Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Pharmacologic inhibition of β-catenin signaling has little effect on the severity of AKI. (A) Experimental design. Cartoon syringes indicate the administration of ICG-001. (B and C) ICG-001 blocks β-catenin activation in the kidney at 1 day after IRI. Kidney lysates were immunoblotted with antibodies against active (dephosphorylated) β-catenin and α-tubulin. (B) Representative western blots and (C) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). (D) qRT-PCR analyses reveal that ICG-001 suppressed PAI-1 mRNA expression at 1 day after IRI. *P<0.05 versus shams, †P<0.05 versus vehicles (n=4–6). (E) Serum creatinine level in the mice with or without ICG-001 at 1 day after IRI, compared with shams. *P<0.05 versus shams (n=4–6). (F) Representative micrographs show morphologic injury and apoptosis at 1 day after IRI. Boxed areas are enlarged and presented. Apoptotic cell death was assessed by TUNEL staining. Arrows indicate apoptotic cells. Scale bar, 100 µm. (G and H) Quantitative data on (G) histologic injury and (H) apoptosis are presented. Data are presented as (G) percentage of injured tubules or (H) numbers of apoptotic cells per high power field (HPF), respectively. *P<0.05 versus shams (n=3). (I–K) Western blot analyses of renal expression of Bax and PCNA proteins at 1 day after IRI. Kidney lysates were immunoblotted with specific antibody against Bax, PCNA, and α-tubulin, respectively. (I) Representative western blot and (J and K) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group (n=6). Sac, sacrifice.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Inhibition, Activation Assay, Western Blot, Quantitative RT-PCR, Expressing, TUNEL Assay, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Specific ablation of β-catenin in fibroblasts reduces kidney injury after AKI. (A–C) Using Gli1-LacZ reporter mice determinates the efficacy and specificity of Gli1-driven gene expression after IRI. X-Gal staining shows the Gli1-LacZ+ cell population and distribution at 1 day after IRI. (A) Boxed area was enlarged. Scale bar, 100 µm. (B) Intensive staining was found in the corticomedullary junction region of the kidney. Arrows indicate positive staining. (C) Pie chart shows the relative distribution of the Gli1-LacZ+ fibroblasts in renal interstitium. (D) Schematic diagram depicts the generation of conditional knockout mice with fibroblast-specific deletion of β-catenin by using Cre-LoxP system. The β-catenin–floxed mice (β-catfl/fl) were crossbred with tamoxifen-inducible Cre transgenic mice under the control of endogenous Gli1 promoter/enhancer elements. Black boxes indicate the exons of the β-catenin gene. Orange boxes denote LoxP site. (E) Experimental design. The mice were injected with tamoxifen at 30 mg/kg body wt for 5 consecutive days to activate Cre recombinase in vivo. After wash out for 2 weeks, the mice were subjected to IRI for 1 day. (F) Representative micrographs show kidney morphology in Gli-β-cat−/− and Gli-β-cat+/+ mice. Sections of PAS staining are shown. Scale bar, 50 µm. (G and H) Ablation of β-catenin does not affect fibroblast survival in the kidney in vivo. Immunostaining for Fsp1 shows similar fibroblast density in Gli-β-cat−/− and Gli-β-cat+/+ kidneys under basal conditions. (G) Representative micrographs and (H) quantitative data are presented. Arrows indicate positive Fsp1 staining. Scale bar, 50 µm. (I) Knockdown of β-catenin in vitro does not affect PCNA and Bax expression in renal fibroblasts. NRK-49F cells were transfected with control or β-catenin–specific siRNA for 3 days. Cell lysates were then immunoblotted with antibodies against β-catenin, PCNA, Bax, and α-tubulin, respectively. (J) Serum creatinine level in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. *P<0.05 (n=9). (K and L) Representative micrographs show the kidneys at 1 day after IRI in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice. Boxed areas were enlarged and presented. Black asterisks in the enlarged boxed areas indicate the injured tubules. Scale bar, 100 µm. (F) Quantitative assessment of injury is presented. *P<0.05 (n=4). (M) qRT-PCR analyses show an increased Klotho mRNA expression in the Gli1-β-cat−/− mice at 1 day after IRI, compared with the Gli1-β-cat+/+ controls. *P<0.05 (n=9). Ctrl, control; PAS, periodic acid–Schiff.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Expressing, Staining, Knock-Out, Transgenic Assay, Injection, In Vivo, Immunostaining, In Vitro, Transfection, Quantitative RT-PCR

