dominant negative human tcf4  (Vector Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Breast Cancer-induced Bone Lesions by ?-Catenin Protein Signaling *

doi: 10.1074/jbc.M111.294595

Figure Lengend Snippet: Modification of β-catenin signaling in TM40D-MB cells. A, 24 h after transfection of β-catenin-responsive plasmid TOPflash and FOPflash, reporter activity assay showed that treatment of Ad-β-catenin had a modest increase in the reporter activity, but treatment of Ad-TCF4_DN significantly decreased luciferase reporter activity. *, p < 0.05; **, p < 0.01. B, MTT assay revealed that proliferation was increased in TM40D-MB cells treated with Ad-TCF4_DN, whereas proliferation was not changed in cells treated with Ad-β-catenin. *, p < 0.001. Error bars, S.D.

Article Snippet: The TM40D-MB cells were treated with adenovirus expressing human β-catenin (Ad-β-catenin), or dominant negative human TCF4 (Ad-TCF4_DN), or a control virus (Ad-control, all from Vector Biolabs) at a multiplicity of infection (m.o.i.) of 100 for 24 h. Cells were resuspended and plated in 6-well plate at 2 × 10 5 /well overnight.

Techniques: Modification, Transfection, Plasmid Preparation, Activity Assay, Luciferase, MTT Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Breast Cancer-induced Bone Lesions by ?-Catenin Protein Signaling *

doi: 10.1074/jbc.M111.294595

Figure Lengend Snippet: Inactivation of β-catenin increases osteoclast differentiation and inhibits osteoblast differentiation in vitro. A, real-time PCR assay showed that both RANKL/OPG ratio and pTHrP were up-regulated in tumor cells treated with Ad-TCF4_DN compared with cells treated with Ad-control. TM40D-MB cells were infected with Ad-TCF4_DN at a m.o.i. of 100 for 24 h; cells were then co-cultured with osteoclast precursors, RAW 264.7, for 5 days. TRAP staining was performed to detect multinucleate osteoclast formation. B and C, TRAP staining showed an increased number of multinucleated osteoclasts in cells treated with Ad-TCF4_DN. **, p < 0.01. Error bars, S.D.

Article Snippet: The TM40D-MB cells were treated with adenovirus expressing human β-catenin (Ad-β-catenin), or dominant negative human TCF4 (Ad-TCF4_DN), or a control virus (Ad-control, all from Vector Biolabs) at a multiplicity of infection (m.o.i.) of 100 for 24 h. Cells were resuspended and plated in 6-well plate at 2 × 10 5 /well overnight.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Control, Infection, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Breast Cancer-induced Bone Lesions by ?-Catenin Protein Signaling *

doi: 10.1074/jbc.M111.294595

Figure Lengend Snippet: Modification of β-catenin pathway affects bone lesion phenotype in vivo. After infection of TM40D-MB cells with various adenoviruses, tumor cells were injected into the mouse tibia. After 5 weeks, animals were examined by radiography, and tissue samples were harvested for histological analysis. A, x-ray examination displayed enlarged lytic lesions in tibiae of animals injected with Ad-TCF4_DN-treated tumor cells. There was no increased osteoblastic activity in animals injected with tumor cells overexpressing β-catenin. B, μCT confirmed phenotype description above. C and D, analysis using μCT revealed that both total bone volume fraction and trabecular bone volume fraction decreased in animals injected with tumor cells expressing TCF4_DN. However, no significant difference was observed in total bone volume fraction and trabecular bone volume fraction between mice injected with control virus and Ad-β-catenin; E, H&E staining demonstrated a mixed bone lesion in tibia of control virus-treated mice, as both tumor bone formation and bone resorption were observed in tumor area. In contrast, a predominantly lytic phenotype was seen in mouse tibia that had been injected with Ad-TCF4_DN-treated tumor cells. The marrow cavity was completely filled with tumor cells; bone formation was hardly detected. In mice injected with tumor cells overexpressing β-catenin, both osteoblastic and osteolytic activity were seen, which is comparable with control virus-treated mice. F, real-time PCR assay showed a decrease in ALP activity in MC3T3 cells co-cultured with TM40D-MB cells treated with Ad-TCF4_DN rather than those co-cultured with control virus-treated tumor cells. *, p < 0.05; **, p < 0.01. Error bars, S.D.

