Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: (A) Western blot analysis showed that only the level of DVL3 but not DVL1 and DVL2 was significantly up-regulated in cervical cancer cell lines when compared with the normal cervix cell lines. (B) Real time q-PCR revealed the mRNA of DVL3 was obviously higher among cervical cancer cell lines when compared with the normal cervix cell lines. The value of NC105 was used to normalize other cell lines. (C) Immunohistochemical analysis showed that both DVL3 and β-catenin were upregulated on cervical cancer tissues when compared with normal cervix samples. DVL3 was distributed in the cytoplasmic region whereas β-catenin was located in both the cytoplasmic and nuclear regions. (Magnification: 20x) (D) Luciferase reporter assay revealed that transient transfection of DVL3-expressing plasmid could increase β-catenin transcriptional activity in HEK293 cells.

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Western Blot, Immunohistochemical staining, Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay

Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: Clinicopathological correlation of DVL3 expression in cervical cancer tissue array (CX1021) (Pantomics, CA, USA).

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: Clinicopathological correlation of β-catenin expression in cervical cancer tissue array (CX1021) (Pantomics, CA, USA).

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: (A) Western blot showed that stably over-expressing GFP-DVL3 elevated expression of β-catenin in C33A (C-G25 and C-G28) and SiHa (S-G18 and S-G20). (B) XTT assay revealed a significant increase of cell proliferation in cervical cancer cells with stable expression of GFP-DVL3 (C-G24 and C-G28; P <0.05) (S-G18 and S-G20; P <0.05). (C) Clonogenic assay showed that twice and thrice the number of colonies were found in GFP-DVL3 ectopically expressing C33A cells (C-G25 and C-G28) ( P <0.05) and SiHa cells (S-G18 and S-G20) ( P <0.05) as compared with their vector controls.

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Western Blot, Stable Transfection, Expressing, XTT Assay, Clonogenic Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: (A) Upon treatment of AICAR, approximately 80% reduction of DVL3 level was observed among C33A, C41 and CaSki. (B) Upon treatment of A769662 at different doses (0, 50 and 100 μM), a 20-30% and 80%–100% reduction of DVL3 in SiHa and C33A could be observed respectively. (C) Upon treatment with A23187 at different doses (0, 1.25 and 2.5 μM) for twenty hours, C33A and SiHa cells showed 80–90% reduction of DVL3 expression in a dose-dependent manner. (D) Upon treatment of 1.25 μM A23187, the decrease of DVL3 could be observed after 10 and 12 hours in C33A and CaSki respectively, while a further 100% reduction of DVL3 was observed at 12 and 24 hours for both cell lines. (E) Upon treatment of metformin, 60–80%reduction of DVL3 was observed in Hela, SiHa, CaSki, C41 and C33A in a dose-dependent manner.

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: (A) Real time q-PCR analysis demonstrated that metformin had no effect on the expression of DVL3 mRNA expression in C33A and HeLa cells. (B) Western blot analysis revealed that compound C could abate the decrement of DVL3 mediated by metformin. (C) XTT cell proliferation assay showed a significant reduction of cell proliferation in C33A ( P >0.001), SiHa ( P >0.001) and HeLa cells ( P >0.001) treated with metformin in a dose-dependent manner. (D) Clonogenic assay showed a remarkable reduction of the number of colonies formed in HeLa and C33A treated with metformin in a dose-dependent manner.

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Proliferation Assay, Clonogenic Assay

Journal: PLoS ONE

Article Title: AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

doi: 10.1371/journal.pone.0053597

Figure Lengend Snippet: (A) Rapamycin inactivated p70S6 kinase activity and aborted protein synthesis of DVL3 in all cervical cancer cell lines (C33A, C41, CaSki, HeLa and SiHa) in a dose-dependent manner. (B) Proteasomal inhibitor, AM114, restored the reduced DVL3 mediated by metformin in C33A. (C) Proteasomal inhibitor, AM114, could not restore the reduced DVL3 mediated by metformin in SiHa and HeLa. (D) In vivo ubiquitination assay showed that DVL3 was degraded as poly-ubiquitinated DVL3 protein ((Ub)n-DVL3) in C33A cell model. C33A cells were transfected with pcDNA-HA(Ub) 8 plasmid. Both MG132 or AM114 and metformin were used to enhance the amount of polyubiquitinated DVL3 protein products ((Ub)n-DVL3). Cell lysates were incubated with anti-DVL3, and the immunoprecipitates were probed with anti-HA. The same membrane was re-probed with anti-DVL3 to detect the expression of DVL3.

