Journal: Oncogene

Article Title: ADP-ribosylation factors 1 and 6 regulate Wnt/β-catenin signaling via control of LRP6 phosphorylation.

doi: 10.1038/onc.2012.373

Figure Lengend Snippet: Figure 3. Fzd, LRP6 and Dvls are necessary for Wnt3a-mediated Arf activation. (a) Arf1 activation was inhibited by the knockdown of Fzds. HEK293T cells were transiently co-transfected with expression vectors for shFzd2, 4 and 5, and the clones stably expressing shFzds were selected using 2 mg/ml of puromycin. Subsequently, Arf activation assay was performed (left panel). Knockdown of Fzd2, 4 and 5 was shown by reverse transcription-PCR (right panel). (b) Activation of Arf1 was attenuated by the addition of Fc-CRD. HEK293T cells were transiently transfected with Fc or Fc-CRD, after which the media were incubated with Wnt3a-CM for 1 h. The mixed media were then incubated with Fc agarose beads and the supernatant was collected. HEK293T cells were treated with the collected media for the indicated times, and the levels of Arf1-GTP and Arf1 were measured (left panel). The expression of Fc or Fc-CRD was shown by western blot (right panel). (c) Arf1 activation was inhibited by the knockdown of LRP6, and the levels of proteins were measured by immunoblotting. (d) Arf1 activation was inhibited by the knockdown of Dvls, and the levels of proteins were measured by immunoblotting.

Article Snippet: ARF1(T31N)seCFP-INT, ARF1(Q71L)-seCFP-INT, ARF6(T27N)-seCFP-INT and ARF6(Q67L)seCFP-INT were generated by modified QuikChange mutagenesis.37 For simultaneous imaging of the rapamycin-induced depletion of PtdIns (4,5)P2 and FRET-based PtdIns (4,5)P2 indicator, the CFP part of CF-INP was replaced with mCherry to generate mCh-INP. pGEX-GGA3 (1–316) and pSuper vector, which was used for the expression of shRNAs, were kindly provided by Drs Parent (Université de Sherbrooke, Quebec, Canada) and Agami (The Netherlands Cancer Institute, Amsterdam, Netherlands), respectively.17,38 Previously published nucleotide sequences for the shRNAs were used in our experiments; GFP,39 Arf1, Arf6,31 Frizzled2, 4, 5, LRP6 and Dvl1, 2, 3.14 A plasmid for FRET-based PtdIns (4,5)P2 indicator (Pippi-PI(4,5)P2) was provided by Professor Michiyuki Matsuda of Kyoto University, Kyoto, Japan.25 For the depletion of PtdIns (4,5)P2, PM-localized FK506-binding protein (FKBP12)-rapamycin-binding (FRB) construct and a cytosolic INP54p enzyme conjugated with FKBP12 (CF-INP) construct were provided by Professor Won Do Heo of the Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.40 Antibodies Anti-b-catenin monoclonal antibody (Transduction Laboratories, Rockville, MD, USA), anti-HA monoclonal antibody, anti-a-tubulin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-b-actin monoclonal antibody (Sigma, St Louis, MO, USA), anti-LRP6 polyclonal antibody (Abcam, Cambridge, MA, USA), anti-phospho LRP6 (Ser 1490) polyclonal antibody (Cell Signaling, Danvers, MA, USA), anti-Arf1 monoclonal antibody (Epitomics, Burlingame, CA, USA) and anti-Arf6 monoclonal antibody (Santa Cruz Biotechnology Inc.) were used to detect their corresponding proteins.

Techniques: Activation Assay, Knockdown, Transfection, Expressing, Clone Assay, Stable Transfection, Reverse Transcription, Incubation, Western Blot

Journal: Oncogene

Article Title: ADP-ribosylation factors 1 and 6 regulate Wnt/β-catenin signaling via control of LRP6 phosphorylation.

doi: 10.1038/onc.2012.373

Figure Lengend Snippet: Figure 4. Knockdown of Arfs inhibits Wnt3a-mediated formation of PtdIns (4,5)P2 and phosphorylation of LRP6. (a) Wnt3a-CM-induced formation of PtdIns (4,5)P2 was inhibited by the knockdown of Arf1 or Arf6. HEK293T cells stably expressing shGFP or shArf1, Arf6 were transfected with Pippi-PI(4,5)P2 FRET-based indicator plasmids and then treated with control-CM or Wnt3a-CM. Treatment with Wnt3a-CM induced the formation of PtdIns (4,5)P2, which reached a maximum at 10B30 min, whereas Arf1, 6-depleted cells did not. (b) The net intensities of CFP and FRET in each cell were measured, and the average emission ratio (FRET/CFP) was calculated as in Figure 4a. The emission ratio values were normalized to those of the record-starting time. (c) The depletion of Arf1 or Arf6 decreased Wnt3a-mediated induction of LRP6 phosphorylation at Serine 1490. HEK293T cells transfected with siGFP, siArf1 or siArf6 were incubated for 72 h and treated with Wnt3a- CM for the indicated times, after which the lysates were immunoblotted with the indicated antibodies. (d) Schematic diagram of the involvement of Arfs in the regulation of Wnt/b-catenin signaling.

Article Snippet: ARF1(T31N)seCFP-INT, ARF1(Q71L)-seCFP-INT, ARF6(T27N)-seCFP-INT and ARF6(Q67L)seCFP-INT were generated by modified QuikChange mutagenesis.37 For simultaneous imaging of the rapamycin-induced depletion of PtdIns (4,5)P2 and FRET-based PtdIns (4,5)P2 indicator, the CFP part of CF-INP was replaced with mCherry to generate mCh-INP. pGEX-GGA3 (1–316) and pSuper vector, which was used for the expression of shRNAs, were kindly provided by Drs Parent (Université de Sherbrooke, Quebec, Canada) and Agami (The Netherlands Cancer Institute, Amsterdam, Netherlands), respectively.17,38 Previously published nucleotide sequences for the shRNAs were used in our experiments; GFP,39 Arf1, Arf6,31 Frizzled2, 4, 5, LRP6 and Dvl1, 2, 3.14 A plasmid for FRET-based PtdIns (4,5)P2 indicator (Pippi-PI(4,5)P2) was provided by Professor Michiyuki Matsuda of Kyoto University, Kyoto, Japan.25 For the depletion of PtdIns (4,5)P2, PM-localized FK506-binding protein (FKBP12)-rapamycin-binding (FRB) construct and a cytosolic INP54p enzyme conjugated with FKBP12 (CF-INP) construct were provided by Professor Won Do Heo of the Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.40 Antibodies Anti-b-catenin monoclonal antibody (Transduction Laboratories, Rockville, MD, USA), anti-HA monoclonal antibody, anti-a-tubulin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-b-actin monoclonal antibody (Sigma, St Louis, MO, USA), anti-LRP6 polyclonal antibody (Abcam, Cambridge, MA, USA), anti-phospho LRP6 (Ser 1490) polyclonal antibody (Cell Signaling, Danvers, MA, USA), anti-Arf1 monoclonal antibody (Epitomics, Burlingame, CA, USA) and anti-Arf6 monoclonal antibody (Santa Cruz Biotechnology Inc.) were used to detect their corresponding proteins.

Techniques: Knockdown, Phospho-proteomics, Stable Transfection, Expressing, Transfection, Control, Incubation

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