Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci80205

Figure Lengend Snippet: Figure 7. Summary of interaction between hedgehog and β-catenin signaling in interzone progeny and articular chondrocytes. In interzone progeny during development, hedgehog (HH) signaling induces the expres- sion 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 car- tilage 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: Dominant Negative Mutation, Expressing, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: MIN6 cells were plated at 5 × 10 5 cells per well in 6-well plates and exposed to a TCF7L2-specific short hairpin RNA (shTCF7L2) or a scrambled shRNA (shScr) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A ) Western blots showing the TCF7L2 protein content after E 2 or P 4 treatment. ( B ) Viable cells. ( C,D ) Basal and stimulated insulin/proinsulin secretions (normalized to viable cell numbers). ( E,F ) Stimulatory indexes. ( G ) Proinsulin-to-insulin ratio. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 shTCF7L2 vs. shScr.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: shRNA, Cell Culture, Western Blot, Control

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: MIN6 cells were plated at 5 × 10 5 cells per well in 6-well plates and exposed to high glucose concentration (33.3 mM) and transfected with TCF7L2-IRES2-EGFP (OE-TCF7L2) or a control vector (CV) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A ) Western blots showing the TCF7L2 protein content after E 2 or P 4 treatment. ( B ) Viable cells. ( C , D ) Basal and stimulated insulin/proinsulin secretions (normalized to viable cell numbers). ( E,F ) Stimulatory indexes. ( G ) Proinsulin-to-insulin ratio. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 OE-TCF7L2 vs. CV.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: Concentration Assay, Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: HepG2 cells (2.5 × 10 5 cells per well) were seeded in 6-well plates and exposed to a TCF7L2-specific short hairpin RNA (shTCF7L2) or a scrambled shRNA (shScr) for 72 h, or transfected with TCF7L2-IRES2-EGFP (OE-TCF7L2) or a control vector (CV) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A,B ) 2-NBDG uptake. ( C,D ) Glucose production. ( E,F ) Western blot. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 shTCF7L2 vs. shScr or OE-TCF7L2 vs. CV.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: shRNA, Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot

Journal: Frontiers in Physiology

Article Title: Placental galectin-3 is reduced in early-onset preeclampsia

doi: 10.3389/fphys.2022.1037597

Figure Lengend Snippet: LGALS3 and LGALS3BP mRNA expression in first trimester placental stem cells differentiated into syncytiotrophoblast and extravillous trophoblasts. First trimester placental cytotrophoblast cells were differentiated into either syncytiotrophoblast or extravillous trophoblast (EVT) cells over 96 h. Syncytiotrophoblast differentiation was confirmed by increased expression of SDC1 (syncytiotrophoblast marker) (A) and decreased expression of CDH2 (cell border marker) (C) across time. LGALS3 (E) and LGALS3BP (G) mRNA expression with syncytiotrophoblast differentiation across 96 h. EVT differentiation was confirmed by increased expression of HLAG (EVT marker) (B) and reduced expression of TEAD4 (cytotrophoblast marker) (D) across time. LGALS3 (F) and LGALS3BP (H) mRNA expression with EVT differentiation over 96 h. All experiments were repeated n = 5 times in duplicate. Data is expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: RNA was converted to cDNA with high-capacity cDNA reverse transcriptase kit (Applied Biosystems, Life Technologies) as per manufacturer’s instructions using iCycler iQ5 machine (Biorad) with run conditions: 25°C for 10 min, 37°C for 60 min and 85°C for 5 min. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) measured the mRNA expression of genes; LGALS3 (Assay ID: Hs00173587_m1), LGALS3BP (Assay ID: Hs00174774_m1), TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1) and HLAG (Human Leukocyte Antigen G, Assay ID: Hs03045108_m1) using Fluorescein amidite (FAM) labelled Taqman gene expression assays (Life Technologies) on the CFX 384 (Biorad, Hercules, CA) with Taqman fast advanced universal PCR mastermix (Applied Biosystems).

