Journal: Diabetes

Article Title: Inhibition of Connective Tissue Growth Factor Overexpression in Diabetic Retinopathy by SERPINA3K via Blocking the WNT/β-Catenin Pathway

doi: 10.2337/db09-1056

Figure Lengend Snippet: Wnt/β-catenin signaling was responsible for the high-glucose–induced CTGF overexpression. A : HTERT RPE-1 cells were exposed to high glucose for different durations as indicated. p-LRP6 levels were measured by Western blot analysis using 100 μg of total proteins with an antibody specific for p-LRPE6, and cytosolic β-catenin levels were determined by Western blot analysis using 20 μg cytosolic proteins. B : HTERT RPE-1 cells were exposed to low glucose and high glucose media with 100 nmol/l DKK1 or BSA. After 24-h treatment of the cells, CTGF levels were measured by Western blot analysis. C and D : The retinas were dissected from nondiabetic rats and diabetic rats. β-Catenin levels in the retinal homogenates were measured by Western blot analysis ( C ) and quantified by densitometry ( D ) (* P < 0.05, means ± SD, n = 6).

Article Snippet: Antibodies for CTGF, LRP6, and β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:400, 1:500, and 1:2,500 dilutions, respectively.

Techniques: Over Expression, Western Blot

Journal: Diabetes

Article Title: Inhibition of Connective Tissue Growth Factor Overexpression in Diabetic Retinopathy by SERPINA3K via Blocking the WNT/β-Catenin Pathway

doi: 10.2337/db09-1056

Figure Lengend Snippet: SERPINA3K inhibited the high-glucose–induced Wnt/β-catenin signaling. A : HTERT RPE-1 cells were exposed to low glucose and high glucose media for 6 h with various concentrations of SERPINA3K. Levels of p-LRP6 and cytosolic β-catenin were determined by Western blot analysis. B and C : The retinas were dissected from the diabetic rats 4 weeks after the intravitreal injection of Ad-LacZ or Ad-SA3K. β-Catenin levels in the retinal homogenates were measured by Western blot analysis ( B ) and quantified by densitometry ( C ) (mean ± SD, n = 6, * P < 0.05).

Article Snippet: Antibodies for CTGF, LRP6, and β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:400, 1:500, and 1:2,500 dilutions, respectively.

Techniques: Western Blot, Injection

Journal: Diabetes

Article Title: Inhibition of Connective Tissue Growth Factor Overexpression in Diabetic Retinopathy by SERPINA3K via Blocking the WNT/β-Catenin Pathway

doi: 10.2337/db09-1056

Figure Lengend Snippet: SERPINA3K blocked the Wnt ligand-induced CTGF overexpression. A and B : HTERT RPE-1 cells were exposed to 50% control L-cell medium (LM) or 50% Wnt3a conditioned medium (WM) for 1 h ( A ) or 2 h ( B ), with 100 nmol/l SERPINA3K or BSA. The same amounts of total cellular proteins (100 μg) ( A ) or cytosolic proteins (20 μg) ( B ) were blotted separately with antibodies specific for p-LRP6 and total LRP6 ( A ), or for β-catenin ( B ). C : The cells were transfected with the TOPFLASH vector, followed by exposure to the LM or WM containing 1,000 nmol/l BSA or SERPINA3K for 24 h. The TOPFLASH activity was measured using luciferase assay (means ± SD, n = 3, * P < 0.05). D : HTERT RPE-1 cells were exposed to LM and WM media with 100 nmol/l SERPINA3K or BSA. After culture for 24 h, CTGF levels were measured by Western blot analysis.

Article Snippet: Antibodies for CTGF, LRP6, and β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:400, 1:500, and 1:2,500 dilutions, respectively.