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Loss of β-catenin in fibroblasts reduces tubular cell apoptosis after AKI. (A) Representative micrographs show apoptotic cells detected by TUNEL staining after IRI at 1 day after IRI. Scale bar, 50 µm. Boxed areas were enlarged. Arrows indicate apoptotic cells. (B) Representative micrographs show renal expression of cytochrome C in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. Scale bar, 50 µm. The images in the blue channel were shown in the bottom panels. (C and D) Quantitative assessments of apoptotic cells are presented. Data are presented as (C) numbers of apoptotic cells per HPF or (D) area of positive staining of cytochrome C. *P<0.05 (n=4). (E–G) Loss of β-catenin in fibroblasts reduced renal Bax protein expression and activated Akt. (E) Representative western blots and (F and G) quantitative analyses are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). (H–J) Fibroblast-specific ablation of β-catenin suppressed FasL and p53 protein expression. (H) Representative western blots and (I and J) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). Ctrl, control; Cyto C, Cytochrome C

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: TUNEL Assay, Staining, Expressing, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Loss of β-catenin in fibroblasts promotes tubular cell proliferation and tubular regeneration after AKI. (A–C) Western blot analyses show that fibroblast-specific ablation of β-catenin promoted renal PCNA and Na+/K+-ATPase expression at 1 day after IRI. (A and B) Representative western blots and (C) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. **P<0.01, *P<0.05 (n=6). (D–F) Representative immunohistochemical micrographs in blue channel show (D) Ki-67 and Na+/K+-ATPase protein expression in Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. Kidney sections were immunostained with specific antibodies against Ki-67 and Na+/K+-ATPase. Boxed areas are enlarged. Scale bar, 50 µm. Quantitative determination of (E) positive Ki-67 cells and (F) areas of positive Na+/K+-ATPase are presented. *P<0.05 (n=4). Ctrl, control.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Western Blot, Expressing, Immunohistochemical staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Loss of β-catenin in fibroblasts attenuates renal inflammation after AKI. (A and B) qRT-PCR demonstrates a decreased mRNA expression of (A) proinflammatory cytokine TNF-α and (B) MCP-1 in Gli1-β-cat−/− kidneys, compared to Gli1-β-cat+/+ controls. *P<0.05 (n=9). (C and D) Western blot analyses show phosphorylated p65 (p-p65) and total p65 expression in the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. Graphic presentations show the p-p65 and p65 protein abundances in different groups. Relative protein levels over the Gli1-β-cat+/+ controls are reported. *P<0.05 (n=6). (E–G) Immunofluorescence staining revealed a decreased infiltration of CD3+ T cells and CD45+ cells in the kidneys at 1 day after IRI. Boxed areas are enlarged. Arrows indicate positive staining. Scale bar, 50 µm. Quantitative data are presented as the percentage of the areas of (F) CD3+ T cells and (G) CD45+ cells. *P<0.05 (n=4). Ctrl, control.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Fibroblast-specific ablation of β-catenin promotes HGF/c-met signaling after AKI in vivo. (A) qRT-PCR revealed an increased expression of HGF mRNA in Gli1-β-cat−/− kidneys at 1 day after IRI, compared with the Gli1-β-cat+/+ controls. *P<0.05 (n=6). (B and C) Western blot analyses show an increased HGF receptor, c-met, phosphorylation at Tyr1234/1235 in the Gli1-β-cat−/− kidneys at 1 day after IRI, compared with the Gli1-β-cat+/+ controls. (B) Representative western blot and (C) quantitative data are presented. Numbers (1–4) indicate each individual animal in a given group. *P<0.05 (n=6). (D and E) Representative micrographs show renal localization of phosphorylated c-met in the Gli1-β-cat+/+ and Gli1-β-cat−/− kidneys at 1 day after IRI. The images in the blue channel were shown in the bottom panels. The phosphorylated c-met protein was detected by immunohistochemical staining. Boxed areas are enlarged. Scale bar, 50 µm. (E) Quantitative data are presented as the percentage of the areas of phosphorylated c-met. *P<0.05 (n=4). (F and G) Western blots demonstrate ERK1/2 phosphorylation in the kidneys of the Gli1-β-cat+/+ and Gli1-β-cat−/− mice at 1 day after IRI. (F) Representative western blot and (G) quantitative data are presented. *P<0.05 (n=4). Ctrl, control.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Pharmacologic inhibition of β-catenin signaling in Ksp-β-cat mice attenuates kidney injury after AKI. (A) Experimental design. ICG-001 was administrated in conditional knockout mice with tubule-specific ablation of β-catenin 2 days before IRI, and the mice were euthanized at 1 day after IRI. (B) Serum creatinine level in Ksp-β-cat+/+ and Ksp-β-cat−/− mice in the absence or presence of ICG-001 at 1 day after IRI. *P<0.05 (n=8–9). (C) Quantitative assessment of renal injury in Ksp-β-cat+/+ and Ksp-β-cat−/− mice after ICG-001 treatments. Injury score (% of injured tubules) is presented (n=4). (D and E) Representative micrographs showed immunohistochemical staining (blue channel) for Bax in the kidneys at 1 day after IRI. Red color indicates Bax-positive tubules. Scale bar, 50 µm. (E) Quantitative data is presented as the percentage of the areas of Bax staining (n=4). (F and G) Western blot analysis showed little difference in Bax expression between Ksp-β-cat+/+ mice and Ksp-β-cat−/− mice administrated with ICG-001. (F) Representative western blot and (G) quantitative data are presented (n=6). (H and I) Western blot analysis demonstrated Cyclin D1 expression also has no changes between Ksp-β-cat+/+ and Ksp-β-cat−/− mice with ICG-001. (H) Representative western blot and (I) quantitative data are presented (n=6). (J and K) Representative micrographs show renal expression of (J) Cyclin D1 in Ksp-β-cat+/+ and Ksp-β-cat−/− mice in the absence or presence of ICG-001. Scale bar, 50 µm. (K) Quantitative data is presented as positive cell number of Cyclin D1 per HPF. *P<0.05; †P<0.05 (n=4). Sac, Sacrifice.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Inhibition, Knock-Out, Immunohistochemical staining, Staining, Western Blot, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Fibroblast-Specific β -Catenin Signaling Dictates the Outcome of AKI