Article Snippet: Membranes were then blotted with 5% skimmed milk for 30 minutes and incubated with primary antibodies; anti-pp70S6kinase, anti-p70S6 kinase, anti-pAMPKα, anti-AMPKα, anti-CyclinD1 (Cell signaling Technology), anti-DVL1, anti-DVL2, anti-DVL3, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-catenin (BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Activity Assay, In Vivo, Ubiquitin Assay, Transfection, Plasmid Preparation, Incubation, Expressing

Journal: International Journal of Medical Sciences

Article Title: Hsa_circ_0087302, a circular RNA, affects the progression of osteosarcoma via the Wnt/β-catenin signaling pathway

doi: 10.7150/ijms.69501

Figure Lengend Snippet: Expression level of Wnt/β-catenin signaling pathway-related proteins after overexpression of hsa_circ_0087302. ( A ) The protein levels of β-catenin, cyclin D1, Dvl 2, Dvl 3, CD44, LRP 6, LEF 1, c-Myc and c-Jun were downregulated by lentiviral transduction in MG63 and U2OS cells, while the protein level of Axin was increased. GAPDH was used as a loading control. The bar chart shows the expression level of each protein in MG63 ( B ) and U2OS ( C ) cells compared to control cells. NC: negative control. Data represent the mean ± SD (n = 3) (*P < 0.05, **P < 0.01 and ***P < 0.001).

Article Snippet: Mouse monoclonal antibodies against β-catenin, cyclin D1, Dvl 2, Dvl 3, CD44, LRP 6, LEF 1, Axin, c-Myc and c-Jun [Cell Signaling Technology (CST), Inc., Danvers, MA, USA] were diluted at 1:1000.

Techniques: Expressing, Over Expression, Transduction, Negative Control

Journal: Journal of Molecular Signaling

Article Title: Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

doi: 10.1186/1750-2187-7-8

Figure Lengend Snippet: Interrogation of the functional status of Y17 and S407 sites of Dvl3 in Lef/Tcf-sensitive transcriptional activation in response to stimulation with Wnt3a. Panel A , F9 cells co-expressed with Rfz1, Super8xTOPFlash (M50) and either wild-type Dvl3 or one of following Dvl3 mutants (Y17D-Dvl3, S407A-Dvl3 and S407D-Dvl3) were stimulated with or without Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Cell lysates were immunoblotted and subsequently stained with anti-GFP (Dvl3 expression) and anti-GAPDH (control) antibody. Results are displayed for transcriptional activity relative to unstimulated cells (set to “1”). Statistical significance is indicated (*, p < 0.005, versus Dvl3 expressed cells). Panel B , F9 cells stably expressing Rfz1 and M50 were treated with siRNA targeting Dvl3 one day before transfection of the cells with either wild-type Dvl3 or Y17D-Dvl3 or S407A-Dvl3 or S407D-Dvl3. On the following day, cells were stimulated with Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Cell lysates were immunoblotted with either anti-GFP or anti-GAPDH antibody (control). Results are displayed for transcriptional activity relative to unstimulated cells (set to “1”). Statistical significance is indicated (*, p < 0.05, versus Dvl3 expressed cells).

Article Snippet: The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1 and anti-Dvl2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β and anti-Dvl3 antibodies from Cell Signaling (Danvers, MA); Immobilon membrane from Millipore (Bedford, MA); purified, biologically active Wnt3a, and anti-Axin antibody from R& D Systems (Minneapolis, MN).

