Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms

doi: 10.1002/stem.1263

Figure Lengend Snippet: Top: Representative images of CM-DiI labeled (red) endothelial progenitor cells (EPC) or mesenchymal stem cells (MSC) in the post-stenotic kidneys of pigs with renal artery stenosis (RAS) 4 weeks after cell delivery. Green shows peanut agglutinin (PA, green arrow), a distal tubular marker, and cyan shows a proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, cyan arrow). EPC showed mainly tubular engraftment (yellow arrow), while MSC tend to integrate into both proximal tubules (yellow arrow) and interstitial area (red arrow). Bottom: Both EPC and MSC improved RBF and GFR in pigs with RAS, yet MSC more effectively restored GFR. *p<0.05 vs. Normal, †p<0.05 vs. RAS. Scale bar=200µm

Article Snippet: Furthermore, frozen kidney sections from pigs infused with cells were stained with the distal tubular marker peanut agglutinin (5ug/ml, Vector) and proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, 5ug/ml, Vector).

Techniques: Labeling, Marker

Journal: Acta Pharmacologica Sinica

Article Title: Involvement of endoplasmic reticulum stress in angiotensin II-induced NLRP3 inflammasome activation in human renal proximal tubular cells in vitro

doi: 10.1038/aps.2015.21

Figure Lengend Snippet: Angiotensin II induced inflammasome activation in tubular epithelial cells. (A) Real-time PCR and (C, E, and G) western blot analysis show that the mRNA and protein expression of caspase-1, IL-1β, and IL-18 were increased after treatment with different amounts (0, 10, 100, or 1000 nmol/L) of angiotensin II for 12 h in serum-free medium. (B) Real-time PCR and (D, F, and H) western blot analysis indicate that the mRNA and protein expression of caspase-1, IL-1β, and IL-18 were increased after treatment with 100 nmol/L angiotensin II for various time periods (0, 6, 12, or 24 h) in serum-free medium. β-Actin served as an internal control gene. The results represent the mean±SD from four experiments. bP<0.05 vs control.

Article Snippet: Human kidney proximal epithelial cells (HK-2) were purchased from the China Center for Type Culture Collection (CCTCC).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: Life Science Alliance

Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

doi: 10.26508/lsa.202000832

Figure Lengend Snippet: (A) A schematic diagram of Src-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length Src protein. SH, Src homology domain. (B) Src-MER–overexpressing MDCK cells were incubated with 10 μM 4-OHT for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (C) Src-MER–overexpressing cysts embedded within the collagen matrix were incubated with 10 μM 4-OHT for 2 d. Activated Src was visualized with an anti-Src pY418 antibody (green). Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (D) Src-MER–overexpressing MDCK cells were incubated with 10 μM 4-OHT for the indicated time periods. Detergent-resistant membrane (DRM) and non-DRM fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using an anti-Src pY418 antibody. (E, F) DRM fractions from Src-MER–activated cells were subjected to immunoprecipitation with an anti-estrogen receptor (ERα) antibody. Immunoprecipitates were confirmed by silver staining. IgG HC, IgG heavy chain. (F) Schematic flow diagram of identification of Src scaffolding proteins. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database and the number of identified proteins is depicted. Expression in proximal renal tubule was surveyed using the anatomical database Kidney Cell Explorer ( https://cello.shinyapps.io/kidneycellexplorer/ ). Source data are available for this figure.

Article Snippet: The kidney sections were blocked with Blocking One (Nacalai Tesque), incubated with primary antibodies for overnight at 4°C, and then incubated with Alexa Fluor 488/594–conjugated secondary antibodies (Molecular Probes) and the fluorescein-conjugated proximal tubule marker, FITC-LTL (FL-1321; Vector Laboratories), for 2 h at room temperature.

Techniques: Modification, Ligand Binding Assay, Incubation, Western Blot, Staining, Immunoprecipitation, Silver Staining, Scaffolding, Mass Spectrometry, Expressing

Journal: Life Science Alliance

Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

doi: 10.26508/lsa.202000832

Figure Lengend Snippet: (A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.