Techniques: Over Expression, Control, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Western Blot

Journal: Endocrine Connections

Article Title: IUGR with catch-up growth programs impaired insulin sensitivity through LRP6/IRS-1 in male rats

doi: 10.1530/EC-21-0203

Figure Lengend Snippet: The expression of LRP6 was decreased in the skeletal muscle of male CG-IUGR. The protein (A) and mRNA (B) levels of LRP6 and β-catenin in skeletal muscle were reduced in the male CG-IUGR rats. Data are presented as mean ± s.e.m. ( n = 5). * P < 0.05, ** P < 0.01 compared with same-age controls.

Article Snippet: LRP6 shRNA, control shRNA, polybrene and puromycin dihydrochloride were purchased from Santa Cruz Biotechnology Inc (TX).

Techniques: Expressing

Journal: Endocrine Connections

Article Title: IUGR with catch-up growth programs impaired insulin sensitivity through LRP6/IRS-1 in male rats

doi: 10.1530/EC-21-0203

Figure Lengend Snippet: LRP6 is requisite for normal expression of the IR-β/IRS-1. LRP6 specific shRNA decreased the protein (A) and mRNA (B) expression of LRP6, IR-β and IRS-1 in C2C12 cells. Data are presented as mean ± s.e.m. ( n = 3). ** P < 0.01 compared with same-age controls.

Article Snippet: LRP6 shRNA, control shRNA, polybrene and puromycin dihydrochloride were purchased from Santa Cruz Biotechnology Inc (TX).

Techniques: Expressing, shRNA

Journal: Endocrine Connections

Article Title: IUGR with catch-up growth programs impaired insulin sensitivity through LRP6/IRS-1 in male rats

doi: 10.1530/EC-21-0203

Figure Lengend Snippet: Wnt3a/LRP6 enhanced the expression of IRS-1 and IGF-1R in muscle cells of male CG-IUGR. (A) The curve of LRP6 mRNA expression in primary muscle cells treated with Wnt3a (30 ng/mL) for different time spans in male CG-IUGR rats. (B and C) Upon Wnt3a stimulation for 12 h, the protein levels of LRP6 (B), β-catenin (B) and IRS-1 (C) were increased in the primary muscle cells of male CG-IUGR rats and the expression of IR-β (C) was not altered. (D) The expression of IGF-1R was reduced in the skeletal muscle of male CG-IUGR rats. (E) The expression of IGF-1R was increased upon Wnt-3a stimulation in the primary muscle cells of male CG-IUGR. Data are presented as mean ± s.e.m. ( n = 3). ** P < 0.01 compared with same age controls.

Article Snippet: LRP6 shRNA, control shRNA, polybrene and puromycin dihydrochloride were purchased from Santa Cruz Biotechnology Inc (TX).

Techniques: Expressing

Journal: Endocrine Connections

Article Title: IUGR with catch-up growth programs impaired insulin sensitivity through LRP6/IRS-1 in male rats

doi: 10.1530/EC-21-0203

Figure Lengend Snippet: Wnt3a/LRP6 increased the expression of mTOR/S6K in skeletal muscle cells of male CG-IUGR rats. (A) The protein levels of mTOR and P70S6K were decreased in skeletal muscle of male CG-IUGR rats ( n = 5). (B) The expression of mTOR and P70S6K was increased upon stimulating with Wnt3a (30 ng/mL) for 12 h in skeletal muscle cells of male CG-IUGR rats.

Article Snippet: LRP6 shRNA, control shRNA, polybrene and puromycin dihydrochloride were purchased from Santa Cruz Biotechnology Inc (TX).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH

doi: 10.3390/ijms23020940

Figure Lengend Snippet: Naca and Lrp6 form part of a common genetic pathway regulating bone mass. Femurs of female mice were analyzed by μCT at 3 months of age. BV/TV, bone volume/tissue volume. * p < 0.05, two-way ANOVA with post hoc tests.

