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

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the phosphorylation of LRP6 in cardiac side population cells (CSPs) by UT/JNK signaling during mechanical stretch. ( A ) Immunofluorescent images revealed that positive signal (green) could be observed in cultured CSPs with antibody to p-LRP6 (1:200); bar: 20 μm. ( B ) Quantitative evaluation of p-LRP6 by analysis of fluorescence intensity. ( C ) P-LRP6 was detected in cultured CSPs by nanofluidic proteomic immunoassay (NIA). Peaks on the traces that represent phosphorylated isoforms of LRP6 are indicated. ( D ) NIA pseudoblot representation of p-LRP6 and bar graph of p-LRP6/GAPDH in cultured CSPs by NIA quantification. Cultured CSPs were pre-treated with PBS, urantide (Ura, 1 μM) or SP600125 (SP6, 5 μM), respectively, for 30 min, then subjected to mechanical stretch (MS) and incubated with PBS or UII (0.1 μM) for 3 hrs. Values are expressed as mean ± SEM. ** P < 0.01 versus control; ## P < 0.01 versus control; # P < 0.05 versus MS; & P < 0.05 versus MS plus UII; && P < 0.01 versus MS plus UII. The experiment was repeated for at least three times. ( E ) P-LRP6 was detected in CSPs isolated from transverse aorta constriction (TAC) or Sham mice by NIA. ( F ) NIA pseudoblot representation of p-LRP6 and bar graph of p-LRP6/GAPDH in isolated CSPs by NIA quantification. Values are expressed as mean ± SEM. Sham: n = 9; Sham+Ura: n = 6; TAC: n = 6; TAC+Ura: n = 7. Ura: urantide. ** P < 0.01 versus Sham mice; ## P < 0.01 versus TAC mice.

Article Snippet: Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion.

Techniques: Phospho-proteomics, Cell Culture, Fluorescence, Incubation, Control, Isolation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the phosphorylation of LRP6 in UT cell line by activating JNK during mechanical stretch. Western blot analysis for p-LRP6 and p-JNK levels. UT cells were pre-treated with Urantide (Ura, 1 μM) for 30 min., then subjected to MS and treated with UII for 30 min. and 3 hrs. ( A ) Representative picture of p-LRP6 and p-JNK level at 30 min. after UII treatment during MS. Quantitative evaluation of p-JNK ( B ) and p-LRP6 ( C ) by analysis of Western blot. ( D ) Representative picture of p-LRP6 and p-JNK level at 3 hrs after UII treatment during MS. Quantitative evaluation of p-JNK ( E ) and p-LRP6 ( F ) by analysis of Western blot. P-JNK and p-LRP6 were analysed in UT cells pre-treated with SP600125 (SP6, 5 μM; G ) or overexpressed LRP6 ( H ) with or without UII (0.1 μM) during MS. UT cells were pre-treated with SP600125 or DMSO for 30 min., then subjected to MS and treated with UII for 3 hrs. UT cells were overexpressed with LRP6-pCS2 or pCS2 for 48 hrs, then subjected to MS and treated with UII for 3 hrs. Values are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 versus control. # P < 0.05, ## P < 0.01 versus MS. & P < 0.05, && P < 0.01 versus MS plus UII. The experiment was repeated for at least three times.

Article Snippet: Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the proliferation of cardiac side population cells (CSPs) by c-Jun N -terminal kinase (JNK)-LRP6 signalling during mechanical stretch. CSPs proliferation was determined by Cell Viability Assay. Cultured CSPs were overexpressed LRP6 ( A ) or pretreated with SP600125 (SP6, 5 μM; B ) with or without UII (0.1 μM) during mechanical stretch (MS). CSPs were pre-treated with SP600125 or DMSO for 30 min., then subjected to MS and treated with UII for 48 hrs. CSPs were overexpressed with LRP6-pCS2 or pCS2 for 48 hrs, then subjected to MS and treated with UII for 48 hrs. Values are expressed as mean ± SEM. ** P < 0.01 versus MS. # P < 0.05 versus MS plus UII. The experiment was repeated for at least three times. ( C ) The ratio of CSPs per mouse was analysed by fluorescence-activated cell sorting (FACS). Representative photographs are shown. ( D ) The ratio of CSPs per mouse was calculated by FACS. SP600125 (SP6) or DMSO were injected intraperitoneally either with 40 mg/kg bodyweight every 3 days for 2 weeks. At 4 weeks after transverse aorta constriction (TAC) or sham operation, the number of CSPs was determined. Values are expressed as mean ± SEM. Sham: n = 5; Sham+SP6: n = 6; TAC: n = 6; TAC+SP6: n = 6. Ura: urantide. * P < 0.05 versus sham mice; # P < 0.05 versus TAC mice.