doi: 10.1681/ASN.2017080903

Figure Lengend Snippet: Wnt/β-catenin suppresses HGF expression in vitro. (A) RT-PCR analyses show that Wnt-enriched conditioned medium suppressed HGF gene expression in cultured NRK-49F fibroblasts, whereas blockade of Wnt/β-catenin signaling by ICG-001 restored, at least partially, HGF expression in vitro. (B) Western blot analyses reveal that Wnt-enriched conditioned medium inhibited HGF protein expression in NRK-49F cells in vitro. (C) RT-PCR analyses show that recombinant Wnt3a and Wnt5a repressed HGF gene expression in cultured NRK-49F fibroblasts in vitro. (D) Western blot analyses demonstrate that constitutive activation of β-catenin also inhibited HGF expression in human cervical cancer cells (C-33A). C-33A cells were transfected with N-terminally truncated, stabilized β-catenin (pDel-β-cat) or empty vector (pcDNA3), respectively. (E) Expression of stabilized β-catenin suppressed HGF gene transcription in luciferase assay. NRK-49F cells were cotransfected with the pGL3-HGF promoter reporter plasmid (HGF promoter region, −1037 to +56) or pGL3-Basic vector, and N-terminally truncated, stabilized β-catenin expression vector (pDel-β-cat). Luciferase activity was assessed and reported as fold induction over the controls. **P<0.01 versus pcDNA3 control (n=3). (F) Schematic diagram depicts the potential mechanisms accounting for fibroblast-specific β-catenin ablation protecting against tubular injury after AKI by promoting HGF signaling. Ctrl, control.

Article Snippet: NRK-49F cells were also transiently transfected with control siRNA or β -catenin–specific siRNA (#6225; Cell Signaling Technology, Danvers, MA) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Western Blot, Recombinant, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

doi: 10.18632/oncotarget.12781

Figure Lengend Snippet: ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.

Article Snippet: Osteosarcoma cells were transfected with 15 nmol/l non-specific (SignalSilence control siRNA, Cell Signaling Technology) or β-catenin-specific siRNA (SignalSilence β-catenin siRNA II, Cell Signaling Technology) for 18 hrs.

Techniques: Western Blot, Expressing, Immunofluorescence, Standard Deviation, Activity Assay

Journal: Oncotarget

Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

doi: 10.18632/oncotarget.12781

Figure Lengend Snippet: (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H&E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in . Higher resolution versions of the lower six panels are shown in . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.

Article Snippet: Osteosarcoma cells were transfected with 15 nmol/l non-specific (SignalSilence control siRNA, Cell Signaling Technology) or β-catenin-specific siRNA (SignalSilence β-catenin siRNA II, Cell Signaling Technology) for 18 hrs.

Techniques: Immunohistochemical staining, Staining