Article Snippet: The kidney sections were blocked with Blocking One (Nacalai Tesque), incubated with primary antibodies for overnight at 4°C, and then incubated with Alexa Fluor 488/594–conjugated secondary antibodies (Molecular Probes) and the fluorescein-conjugated proximal tubule marker, FITC-LTL (FL-1321; Vector Laboratories), for 2 h at room temperature.

Techniques: Immunofluorescence, Marker, Knock-Out, Staining

Journal: Nature Communications

Article Title: Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

doi: 10.1038/ncomms12887

Figure Lengend Snippet: ( a ) Parafibromin knockdown results in reduced Hh signal activity. Hh-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( b ) Parafibromin interacts with Gli1. TCLs prepared from SKES cells were sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. ( c ) Phospho-resistant (PR)-parafibromin shows increased ability to activate Hh signalling. Hh-responsive luciferase reporter assays were performed in HEK293T cells ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( d ) SHP2 expression promotes Hh signalling. Hh-responsive luciferase reporter assays were performed in A549 cells ( n =3, mean±s.d. ** P <0.01; a two-tailed unpaired Student's t -test). ( e , f ) PR-parafibromin shows increased binding to β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. ( g , h ) Non-phosphorylated parafibromin specifically interacts with β-catenin and Gli1. HEK293T cells were transfected with the indicated vectors. TCLs were immunoprecipitated with the indicated antibodies and immunoprecipitates (IP) were incubated with recombinant phosphorylated/non-phosphorylated parafibromin. The reaction mixtures were subjected to immunoblotting.

Article Snippet: HEK293T human embryonic kidney cells (ATCC CRL3216), A549 lung adenocarcinoma-derived epithelial cells (Japanese Cancer Research Resources Bank), MCF7 human mammary carcinoma-derived epithelial cells (ATCC HTB22) and Hrpt2 flox/flox /CAG-CreER MEF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Knockdown, Activity Assay, Luciferase, Western Blot, Immunoprecipitation, Expressing, Two Tailed Test, Binding Assay, Transfection, Incubation, Recombinant

Journal: Nature Communications

Article Title: Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

doi: 10.1038/ncomms12887

Figure Lengend Snippet: ( a ) Parafibromin knockdown results in reduced Notch signal activity. Notch-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( b ) Parafibromin forms a complex with NICD. TCLs prepared from MCF7 cells were sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. ( c ) A γ-secretase inhibitor (DAPT) treatment blocked the parafibromin-dependent Notch reporter activation. Notch-responsive luciferase reporter assays were performed in HEK293T cells. Cells were incubated with medium containing 100 μM DAPT for 48 h ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( d ) Phospho-resistant (PR)-parafibromin shows increased NICD binding. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. ( e ) Non-phosphorylated parafibromin specifically interacts with NICD. HEK293T cells were transfected with the indicated vectors. TCLs were immunoprecipitated with the indicated antibodies and immunoprecipitates (IP) were incubated with recombinant phosphorylated/non-phosphorylated parafibromin. The reaction mixtures were subjected to immunoblotting. ( f ) PR-parafibromin shows increased ability to activate Notch signalling. Notch-responsive luciferase reporter assays were performed in HEK293T cells ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( g ) MCF7 cells were transiently transfected with a parafibromin expression vector. At 48 h after transfection, TCLs were subjected to immunoblotting. ( h ) Parafibromin inhibits proteasome-dependent degradation of NICD. HEK293T cells were transfected with the indicated vectors and incubated with or without 10 μM MG132 for 8 h. TCLs were immunoblotted with the indicated antibodies. ( i ) Expression of PR-parafibromin induces transactivation of Notch target genes. MCF7 cells were transfected with the indicated vectors and mRNA expression levels of HEY1 and HES1 were measured by RT–qPCR analyses ( n =4, mean±s.d. * P <0.05, ** P <0.01; a two-tailed unpaired Student's t -test). ( j ) Conditional deletion of parafibromin in Hrpt2 flox/flox MEFs was induced by 4-OHT treatment and expression levels of Hey1 and Hes1 were measured by RT–qPCR analyses ( n =3, mean±s.d. ** P <0.01; a two-tailed unpaired Student's t -test). Parafibromin knockout was confirmed by immunoblotting.