Techniques: Functional Assay, Activation Assay, Staining, Expressing, Activity Assay, Stable Transfection, Transfection

Journal: Journal of Molecular Signaling

Article Title: Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

doi: 10.1186/1750-2187-7-8

Figure Lengend Snippet: Interrogation of functional roles of Y17 and S407 sites of Dvl3 in assembly of very large Dvl3-based supermolecular complexes in response to stimulation with Wnt3a. Y17D-Dvl3 and S407A-Dvl3 abolished the assembly of Dvl3-based supermolecular complexes in response to Wnt3, whereas S407D-Dvl3 enhances the assembly of Dvl3-based supermolecular complexes. Cells expressing either wild-type Dvl3 or Y17D-Dvl3 or S407A-Dvl3 or S407D-Dvl3 were stimulated with or without Wnt3a for 30 min. Cells were lysed and extracts (20 mg protein) were applied to Sephacryl S-400 gel filtration column (AKTA, GE Health Care). Fractions were analyzed by SDS-PAGE and immunoblotted, subsequently stained with anti-Dvl3 antibody. Dvl3 blots were quantified by the calibrated scanner and results were displayed . The Dvl3-based supermolecular complexes in F9 cells expressing Rfz1 were also displayed as a control. The calculated, relative molecular weight ( M r ) positions from the calibration curve are labeled at the top. The bottom numbers indicate fraction number. Arrow indicates the precise position at which calibration proteins elute from Sephacryl S-400. Results are representative of at least 2 independent experiments.

Article Snippet: The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1 and anti-Dvl2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β and anti-Dvl3 antibodies from Cell Signaling (Danvers, MA); Immobilon membrane from Millipore (Bedford, MA); purified, biologically active Wnt3a, and anti-Axin antibody from R& D Systems (Minneapolis, MN).

Techniques: Functional Assay, Expressing, Filtration, SDS Page, Staining, Molecular Weight, Labeling

Journal: Journal of Molecular Signaling

Article Title: Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

doi: 10.1186/1750-2187-7-8

Figure Lengend Snippet: Interrogation of functional roles of Y17 and S407 sites of Dvl3 in formation of macromolecular Dvl3-based punctae in response to stimulation with Wnt3a. Panel A, live cell images of expression of either wild-type Dvl3 or Y17D-Dvl3 or S407A-Dvl3 or S407D-Dvl3 in response to Wnt3a. F9 cells were co-transfected with Rfz1 and either wild-type Dvl3 or Y17D-Dvl3 or S407A-Dvl3 or S407D-Dvl3. One day post transfection, cells were stimulated with Wnt3a and cells images were taken at 0, 15, and 30 min post stimulation with Wnt3a. Panel B, live cell images of cells expressing either wild-type or Dvl3 mutants (Y17D-Dvl3 or S407D-Dvl3 or S407D-Dvl3) in Dvl3-deficient cell: response to Wnt3a. F9 cells stably expressing with M50 and Rfz1 were treated with siRNA targeting Dvl3. On the following day, cells were transfected with either wild-type Dvl3 or one of the Dvl3 mutants. Post 24 hr of transfection, cells were stimulated with Wnt3a and live cell images were captured during Wnt stimulation. Results were displayed at 0, 5, and 10 min time points. The images shown are representative of three or more separate experiments.

Article Snippet: The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1 and anti-Dvl2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β and anti-Dvl3 antibodies from Cell Signaling (Danvers, MA); Immobilon membrane from Millipore (Bedford, MA); purified, biologically active Wnt3a, and anti-Axin antibody from R& D Systems (Minneapolis, MN).

Techniques: Functional Assay, Expressing, Transfection, Stable Transfection

Journal: Journal of Molecular Signaling

Article Title: Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

doi: 10.1186/1750-2187-7-8

Figure Lengend Snippet: Formation of Dvl3-based punctae by wild-type versus mutant forms of Dvl3. Y17D-Dvl3 forms punctae in cells co-expressing wild-type Dvl3 in untreated and Dvl3-deficient F9 cells . Panel A , F9 cells were co-transfected with GFP- and HA-tagged Y17D-Dvl3 and increasing amount of co-expressed untagged wild-type Dvl3 (ratio = 1 : 0.2, or 1 : 2). Post 1 day transfection, cells were stimulated with Wnt3a. Cell images were taken during Wnt stimulation. Results were displayed at 0, 7.5, and 15 min time-points. Panel B , parallel experiments were performed in F9 cells made deficient of endogenous wild-type Dvl3 by siRNA. The images shown are representative of three or more separate experiments.

Article Snippet: The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1 and anti-Dvl2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β and anti-Dvl3 antibodies from Cell Signaling (Danvers, MA); Immobilon membrane from Millipore (Bedford, MA); purified, biologically active Wnt3a, and anti-Axin antibody from R& D Systems (Minneapolis, MN).