Article Snippet: Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion.

Techniques: Viability Assay, Cell Culture, Fluorescence, FACS, Injection

Journal: Translational Vision Science & Technology

Article Title: SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy

doi: 10.1167/tvst.11.9.19

Figure Lengend Snippet: FZD4 and LRP5, primary receptors for Wnt signaling in vascular endothelial cells. (A) FZD1–10 and LRP5 and LRP6 mRNA expression in HRMECs, bEnd.3 cells, and HUVECs quantified by quantitative polymerase chain reaction (qPCR) and normalized by the housekeeping gene Actin . Bars represent mean ± SD. (B) Data from Macosko et al. 31 were reanalyzed to derive scaled average gene expression by cell type of Wnt receptors in mouse retina. (C) Data from Menon et al. were reanalyzed to derive scaled average gene expression by cell type of Wnt receptors in human retina.

Article Snippet: RNA was quantified using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) with the following Thermo Fisher Scientific probes: Mm00445405_s1 Fzd1 , Mm02524776_s1 Fzd2 , Mm00445423_m1 Fzd3 , Mm00433382_m1 Fzd4 , Mm00445623_s1 Fzd5 , Mm00433387_m1 Fzd6 , Mm00433409_s1 Fzd7 , Mm01234717_s1 Fzd8 , Mm01206511_s1 Fzd9 , Mm00558396_s1 Fzd10 , Mm01227476_m1 Lrp5 , Mm00999795_m1 Lrp6 , Hs00268943_s1 FZD1 , Hs00361432_s1 FZD2 , Hs00907280_m1 FZD3 , Hs00201853_m1 FZD4 , Hs00258278_s1 FZD5 , Hs01095627_m1 FZD6 , Hs00275833_s1 FZD7 , Hs00259040_s1 FZD8 , Hs00268954_s1 FZD9 , Hs00273077_s1 FZD10 , Hs00182031_m1 LRP5 , Hs00233945_m1 LRP6 , Hs01547250_m1 LEF1 , Hs00293017_m1 MFSD2A , Hs00610344-m1 AXIN2 , and Mm00443610_m1 Axin2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: The Journal of Infectious Diseases

Article Title: Anthrax Lethal and Edema Toxins Fail to Directly Impair Human Platelet Function

doi: 10.1093/infdis/jir763

Figure Lengend Snippet: Assessment of anthrax toxin binding to human platelet and platelet surface expression of anthrax toxin receptors. A, B, Flow cytometry measuring protective antigen (PA) binding and internalization by human platelets (A) and by THP-1 cells (B) after 2 hours of treatment with 1000 ng/mL PA-biotin at 4°C, 37°C, or incubation in buffer alone. Cells were fixed in a 1:1 ratio of 3.7% formaldehyde, probed with fluorescein isothiocyanate (FITC)–streptavidin, and analyzed using FACScan (BD) and FCS Express (DeNovo) software. Data are representative of 4 experiments from 3 platelet donors and 3 experiments using separate THP-1 cell preparations. C, D, Flow cytometry measuring anthrax receptor surface expression in human platelets (C) and THP-1 cells (D). Cells were fixed, probed for tumor endothelial marker 8 (TEM8)/capillary morphogenesis gene 2 (CMG2), LRP6, or an isotype control, followed by secondary goat anti-rabbit Alexa Fluor 488–conjugated antibody, and analyzed using Accuri C6 (BD Biosciences) and FCS Express (De Novo). Data are representative of 3 experiments using 3 donors. E, Fold changes in peak fluorescent intensity of bound polyclonal antibodies directed against anthrax receptors: TEM8/CMG2 and LRP6 in human platelets and THP-1 cells. Brackets show the standard error of the mean for 3 donors. The fold change in peak fluorescence intensity for each condition was calculated by comparing each sample with the peak fluorescence intensity of an isotype control polyclonal primary rabbit antibody.

Article Snippet: Antibodies used were reactive against MEK1 (Upstate), TEM8/CMG2 (Abcam), low density lipoprotein receptor-related protein 6 (LRP6) (Santa Cruz), Alexa Fluor 488 goat anti-rabbit immunoglobulin G (H+L) (Invitrogen), isotype control/VASP antibody (Cell Signaling), total HSP27 (Cell Signaling), phosphorylated serine 82 HSP27 (Cell Signaling), or β-actin (Sigma).