Article Snippet: The TM40D-MB cells were treated with adenovirus expressing human β-catenin (Ad-β-catenin), or dominant negative human TCF4 (Ad-TCF4_DN), or a control virus (Ad-control, all from Vector Biolabs) at a multiplicity of infection (m.o.i.) of 100 for 24 h. Cells were resuspended and plated in 6-well plate at 2 × 10 5 /well overnight.

Techniques: Modification, In Vivo, Infection, Injection, Activity Assay, Expressing, Control, Virus, Staining, Real-time Polymerase Chain Reaction, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Hedgehog inhibits β-catenin activity in synovial joint development and osteoarthritis

doi: 10.1172/JCI80205

Figure Lengend Snippet: Primary mouse costal chondrocytes (A–D) or human knee chondrocytes (E–K) were isolated and cultured. Protein levels of TCF7L2/TCF4 isoforms were determined by immunoblotting (A and E), and RNA levels of Axin2 (B and G), Fgf18 (C and H), Gli1 (D), total TCF4 (F), MMP13 (I), MMP3 (J), and ADAMTS4 (K) were determined by qPCR relative to Actb (B–D) or HPRT (F–K). (A–D) Primary mouse chondrocytes were transfected with scrambled (DMSO or PM) or siRNA mix (PM + siRNA) that selectively targets all isoforms of Tcf7l2 and treated with DMSO or PM (10 μM) for 48 hours. (B–D) Bars represent mean ± SEM normalized to DMSO-treated cultures. Data were log transformed prior to analysis by repeated-measures ANOVA followed by Tukey’s post-hoc tests. a, significantly different (P < 0.05) compared with unlabeled bars. (E–K) Primary human chondrocytes were isolated and infected with adenoviral vectors expressing GFP or a dominant negative form of TCF4. (E–K) Bars represent mean ± SEM. Data were log transformed prior to analysis by paired t test. *P < 0.05 compared with GFP-infected cells. See also Supplemental Figure 4.

Article Snippet: Human chondrocytes were infected with an adenovirus expression vector for human dominant negative TCF4 (Vector Biolabs) or an adenovirus-GFP (control, Vector Biolabs) for 24 hours at 50 MOI and replaced in fresh medium.

Techniques: Isolation, Cell Culture, Western Blot, Transfection, Transformation Assay, Infection, Expressing, Dominant Negative Mutation

Journal: The Journal of Clinical Investigation

Article Title: Hedgehog inhibits β-catenin activity in synovial joint development and osteoarthritis

doi: 10.1172/JCI80205

Figure Lengend Snippet: In interzone progeny during development, hedgehog (HH) signaling induces the expression of TCF7L2 (and human TCF4) isoforms, including dominant negative isoforms. Increased expression of TCF7L2 protein isoforms limits signaling by β-catenin (β-cat), resulting in an inhibition of expression of FGF18, leading to ectopic cartilage formation. In adult chondrocytes, HH signaling activity induces cartilage degeneration. Expression of dnTCF7L2 and other TCF7L2 isoforms induces the expression of catabolic enzymes, including ADAMTS4 and MMP13, which are involved in cartilage degeneration as part of OA. Increasing β-catenin activity rescues hedgehog-induced ectopic cartilage formation and cartilage degradation, likely by restoring the balance between HH and β-catenin signaling.

Article Snippet: Human chondrocytes were infected with an adenovirus expression vector for human dominant negative TCF4 (Vector Biolabs) or an adenovirus-GFP (control, Vector Biolabs) for 24 hours at 50 MOI and replaced in fresh medium.

Techniques: Expressing, Dominant Negative Mutation, Inhibition, Activity Assay