Journal: Journal of Biological Chemistry

Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function

doi: 10.1074/jbc.m110.190330

Figure Lengend Snippet: FIGURE 3. LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 m. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transducedwithlentiviralparticlesharboringshRNAagainstLrp4.ReductionofLrp4RNAlevelswasassessedbyreal-timequantitativePCR,priortotheaddition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

Article Snippet: Recombinant human DKK1 and LRP6 were purchased from R&D Systems.

Techniques: Inhibition, In Vitro, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Activity Assay

Journal: Experimental cell research

Article Title: WNT/β-catenin signaling promotes VSMCs to osteogenic transdifferentiation and calcification through directly modulating Runx2 gene expression.

doi: 10.1016/j.yexcr.2016.06.007

Figure Lengend Snippet: Fig. 1. β-GP stimulates Runx2 induction and activates β-catenin signaling in VSMCs. (A-C) β-GP induces Runx2 expression in VSMCs. VSMCs were treated with 10 mM β-GP for various periods of time as indicated, and the cell lysates were immunoblotted with antibodies against Runx2 and β-actin, respectively. A representative Western blot (A) and the quantitative data (B) are shown. *, po0.05, **, po0.01 versus without β-GP (n¼3). Immunofluorescence staining shows Runx2 expression and localization in VSMCs incubated with 10 mM β-GP for 24 h. Arrowheads indicate positive nuclear staining of Runx2 (C). (D, E) β-GP induces β-catenin activation in VSMCs. VSMCs were treated with 10 mM β-GP for various periods of time as indicated, and the cell lysates were immunoblotted with antibodies against active β-catenin (D), phospho-β-catenin (Ser675) (E), and total β-catenin (D, E), respectively. (F-H) High-phosphate induces β-catenin nuclear translocation in VSMCs. VSMCs were treated with 10 mM β-GP for 3 or 6 h, and nuclear and cytoplasmic protein from VSMCs were prepared and immunoblotted with antibodies against β-catenin, TBP or GAPDH, respectively. The ratios of β- catenin per control proteins (TBP for nuclear protein and GAPDH for cytoplasmic protein) are shown (F, G). Immunofluorescence staining shows β-catenin expression in VSMCs treated with 10 mM β-GP for 6 h. Arrowheads denote nuclear localization of β-catenin (H). (I) β-GP induces LRP6 phosphorylation. VSMCs were treated with 10 mM β- GP for various periods of time as indicated, and the cell lysates were immunoblotted with antibodies against phospho-LRP6 (Ser1490) and LRP6 (C5C7), respectively. (J) Inhibition of PORCN abolishes Runx2 induction stimulated by β-GP. VSMCs were pretreated with LGK 974 (1 nM) for 30 min, then incubated with β-GP (10 mM) for 24 h, and the cell lysates were immunoblotted with antibodies against Runx2 and β-actin, respectively. β-GP, β-glycerophosphoric acid; CTL, control; DAPI, 4′, 6-diamidino-2- phenylindole-HCl; TBP, TATA-binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LRP, low density lipoprotein receptor-related protein.

Article Snippet: The primary antibodies were obtained from the following sources: anti-Runx2 (ab76956; Abcam, Cambridge, MA), anti-dephosphorylated, active β-catenin (anti-ABC, clone 8E7, cat. no. 05-665; Merck Millipore, Billerica, MA), anti-phospho-β-catenin (Ser675) (#9567; Cell Signaling Technology, Beverly, MA), antiβ-catenin (cat. no. 610154; BD Transduction Laboratories, San Jose, CA), anti-Flag (M2, F3165; Sigma), anti-phospho-LRP6 (Ser1490) (#2568; Cell Signal), anti-LRP6 (C5C7) (#2560; Cell Signal), anti-β- actin (sc-1616; Santa Cruz Biotechnology, Dallas, TX), anti-TATAbinding protein (TBP) (ab181-100; Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-25778; Santa Cruz).

Techniques: Expressing, Western Blot, Staining, Incubation, Activation Assay, Translocation Assay, Control, Phospho-proteomics, Inhibition, Binding Assay

Journal: PLOS One

Article Title: Deciphering the synergistic mechanism of a novel flavonoid-antioxidant combination for asthma by combining systems pharmacology and experimental validation

doi: 10.1371/journal.pone.0346165

Figure Lengend Snippet: (A) . Transwell analysis the migrated cells after TGF-β1 (5ng/mL), 74DHF (100nM), and VC (500μM) treatment. Scale bar: 100μm. The quantification of the stained cells was shown on the right (n = 3). (B) . Real time PCR of EMT marker Acta2 and Cdh1 expression in BEAS-2B cells treated by TGF-β1 (5ng/mL), 74DHF (100nM) or VC (500μM) (n = 3). (C) WB analysis of α-SMA, CDH1, COL1A1 and AKT1 in BEAS-2B cells treated by TGF-β1 (5ng/mL), 74DHF (100nM) or VC (500μM) for 24 hours (n = 3). The grey intensity was quantified on the right. Data are presented as the means ± SEM compared by one-way ANOVA with Bonferroni's Multiple Comparison Test. (*, P < 0.05, **, P < 0.01, ***, P < 0.001).