Article Snippet: Real-time qPCR using TaqMan universal PCR master mix and gene-specific Taqman assays for Lrp6 (Mm00521783_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), and the alpha 1 chain of type I collagen ( Col1a1 , Mm00801666_g1) was performed using a QuantStudio 7 real-time PCR system (Life Technologies Applied Biosystems).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH

doi: 10.3390/ijms23020940

Figure Lengend Snippet: The osteoanabolic response to iPTH is inhibited in Naca / Lrp6 compound heterozygotes. Four-month-old female mice were treated once daily, 5 days/week for 1 month with 100 μg/kg of PTH(1-34). Vertebrae were analyzed by μCT. BV/TV, bone volume/tissue volume. Results are expressed as change from vehicle-treated animals, which are ascribed a value of 100%. * p < 0.05, two-way ANOVA with post hoc tests.

Article Snippet: Real-time qPCR using TaqMan universal PCR master mix and gene-specific Taqman assays for Lrp6 (Mm00521783_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), and the alpha 1 chain of type I collagen ( Col1a1 , Mm00801666_g1) was performed using a QuantStudio 7 real-time PCR system (Life Technologies Applied Biosystems).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH

doi: 10.3390/ijms23020940

Figure Lengend Snippet: The increase in biomechanical properties induced by iPTH treatment is blunted in Naca / Lrp6 compound heterozygotes. Four-month-old female mice were treated once daily, 5 days/week for 1 month with 100 μg/kg of PTH(1-34). L2 vertebrae were analyzed by compression testing to measure stiffness ( A ), load at yield ( B ) and maximum load ( C ). Results are expressed as change from vehicle-treated animals, which are ascribed a value of 100%. * p < 0.05; ** p < 0.01; # p < 0.0001, two-way analysis of variance (ANOVA) with post hoc tests.

Article Snippet: Real-time qPCR using TaqMan universal PCR master mix and gene-specific Taqman assays for Lrp6 (Mm00521783_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), and the alpha 1 chain of type I collagen ( Col1a1 , Mm00801666_g1) was performed using a QuantStudio 7 real-time PCR system (Life Technologies Applied Biosystems).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH

doi: 10.3390/ijms23020940

Figure Lengend Snippet: Gene expression monitoring in control ( Naca 99S/S ; Lrp6 +/+ ;OCN-Cre) and compound heterozygous ( Naca 99S/A ; Lrp6 +/fl ;OCN-Cre) littermates. RNA was extracted from marrow-flushed tibial diaphyses, reverse-transcribed and analyzed by RT-qPCR. Relative expression of Lrp6 ( A ), Alpl ( B ) and Col1a1 ( C ) is shown. * p < 0.05; ** p < 0.01; *** p < 0.001, two-way ANOVA with post hoc tests.

Article Snippet: Real-time qPCR using TaqMan universal PCR master mix and gene-specific Taqman assays for Lrp6 (Mm00521783_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), and the alpha 1 chain of type I collagen ( Col1a1 , Mm00801666_g1) was performed using a QuantStudio 7 real-time PCR system (Life Technologies Applied Biosystems).

Techniques: Gene Expression, Control, Reverse Transcription, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH

doi: 10.3390/ijms23020940

Figure Lengend Snippet: Parallel PTH signaling pathways in control ( Naca 99S/S ; Lrp6 +/+ ;OCN-Cre) and compound heterozygous ( Naca 99S/A ; Lrp6 +/fl ;OCN-Cre) littermates. Protein extracts were prepared from tibial shafts and immunoprobed for β-Catenin ( A ) and SIK2 ( B ). Equivalent loading was assessed by probing for α-Tubulin ( C ).

Article Snippet: Real-time qPCR using TaqMan universal PCR master mix and gene-specific Taqman assays for Lrp6 (Mm00521783_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), and the alpha 1 chain of type I collagen ( Col1a1 , Mm00801666_g1) was performed using a QuantStudio 7 real-time PCR system (Life Technologies Applied Biosystems).

Techniques: Protein-Protein interactions, Control