Article Snippet: HEK293T human embryonic kidney cells (ATCC CRL3216), A549 lung adenocarcinoma-derived epithelial cells (Japanese Cancer Research Resources Bank), MCF7 human mammary carcinoma-derived epithelial cells (ATCC HTB22) and Hrpt2 flox/flox /CAG-CreER MEF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Knockdown, Activity Assay, Luciferase, Western Blot, Immunoprecipitation, Activation Assay, Incubation, Binding Assay, Transfection, Recombinant, Expressing, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Knock-Out

Journal: Nature Communications

Article Title: Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

doi: 10.1038/ncomms12887

Figure Lengend Snippet: ( a , b ) β-catenin competitively inhibits parafibromin-dependent Hh signalling. Hh-responsive luciferase reporter assays were performed in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). For b , transfected cells were treated with 25 mM LiCl for 24 h. ( c ) Gli1 suppresses parafibromin-dependent Wnt signalling. Wnt-responsive luciferase reporter assays were performed in HEK293T cells ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( d ) β-catenin competitively inhibits parafibromin/Gli1 interaction. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted. ( e ) A parafibromin mutant lacking β-catenin-binding site (Δβ) failed to potentiate Hh signal. Hh-responsive luciferase reporter assays were performed in HEK293T cells transfected with the Gli1 expression vector. Total cell lysates (TCLs) were subjected to immunoblotting ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( f ) Proximity ligation assay (PLA) in HEK293T cells. Transfected cells were treated with 25 mM LiCl. Red spots indicate the parafibromin/β-catenin interaction. Nuclei and Gli1-expressing cells were stained in blue (DAPI) and green, respectively. Scale bar, 10 μm. Protein expression levels in control and Gli1-expressing cells were indicated by immunoblotting. ( g ) Data for quantitative analysis of the PL spots; n =17. ** P <0.01; Wilcoxon rank sum test.

Article Snippet: HEK293T human embryonic kidney cells (ATCC CRL3216), A549 lung adenocarcinoma-derived epithelial cells (Japanese Cancer Research Resources Bank), MCF7 human mammary carcinoma-derived epithelial cells (ATCC HTB22) and Hrpt2 flox/flox /CAG-CreER MEF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Luciferase, Western Blot, Transfection, Immunoprecipitation, Mutagenesis, Binding Assay, Expressing, Plasmid Preparation, Proximity Ligation Assay, Staining, Control

Journal: Nature Communications

Article Title: Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

doi: 10.1038/ncomms12887

Figure Lengend Snippet: ( a , b ) Wnt and Notch signals mutually potentiate parafibromin-dependent signal activation. Luciferase reporter assays using the indicated reporter plasmids in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting ( n =3, mean±s.d. ** P <0.01; ANOVA with Bonferroni's post hoc test). ( c ) β-catenin stabilizes parafibromin/NICD interaction. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted with the indicated antibodies. ( d ) Endogenous interaction between parafibromin, NICD and β-catenin was increased on endogenous Wnt signal activation. HEK293T cells treated with or without 25 mM LiCl were lysed and sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. ( e ) Parafibromin forms a trimeric complex with β-catenin and NICD. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies and immunoblotted. ( f ) A schematic view of Flag-tagged wild-type parafibromin and its derivative, parafibromin-357X. The cdc73 core homology domain (aa 357–531) and β-catenin-binding site (aa 218–263) are indicated. ( g ) The C-terminal domain of parafibromin is required for NICD binding. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies.

Article Snippet: HEK293T human embryonic kidney cells (ATCC CRL3216), A549 lung adenocarcinoma-derived epithelial cells (Japanese Cancer Research Resources Bank), MCF7 human mammary carcinoma-derived epithelial cells (ATCC HTB22) and Hrpt2 flox/flox /CAG-CreER MEF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Luciferase, Western Blot, Transfection, Immunoprecipitation, Binding Assay