Article Snippet: The membrane was incubated with primary antibody against α-SMA (Abcam, ab7817), CDH1 (CST, 1065), COL1A1 (CST, 3395), AKT1 (CST, 2938), and GAPDH (Abcam, ab70699) over night.

Techniques: Staining, Real-time Polymerase Chain Reaction, Marker, Expressing, Comparison

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: PeerJ

Article Title: CAV1 alleviated CaOx stones formation via suppressing autophagy-dependent ferroptosis.

doi: 10.7717/peerj.14033

Figure Lengend Snippet: Figure 5 GSEA analysis of pathways genes derived from CaOx stones and Control patients mostly involved in. (A) GSEA analyses indicated that Control group was significantly correlated with the WNT signaling pathway in GSE73680 datasets. (B, C) The expression of the top 10 core genes. (D) Western Blot analysis of the expression of CAV1, LRP6 and Wnt/β-Catenin in cells with or without the stimulation of CaOx crystal. The data were expressed as mean ± SE, P < 0.05 compared with the control group. Full-size

Article Snippet: The PVDF membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with primary antibodies against LC3 (14600-1-AP, 1:2000; proteintech, Rosemont, IL, USA), β-actin (66009-1-Ig, proteintech, China, 1:4000), BECN1 (Boster, Wuhan, China, 1:2000), P62 (ABclony, Wuhan, China, 1:2000), caveolin-1 (CAV1) (Santa Cruz, USA, 1:2000), LRP6 (Santa Cruz, USA, 1:2000), and β-Catenin (Santa Cruz, USA, 1:2000) at 4 C overnight.

Techniques: Derivative Assay, Control, Expressing, Western Blot

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 prevents cardiac dysfunction induced by pressure overload. ( A ), Echocardiographic analysis of fraction shortening (FS) in mice at different time-points (3d, 1w, 2w, and 4w) after TAC. * p < 0.05 vs Sham group; n = 6-10 mice/each group. ( B ), Western blot analysis of p-LRP6 and LRP6 levels in left ventricular tissues from mice at different time-points (3d, 1w, 2w, and 4w) after TAC. * p < 0.05; ** p < 0.01 vs sham group; n = 4 mice/ each group. ( C ), Summary of the generation of tamoxifen-inducible cardiac-specific LRP6 overexpressing mice. ( D ), Representative images of Western blot analysis of LRP6 expression in heart and kidney tissues from LRP6 CAG /MCM after injection of tamoxifen or DMSO for 3 consecutive days. ( E ), Representative images of Western blot analysis of LRP6 expression in isolated CMs and non-CMs from tamoxifen-injected LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice. CMs: cardiomyocytes. ( F ), Overall strategy of exploring the effects of cardiac LRP6 overexpression in TAC mice. ( G ), Echocardiographic analysis of cardiac function in tamoxifen-injected LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice at 2 weeks and 4 weeks after TAC or sham operation. FS: fraction shortening; EF: ejection fraction; LVID;d: left ventricular diastolic internal diameter. HR: heart rate; * p < 0.05; ** p < 0.01; n = 7-12/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Western Blot, Expressing, Injection, Isolation

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 attenuates cardiac hypertrophy and fibrosis induced by pressure overload. ( A ), HE and Masson staining of cross heart sections. Scale bars, 2 mm. ( B ), Representative images of Masson staining heart tissue were showed and the cardiac interstitial and vascular fibrosis were quantitatively analyzed. Scale bars, 100 um. ( C ), Western blot analysis of p-LRP6, LRP6, Col1, Col3, α-SMA, MMP2, MMP9, TGF-β1, p-smad2/3 in heart tissues. * p < 0.05; ** p < 0.01; *** p <0.001; n = 3-5/each group. ( D ), Quantitative analysis of the ratio of heart weight/body weight (HW/BW) and heart/tibia (HW/TL). * p < 0.05; ** p < 0.01; *** p < 0.001; n = 4-7/each group. Tamoxifen-injected-LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice were analyzed at 4 weeks after TAC or sham operation.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Staining, Western Blot, Injection

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 inhibits the expression of cardiomyocyte-derived-Wnt5a/11 under pressure overload. ( A ), Western blot analysis of LRP6 expression in CMs, and Col1, MMP2, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in CFs. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON), and the culture medium from these CMs was used to stimulate CFs. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 4/each group. ( B ), Western blot analysis of Wnt5a and Wnt11 expression in CMs and the culture medium. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON). ** p < 0.01; *** p < 0.001; n = 5-6/each group. ( C ), Western blot analysis of Wnt5a and Wnt11 expression in tamoxifen-injected-LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice after TAC or sham operation. ** p < 0.01; n = 5/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Expressing, Derivative Assay, Western Blot, Control, Transfection, Injection

