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: 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