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Software, Marker, Fluorescence

Journal: Life

Article Title: The Relationship between LRP6 and Wnt/β-Catenin Pathway in Colorectal and Esophageal Cancer

doi: 10.3390/life13030615

Figure Lengend Snippet: LRP6 expression in normal colon and CRC tissues. ( A ) LRP6 expression in normal colon mucosa. ( B , C ) Classification of the positivity of LRP6 expression in CRC. ( D , E ) Classification of LRP6 expression intensity in CRC. ( F ) Distribution of the staining score, calculated by multiplying the intensity and positivity scores.

Article Snippet: The product numbers of the TaqMan Gene Expression assay were as follows: ACTB ID Hs01060665_g1 and LRP6 ID Hs00999795_m1.

Techniques: Expressing, Staining

Journal: Life

Article Title: The Relationship between LRP6 and Wnt/β-Catenin Pathway in Colorectal and Esophageal Cancer

doi: 10.3390/life13030615

Figure Lengend Snippet: LRP6 and β-catenin expression in normal esophageal and ESCC tissues. ( A ) LRP6 expression in normal esophageal tissues. ( B , C ) Distribution of the LRP6 staining score, calculated by multiplying the intensity and positivity scores. ( D , E ) Distribution of the β-catenin staining score, calculated by multiplying the intensity and positivity scores.

Article Snippet: The product numbers of the TaqMan Gene Expression assay were as follows: ACTB ID Hs01060665_g1 and LRP6 ID Hs00999795_m1.

Techniques: Expressing, Staining

Journal: Life

Article Title: The Relationship between LRP6 and Wnt/β-Catenin Pathway in Colorectal and Esophageal Cancer

doi: 10.3390/life13030615

Figure Lengend Snippet: Effects of LRP6 on Wnt/β-catenin signaling activity. ( A ) Expression of LRP6 mRNA in CRC and ESCC cell lines, as measured by qRT-PCR. We used ACTB as endogenous control. * p < 0.01. ( B ) The protein level of LRP6 in CRC and ESCC cell lines. We used ACTB as endogenous control. ( C ) Wnt/β-catenin signaling activity under unstimulated conditions in CRC and ESCC cell lines. ( D ) Experimental plan for investigating signaling activity by adding LRP6-siRNA or Wnt3a to the CRC and ESCC cell lines. ( E – H ) Wnt/β-catenin signaling activity using CRC and ESCC cell lines. We used Renilla as endogenous control. * p < 0.05, ** p < 0.01; n.s.: not significant.

Article Snippet: The product numbers of the TaqMan Gene Expression assay were as follows: ACTB ID Hs01060665_g1 and LRP6 ID Hs00999795_m1.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Control

Journal: Life

Article Title: The Relationship between LRP6 and Wnt/β-Catenin Pathway in Colorectal and Esophageal Cancer

doi: 10.3390/life13030615

Figure Lengend Snippet: Comparison of clinicopathological status stratified by LRP6 or β-catenin expression in CRC tissue samples.

Article Snippet: The product numbers of the TaqMan Gene Expression assay were as follows: ACTB ID Hs01060665_g1 and LRP6 ID Hs00999795_m1.

Techniques: Comparison, Expressing

Journal: Life

Article Title: The Relationship between LRP6 and Wnt/β-Catenin Pathway in Colorectal and Esophageal Cancer

doi: 10.3390/life13030615

Figure Lengend Snippet: Comparison of clinicopathological status stratified by LRP6 or β-catenin expression in ESCC tissue samples.

Article Snippet: The product numbers of the TaqMan Gene Expression assay were as follows: ACTB ID Hs01060665_g1 and LRP6 ID Hs00999795_m1.

Techniques: Comparison, Expressing

Journal: PLoS ONE

Article Title: Effects of pharmacologic sclerostin inhibition or testosterone administration on soleus muscle atrophy in rodents after spinal cord injury

doi: 10.1371/journal.pone.0194440

Figure Lengend Snippet: A-C. LDL receptor related protein 5 (LRP5) and 6 (LRP6), and androgen receptor (AR) gene expressions in tibia, levator ani/bulbocavernosus (LABC), and soleus from animals receiving sham surgery (T 8 laminectomy). Values are means ± SEM [corrected for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene] and expressed relative to the tibia, n = 6/tissue. Letters a-c indicate differences from respectively labeled groups at p<0.05 or *p<0.01 (a = vs Tibia; b = vs LABC; c = vs Soleus).

Article Snippet: The following commercially-available gene-specific primers were used (ThermoFisher Scientific, Carlsbad, CA): rat LRP5 (#Rn01451428_m1), rat LRP6 (#Rn01492711_m1), rat AR (#Rn00560747_m1), and rat GAPDH (#Rn01775763_m1).

Techniques: Labeling