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Knockdown of Wnt5a and Wnt11 inhibits cardiac fibrosis in mice under pressure overload. ( A ), Overall strategy to explore cardiac remodeling after knockdown of Wnt5a and Wnt11 in mice under pressure overload. ( B ), Fraction shortening (FS) in mice at 4 weeks after TAC. Mice were injected with sh-Wnt5a/Wnt11-AAV9 (sh-Wnt5a/Wnt11) or sh-scramble-AAV9 (shNC) by tail vein two weeks before TAC or sham operation. * p < 0.05; n = 6-10/each group. ( C ), Masson staining of heart tissue from mice in (B) and fibrosis area was quantitatively analyzed. Bar = 100 µm. * p < 0.05; *** p < 0.001. n = 4-9/each group. ( D ), Western blot analysis of Wnt5a, Wnt11, Col1, Col3, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in heart tissue from TAC or sham mice pre-injected with sh-Wnt5a/Wnt11-AAV9 (sh-Wnt5a/Wnt11) or sh-scramble-AAV9 (sh-NC) by tail vein. ** p < 0.01; *** p < 0.001. n = 4-6 mice/each group. (E), Western blot analysis of Col1, MMP2, MMP9, TGF-β1 and α-SMA expression in CFs stimulated with the culture medium from control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6), Wnt5a, Wnt11, Wnt5a+Wnt11 at 10 ng/mL, or PBS were supplemented with the culture medium from Ad-LRP6-CMs under MS and treated with CFs. * p < 0.05; ** p < 0.01; *** p < 0.001. n = 3/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Knockdown, Injection, Staining, Western Blot, Expressing, Control

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 promotes the degradation of Wnt5a and Wnt11 by interaction with LRP6 and CTSD during pressure overload. ( A ), Western blot analysis of LRP6 and CTSD expression in tamoxifen-injected LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice at 4 weeks after TAC or sham operation. * p < 0.05; ** p < 0.01; n = 4 mice/each group. ( B ), Western blot analysis of LRP6 and CTSD expression in control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) with or without MS. *** p < 0.001; n = 4-6/each group. ( C ), Interaction of LRP6 and CTSD was analyzed by IP analysis in control (Ad-CON) or LRP6-overexpressing CMs (Ad-LRP6) with or without MS. M: marker. ( D ), Western blot analysis of Wnt5a, Wnt11, and CTSD expression in stretched control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) after treatment with si-CTSD or si-scramble. * p < 0.05; *** p < 0.001; n = 3/each group. ( E ), Western blot analysis of Wnt5a and Wnt11 expression in stretched control (Ad-CON) or LRP6-overexpressing CMs (Ad-LRP6) after treatment with PBS or leupeptin (50 µg/ml). * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3 /each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Western Blot, Expressing, Injection, Control, Marker

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Protease inhibitor attenuates the interaction of LRP6 and CTSD in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. ( A ), Schematic of the experimental procedure in (B). A protease inhibitor, leupeptin (40 mg/kg), was intraperitoneally injected into LRP6 Over or -MCM mice from 2 to 4 weeks (every other day) after TAC; PBS treatment was used as a control. ( B ), IP analysis of the interaction among LRP6, CTSD, Wnt5a, and Wnt11. The experiment was repeated for at least 3 times.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Protease Inhibitor, Injection, Control

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Protease inhibitor attenuates cardiac protection in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. ( A ), Western blot analysis of p-LRP6, LRP6, Wnt5a, Wnt11, Col1, Col3, MMP9 and TGF-β1 in the heart tissue. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3/each group. ( B ), Masson staining of heart tissue and quantitative analysis of fibrosis area. The upper lane: bar = 2 mm; The lower lane: bar = 100 µm, * p < 0.05; ** p < 0.01; n = 7-10 /each group. ( C ), Fraction shortening (FS) in mice. * p < 0.05; ** p < 0.01; n = 8/ each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Protease Inhibitor, Western Blot, Staining

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Mutants of LRP6 or CTSD inhibit the interaction of LRP6 and CTSD. ( A ), Sequencing results of LRP6 and CTSD mutations. ( B ), Schematic diagram of LRP6 and CTSD showing approximate locations of the mutants. ( C ), IP analysis of the interaction among LRP6 WT, LRP6 (P1104S), or LRP6 (P1427Q) and CTSD WT or CTSD (G136R). LRP6 WT and CTSD WT or their mutant plasmids were transfected into 293T cells for 48 h. The experiment was repeated 4 times. ( D ), Western blot analysis of LRP6, CTSD, Wnt5a, Wnt11, and MESD in stretched COS7 cells transfected with LRP6 WT and CTSD WT or their mutant plasmids as in (C). **** P < 0.0001, n = 3/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Sequencing, Mutagenesis, Transfection, Western Blot

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Proposed model of cardiac protection mediated by cardiac-specific LRP6 overexpression in response to pressure overload. LRP6 overexpression suppresses β-catenin activation, which may contribute to the inhibition of cardiac hypertrophy. LRP6 overexpression inhibits cardiomyocyte-derived Wnt5a/Wnt11 to inhibit cardiac fibrosis. Cardiomyocyte-expressed LRP6 interacts with CTSD, which may promote the degradation of Wnt5a and Wnt11.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Activation Assay, Inhibition, Derivative Assay