Techniques: Expressing, Marker

Journal: Frontiers in Oncology

Article Title: Syndecan-1-Dependent Regulation of Heparanase Affects Invasiveness, Stem Cell Properties, and Therapeutic Resistance of Caco2 Colon Cancer Cells

doi: 10.3389/fonc.2020.00774

Figure Lengend Snippet: Heparanase regulates the cancer stem cell phenotype of Caco2 cells. (A) Sdc-1 siRNA knockdown and heparanase inhibition by SST0001 affect the stem cell marker side population in opposite directions. ** p < 0.01 vs. all groups. * p < 0.05 vs. untreated control. (B) The HPSE inhibitor SST0001 (10 μg/ml) reduces sphere formation as a readout of stem cell acivity. *** p < 0.001, * p < 0.05 vs. untreated control. (C,D) Overexpression of native and enzymatically inactive forms of HPSE markedly increases the Caco2 side population. *** p < 0.001 vs. vector control. (C) Quantification of flow cytometric data. (D) representative flow cytometric measurements. Verapamil = inhibitor control. (E,F) Overexpression of native and enzymatically inactive forms of HPSE differentially affect the expression of the stem cell markers NANOG, KLF4, NOTCH1, NOTCH3, and TCF4. (E) qPCR, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. vector control, # p < 0.05 vs. HPSE. (F) Western-Blot. (G) The Wnt pathway inhibitor IWP2 reduces the enhancing effect of HPSE overexpression on the side population phenotype. ** p < 0.01, * p < 0.05 vs. control, # p < 0.05 vs. untreated HPSE. All panels N ≥ 3. Error bars = SEM. (D,F) representative example of three independent experiments.

Article Snippet: Immunoblotting was performed exactly as previously described ( , ), using the following primary antibodies (1:1,000): rabbit polyclonal anti-phospho FAK Y925 (Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-FAK (Cell Signaling), rabbit monoclonal anti-human TCF4 (Cell Signaling), mouse anti-E-cadherin (1:2,000; BD Biosciences), mouse anti-human α-Tubulin (Sigma-Aldrich) and appropriate secondary antibodies (diluted 1:5,000): HRP-conjugated goat-anti-mouse or goat-anti-rabbit IgG (Merck-Millipore, Darmstadt, Germany).

Techniques: Knockdown, Inhibition, Marker, Control, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: TCF-4 binds to its putative binding sites on the HIV LTR in vitro. (a) HIVBal was sequenced using primers that spanned from the LTR into the start of the gag gene. AliBaba2 software was used to predict putative TCF-4 binding sites on the HIV LTR using the consensus sequence 5′-(A/T)(A/T)CAAAG-3′. Alignment tools in Vector NTI and OMIGA software were used to find putative binding sites containing single-base substitutions. (b) Multiple-LTR-sequence alignment to scan for putative TCF-4 binding sites in 500 HIV-1 isolates from the Los Alamos gene bank. A representative sample shows the presence of two TCF-4 sites at nt −143 and +66 in several isolates. The yellow highlights TCF-4 bases that are highly conserved (unchanged), while less-conserved bases are in turquoise. The boxes show TCF-4 positions at −143 and +66. The asterisk denotes sequenced HIVBal from panel a that contains point mutations not found in the Bal isolate from the database (pBa_L; AB221005). (c) EMSA indicating binding of TCF-4 at these four sites. Nuclear extract (lysate) from U87MG cells was incubated with the indicated biotinylated DNA probe for 15 min. DNA-protein complexes were run on a 6% native gel and detected with SYBR green dye. For supershift assays, nuclear extract was incubated with IgG control (isotype) or TCF-4 antibody for 15 min prior to incubation with DNA probes. The data in panel c are representative of three independent experiments.

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: Binding Assay, In Vitro, Software, Sequencing, Plasmid Preparation, Incubation, SYBR Green Assay

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: TCF-4 exhibits stronger binding at the −143 site on the HIV LTR. (a) Nuclear extract from U87MG cells was mixed with the indicated biotinylated DNA probe for 5 min, followed by incubation with streptavidin-conjugated agarose beads. The beads were pelleted and washed three times with TNE100 or TNE300. Samples were then run on an SDS-PAGE gel and immunoblotted to detect TCF-4. (b) Densitometric analysis of bands detected in panel a showing relative binding of TCF-4 following washing with TNE300. The data are normalized to scrambled control and represent three independent experiments. The error bars represent standard deviations (SD).

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: Binding Assay, Incubation, SDS Page

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: Knockdown of β-catenin or TCF-4 enhances Tat-mediated transactivation of the LTR independent of the nt −143 site. (a) U87MG cells were transfected with scrambled siRNA, β-catenin siRNA, or TCF-4 siRNA; 48 h postknockdown, the cells were transfected with WT-LTR or Δ-143-LTR luciferase constructs with either vector control (pcDNA3.1) or Tat expression plasmid for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. (b) U87MG cells that stably express an integrated copy of the WT or a Δ-143-LTR-luciferase construct were transfected with the indicated siRNAs for 48 h, followed by transfection with vector control or Tat for an additional 24 h before the cells were lysed and a dual-luciferase assay was performed. Luciferase readings from panels a and b were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P < 0.05 relative to control siRNA; †, P < 0.05 relative to control plus Tat. The error bars represent SD.