Techniques: Mutagenesis, Expressing, Transfection

Journal: Journal of Molecular Signaling

Article Title: Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

doi: 10.1186/1750-2187-7-8

Figure Lengend Snippet: CK1δ/ϵ regulates formation of Dvl3-based very large punctae. Treatment with CK1δ/ϵ inhibitor decreases number as well as apparent size of the punctae. F9 cells were co-transfected with Rfz1 and GFP- and HA-tagged wild-type Dvl3. Cells were stimulated with Wnt3a and images of live cells were captured at the times indicated. After 10 min post Wnt stimulation, a CK1δ/ϵ selective inhibitor (IC261, 5 μM) was added and cell images captured continuously. Results displayed are images captured at 5, 10, and 15 min post treatment with IC261. The images displayed are representative of two or more separate experiments.

Article Snippet: The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1 and anti-Dvl2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β and anti-Dvl3 antibodies from Cell Signaling (Danvers, MA); Immobilon membrane from Millipore (Bedford, MA); purified, biologically active Wnt3a, and anti-Axin antibody from R& D Systems (Minneapolis, MN).

Techniques: Transfection

Journal: Journal of Molecular Signaling

Article Title: Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

doi: 10.1186/1750-2187-7-8

Figure Lengend Snippet: Expression of Axin M3 mutant precludes assembly of very large Dvl3-based supermolecular complexes in response to Wnt3a. Control cells , Wnt3a stimulates formation of very large M r -complexes . SEC chromatogram of Dvl3-based complexes prepared from untreated cells. Axin-deficient cells, knockdown of Axin attenuates assembly of Dvl3-based supermolecular complexes in response to Wnt3a. F9 cells were transfected with siRNA targeting Axin one day before transfection with Rfz1. Twenty four hr later, cells were either unstimulated or stimulated with Wnt3a for 30 min. Axin expression , expression of w ild-type Axin alone stimulates assembly of Dvl3-based supermolecular complexes. F9 cells were co-transfected with Rfz1 and expression vectors harboring human wild-type Axin. Two days post transfection, F9 cells were treated either without or with Wnt3a for 30 min. Polymerization-defective Axin expression , expression of M3 Axin mutant blocks assembly of very large Dvl3-based supermolecular complexes in response to Wnt3a. F9 cells were co-transfected with Rfz1 and expression vectors harboring human M3 Axin mutant. Two days post transfection, F9 cells were treated either without or with Wnt3a for 30 min. Rescue of Axin-depletion by expression of wild-type Axin , expression of w ild-type Axin rescues the inability of Axin-deficient cells to assemble Dvl3-based supermolecular complexes in response to Wnt3a. Cells were treated with siRNA targeting Axin one day before subsequent co-transfection with Rfz1 and human wild-type Axin for an additional day. Twenty four hr after this final transfection, cells were either unstimulated or stimulated with Wnt3a for 30 min. Axin rescue by polymerization-defective Axin , expression of M3 Axin fails to rescue the inability of Axin-deficient cells to assembly of Dvl3-based supermolecular complexes in response to Wnt3a. Cells were treated with siRNA targeting Axin one day before subsequent co-transfection with Rfz1 and M3 Axin mutant for an additional day. Twenty four hr after this final transfection, cells were either unstimulated or stimulated with Wnt3a for 30 min. For all experiments, cell lysates were subjected to Sephacryl S-400 gel filtration column chromatography (SEC) and resolved fractions were analyzed by SDS-PAGE, immunoblotting and eventual staining with anti-Dvl3 antibody. Dvl3 blots were subjected to quantification by calibrated scanning and the results displayed are representative of at least 2 independent experiments, which yielded quantitatively similar results. The calculated, relative molecular weight ( M r ) positions from the calibration curve are labeled at the top of each SEC chromatogram. The bottom labels indicate fraction number. Arrows indicate the precise position at which calibration protein elute from Sephacryl S-400.

Article Snippet: The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1 and anti-Dvl2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β and anti-Dvl3 antibodies from Cell Signaling (Danvers, MA); Immobilon membrane from Millipore (Bedford, MA); purified, biologically active Wnt3a, and anti-Axin antibody from R& D Systems (Minneapolis, MN).

Techniques: Expressing, Mutagenesis, Transfection, Cotransfection, Filtration, Column Chromatography, SDS Page, Western Blot, Staining, Molecular Weight, Labeling