-2 and CTGF protein expression in PTECs on fibroblast proliferation in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium. Data are the mean ± SEM (n=3). ** P <0.01, vs NRK52E-si- Cldn2 -si-NC+NRK49F+NG; ## P <0.01, vs NRK52E-pcDNA3.1- Cldn2 -pcDNA3.1-NC+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Activation Assay, Immunocytochemistry, Co-Culture Assay, Staining, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Claudin-2 deficiency induced tubular epithelial CTGF facilitates fibroblasts to product matrix protein. ( A ) Western blot demonstrated CTGF inhibition in NRK52E-si- Cldn2 cells decreased α-SMA and collage I expression in NRK49F cells, when the cells were co-cultured in 5.5mM D-glucose (NG) medium. Overexpression of CTGF in NRK52E-pcDNA3.1- Cldn2 cells increased α-SMA and collage I expression in NRK49F cells, when the cells were co-cultured in 30mM D-glucose (HG) medium. ( B and C ) The interference efficiency of Claudin-2 and CTGF protein expression in NRK52E cells were confirmed by Western blot analyses. Quantitative analysis of Claudin-2 and CTGF are shown above. ( D and E ) Quantitative analysis of α-SMA and collagen I in NRK49F cells are shown above. Data are expressed as mean data ± SEM (n = 3), ** P <0.01, NRK52E-si- Cldn2 -si-NC+NRK49F+NG; ## P <0.01, vs NRK52E-pcDNA3.1- Cldn2 -pcDNA3.1-NC+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Western Blot, Inhibition, Expressing, Cell Culture, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation

doi: 10.1111/j.1582-4934.2012.01610.x

Figure Lengend Snippet: Effects of conditioned MSCs on expression of α-SMA and fibronectin in renal interstitial fibroblast NRK-49F. NRK-49F cells were cultured without or with 15 ng/ml TGF-β1 alone or co-culture with conditioned MSCs and/or ascorbic acid 2- phosphate for 3 days. ( A ) Representative Western blot analysis and relative bar graph analysis for α-SMA and β-tubulin level. ( B ) Representative Western blot and relative bar graph analysis of fibronectin protein level in NRK-49F after various treatments. * P < 0.05 versus normal control; ** P < 0.05 versus TGF-β1 treated.

Article Snippet: Rat renal proximal tubular cells (NRK-52E) and normal rat kidney interstitial fibroblast cells (NRK-49F) were purchased from the Bioresource Collection and Research Center of the Food Industry Research Institute, Taiwan.

Techniques: Expressing, Cell Culture, Co-Culture Assay, Western Blot, Control

Journal: Cytoskeleton (Hoboken, N.j.)

Article Title: A homozygous genome‐edited Sept2‐EGFP fibroblast cell line

doi: 10.1002/cm.21518

Figure Lengend Snippet: Design and initial characterization of a genome‐edited NRK49F‐Sept2 EGFP cell‐line. (a) Strategy for the integration of EGFP into the rat Sept2 locus. Exons shown in thick black. Recombination site used by the integration matrix represented by a gray box. Left and right TAL effector binding domains (BDs) framed. Sept2 exon given in the uppercase. The integration matrix contains left (LHA) and right (RHA) homology arms for homologous recombination. EGFP is inserted directly before and in frame with Sept2 start codon (ATG, green). (b) Genomic PCR on the Sept2 locus. Successful integration of EGFP into the Sept2 locus results in a longer PCR product. Outcome for the wild type locus and single‐ and double‐allelic integration shown. (c) Western blot analysis on total cell extracts from wild type and genome‐edited cells immunoblotted for Sept2. The same amount of protein was loaded into each lane. (d) Confocal microscopy image of live genome‐edited NRK49F‐Sept2‐EGFP cells and fixed wild type NRK49F cells immunostained for Sept2 (inset). Scale bars are 10 μm. (e) Immunofluorescence micrograph of NRK49F‐Sept2‐EGFP cell‐line showing EGFP fluorescence (green) and actin or tubulin staining (red). Sept2‐EGFP decorates actin cables, but does not co‐localize with tubulin in genome‐edited NRK49F cells. Scale bars are 10 μm and 1 μm in the insets

Article Snippet: Rat kidney fibroblasts (NRK49F) were purchased from the German collection of microorganisms and cell cultures (DSMZ) and maintained in indicator‐free Dulbeccos's modification of Eagle's medium (DMEM, Invitrogen) supplemented with 4.5 g/L glucose, 100 mM glutamax, and 10% fetal bovine serum (Labforce).

Techniques: Binding Assay, Homologous Recombination, Western Blot, Confocal Microscopy, Immunofluorescence, Fluorescence, Staining

Journal: Cytoskeleton (Hoboken, N.j.)

Article Title: A homozygous genome‐edited Sept2‐EGFP fibroblast cell line

doi: 10.1002/cm.21518

Figure Lengend Snippet: Septin expression in homozygous genome‐edited NRK49F‐Sept2‐EGFP cells. (a) Immunofluorescence micrographs showing septin immunofluorescence staining (left), Sept2‐EGFP fluorescence (right) and merged images. Scale bars: 10 μm and 1 μm. (b) Western blot detection of septins in a total lysate from the genome‐edited and wild type cell lines. (c) Real‐time PCR analysis of cell lysate from wt and homozygous genome‐edited cell lines. Shown is the detected mRNA level in homozygous genome‐edited cell line relative to the wt expression level. Error bars are standard deviation

Article Snippet: Rat kidney fibroblasts (NRK49F) were purchased from the German collection of microorganisms and cell cultures (DSMZ) and maintained in indicator‐free Dulbeccos's modification of Eagle's medium (DMEM, Invitrogen) supplemented with 4.5 g/L glucose, 100 mM glutamax, and 10% fetal bovine serum (Labforce).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cytoskeleton (Hoboken, N.j.)