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Stable Transfection, BIA-KA

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: TCF-4 docks on HIV LTR in vivo and exhibits stronger binding affinity at nt −143. (a) U87MG cells were transfected with a wild-type LTR construct. Twenty-four hours later, ChIP was performed using the indicated immunoprecipitating antibodies. Eluted DNA was analyzed by real-time PCR using primers encompassing nt −143 (Sn57 and Sn58) and nt +186 (Sn59 and Sn60) for quantitative amplification. (b) U87MG cells were transfected with an HIV LTR with the nt −143 TCF-4 binding site deleted (Δ-143-LTR). Twenty-four hours later, chromatin immunoprecipitation was performed, followed by real-time PCR, as shown in panel a. All data are expressed as mean fold change relative to a control IgG antibody, as described in Materials and Methods. The data represent at least three independent experiments. *, P < 0.05 compared to IgG control; ND, not detected. The error bars represent SD.

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: In Vivo, Binding Assay, Transfection, Construct, Real-time Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: Deletion of TCF-4 binding sites at nt −143 enhances LTR activity, in combination with β-catenin/TCF-4 knockdown. (a) Schematic representation of HIVBal LTR constructs inserted into the pGL4.19 luciferase vector. The bold X denotes the deletion of the −143 site from the HIV LTR. (b) U87MG cells were transfected with the indicated siRNAs; 48 h later, the cells were transfected with WT LTR or Δ-143-LTR for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (c) U87MG cells containing integrated WT LTR-luciferase or Δ-143-LTR-luciferase plasmid were transfected with the indicated siRNAs; 72 h postknockdown, the cells were lysed and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (d) U87MG cells containing integrated WT LTR-luciferase were transfected with control siRNA, β-catenin siRNA, TCF-4 siRNA, or both β-catenin and TCF-4 siRNA; 72 h postknockdown, the cells were lysed and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P < 0.05 relative to control siRNA. The typical knockdown efficacy was >90%, as indicated previously (26). The error bars represent SD.

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: Binding Assay, Activity Assay, Construct, Luciferase, Plasmid Preparation, Transfection, BIA-KA

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: The nuclear matrix protein SMAR1 binds to the HIV LTR in astrocytes and associates with TCF-4 in vivo. (a) U87MG-LTR-luc containing an integrated HIV LTR-luciferase construct (integrated) or U87MG astrocytoma cells (episomal) was transfected with SMAR1 siRNA. After 48 h, U87MG astrocytoma cells were transfected with the LTR-luciferase construct for an additional 24 h. After 72 h of knockdown, all cells were lysed, and a luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (b) U87MG astrocytoma cells were transfected with the indicated LTR-luciferase plasmid for 24 h. Chromatin immunoprecipitation was performed using antibodies to SMAR1 or rabbit IgG, followed by real-time PCR using primers encompassing nt −143, as described in Materials and Methods. (c to h) TCF-4, SMAR1, or β-catenin was immunoprecipitated from nuclear extracts generated from U87MG cells, followed by Western blotting to detect coimmunoprecipitated proteins, as indicated. For blocking experiments (e and f), SMAR1 antibody was incubated with a 5-fold excess of SMAR1-blocking peptide for 30 min prior to Western blotting to show the specificity of binding. The data are representative of three independent experiments. *, P < 0.05 relative to control. The error bars represent SD.

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: In Vivo, Luciferase, Construct, Transfection, BIA-KA, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Generated, Western Blot, Blocking Assay, Incubation, Binding Assay

Journal: Journal of Virology

Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

doi: 10.1128/JVI.00486-12

Figure Lengend Snippet: Proposed model of SMAR1/TCF-4/β-catenin repression of HIV transcription. Based on data presented here and by others (26), we propose that β-catenin/TCF-4/SMAR form a complex at nt −143 on the HIV LTR. This complex pulls HIV DNA spanning this region into the nuclear matrix and away from transcription machinery. Inhibition of β-catenin/TCF-4 by siRNA disrupts this chromatin repression complex and allows Pol II docking and recruitment of TCoA, such as NF-κB and C/EBP, to drive basal LTR activity.

Article Snippet: The proteins were then transferred onto a nitrocellulose membrane and immunoblotted with a TCF-4 antibody (mouse monoclonal antibody [MAb]; Cell Signaling, Boston, MA).

Techniques: Inhibition, Activity Assay