Article Title: A homozygous genome‐edited Sept2‐EGFP fibroblast cell line

doi: 10.1002/cm.21518

Figure Lengend Snippet: Co‐immunoprecipitation of endogenous septins with Sept2‐EGFP. (a) Western blot of septins coimmunoprecipitated from NRK49F‐Sept2‐EGFP cells using anti‐GFP nanobody beads. (b) Silver‐stained gel of immunoprecipitated septin complexes. E, elution; FT, unbound fraction (flow‐through); S, supernatant incubated with anti‐GFP nanobody beads; W1–W3, washing steps. Indicated septins were identified by western blotting and mass spectrometry analysis of excised bands (see Section )

Article Snippet: Rat kidney fibroblasts (NRK49F) were purchased from the German collection of microorganisms and cell cultures (DSMZ) and maintained in indicator‐free Dulbeccos's modification of Eagle's medium (DMEM, Invitrogen) supplemented with 4.5 g/L glucose, 100 mM glutamax, and 10% fetal bovine serum (Labforce).

Techniques: Immunoprecipitation, Western Blot, Staining, Incubation, Mass Spectrometry

Journal: Cytoskeleton (Hoboken, N.j.)

Article Title: A homozygous genome‐edited Sept2‐EGFP fibroblast cell line

doi: 10.1002/cm.21518

Figure Lengend Snippet: Comparison of Sept2 distribution during cell division in wild type and genome‐edited cells. (a) Confocal images of wild type (left) and genome‐edited (right) NRK49F fibroblast cells in different phases of the cell cycle. Sept2 antibody and Sept2‐EGFP signal in green. Tubulin staining in red. (b) Individual frames from a live‐cell time‐lapse acquisition of dividing NRK49‐Sept2‐EGFP cells. Scale bars 10 μm

Article Snippet: Rat kidney fibroblasts (NRK49F) were purchased from the German collection of microorganisms and cell cultures (DSMZ) and maintained in indicator‐free Dulbeccos's modification of Eagle's medium (DMEM, Invitrogen) supplemented with 4.5 g/L glucose, 100 mM glutamax, and 10% fetal bovine serum (Labforce).

Techniques: Comparison, Staining

Journal: The Journal of Physiology

Article Title: RAAS inhibitors directly reduce diabetes‐induced renal fibrosis via growth factor inhibition

doi: 10.1113/JP277002

Figure Lengend Snippet: A , C‐terminal of fibronectin (FBN‐C) turnover marker secretion by HK‐2 proximal tubular epithelial cells on HG and RAASi treatment. B , type IV collagen formation biomarker (PRO‐C4) secretion by HK‐2 cells on HG and RAASi treatment. C and D , PRO‐C4 secretion by NRK‐49F cells treated with platelet‐derived growth factor (PDGF; C ) or connective tissue growth factor (CTGF; D ). Values are presented as means ± 95% confidence intervals; n = 6 wells/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05 vs . control; §§ P < 0.01 vs . HG.

Article Snippet: Human kidney 2 proximal tubular epithelial (HK‐2) cells (LGC Standards, ATCC Cat. No. CRL‐2190, RRID:CVCL_0302) were cultured in DMEM containing 5.5 mM glucose (Gibco, supplied by Life Technologies, Carlsbad, CA, USA) and normal rat kidney fibroblast (NRK‐49F) cells (LGC Standards, ATCC Cat. No. CRL‐1570, RRID:CVCL_2144) were maintained in DMEM containing 25 mM glucose (Gibco), both supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin and 1% L‐glutamine, and incubated at 5% CO 2 and 37°C.

Techniques: Marker, Biomarker Assay, Derivative Assay

Journal: The Journal of Physiology

Article Title: RAAS inhibitors directly reduce diabetes‐induced renal fibrosis via growth factor inhibition

doi: 10.1113/JP277002

Figure Lengend Snippet: A , representative picture of platelet‐derived growth factor receptor β (PDGFR‐β) stained NRK‐49F cells. 1000× magnification; red, PDGFRβ; blue, nucleus; scale bar = 20 μm. B – D , representative pictures of phalloidin‐TRITC immunostained NRK‐49F cells (control, B ) treated with platelet‐derived growth factor (PDGF; C ) or connective tissue growth factor (CTGF/CCN2; D ). 1000× magnification; red, F‐actin; blue, nucleus; scale bar = 20 μm. E and F , alpha‐smooth muscle actin (αSMA) protein levels in NRK‐49F cells treated with PDGF ( E ) or CTGF/CCN2r ( F ) and RAASi. Representative gel image examples shown above the panels. Samples might be from different gels but were derived at the same time and processed in parallel. On each graph values are presented as means ± 95% confidence intervals; n = 6 wells/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05, *** P < 0.001 vs . control; § P < 0.05, §§§ P < 0.001 vs . PDGF or CTGF.

Article Snippet: Human kidney 2 proximal tubular epithelial (HK‐2) cells (LGC Standards, ATCC Cat. No. CRL‐2190, RRID:CVCL_0302) were cultured in DMEM containing 5.5 mM glucose (Gibco, supplied by Life Technologies, Carlsbad, CA, USA) and normal rat kidney fibroblast (NRK‐49F) cells (LGC Standards, ATCC Cat. No. CRL‐1570, RRID:CVCL_2144) were maintained in DMEM containing 25 mM glucose (Gibco), both supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin and 1% L‐glutamine, and incubated at 5% CO 2 and 37°C.

Techniques: Derivative Assay, Staining