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: 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: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Viability curves of A2780 cell lines transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2). Each experimental group was exposed to six different CDDP concentrations for 48h. Data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each cell line analyzed. (B) Viability of A2780 cell lines transfected with pCMV6 (R-Ø) and with the overexpression vectors (R-ITF2). (C) Relative expression levels of ITF2 measured by quantitative RT-PCR, represented in Log10 scale; In each experimental group, the sensitive cell line transfected with pCMV6 plasmid was used as a calibrator. Each bar represents the combined relative expression of two independent experiments measured in triplicate. (D) β-catenin transcriptional activity was measured in A2780 cells after ITF2 overexpression and treatment with LiCl (10mM) for 24 hours, transfecting with Super8xTopFlash (Top) or Super8xFopFlash (Fop). The results show the fold induction of the Top/Fop ratio with respect to untreated cells (=1). Values represent the mean of three independent experiments measured by triplicate ± SD. (E) Expression analysis of the downstream gene DKK1 regulated by ITF2 in A2780 cell line transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2) for 24 and 72 hours. Representative images of DKK1 and GAPDH measured by RT-PCR. (F), Expression levels of DKK1 measured by qRT-PCR. Each assay was performed at least three times to confirm the results. *** p < 0.001; * p < 0.05 (Students T-test).

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Transfection, Over Expression, Plasmid Preparation, Concentration Assay, Expressing, Quantitative RT-PCR, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Correlation between ITF2 expression and HOXD9 in tumor and non-tumor samples from the complete cohort of 25 NSCLC patients. Pearson coefficient was used for linear correlation of the quantitative variables. (B-C) Effect of ITF2 overexpression on HOXD9 levels (B) Validation of the transfection efficiency of ITF2 at mRNA levels. Relative expression levels of ITF2 measured by qRT-PCR, in the cell line H23R, at 24 and 72 hours after transfectionrepresented in Log10 the 2-ΔΔCt. (C) Relative expression levels of HOXD9 measured by quantitative RT-PCR after ITF2 overexpression. For both (B) and (C) the resistant cell line transfected with pCMV6 plasmid was used as a calibrator (R-Ø). H23R cells were also transfected with ITF2 cDNA (R-ITF2). Each bar represents the combined relative expression of two independent experiments measured in triplicate. *** p < 0.001; ** p < 0.01 (Students T-test). (D) Correlation between ITF2 and HOXD9 expression levels with the overall survival analyzed in NSCLC patients selected form the RNA-seq analysis. Data represents the quantitative expression levels of the two genes measured by qRT-PCR and represented as 2-ΔΔCt for ITF2 (referred to the NLM) and 2-ΔCt for HOXD9 . (E) Survival analysis in NSCLC samples according to the mean of HOXD9 . LogRank, Breslow and Tarone-Ware test were used for comparisons and p < 0.05 was considered as a significant change in OS. (F-H) Survival analysis in 1,926 patients from the Kaplan Meier online tool (F) and the TCGA data sets of lung Adenocarcinoma (G, High=23; No Change=203) and SCC (H, High=28; No Change=441) for HOXD9 .

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Expressing, Over Expression, Transfection, Quantitative RT-PCR, Plasmid Preparation, RNA Sequencing Assay

Journal: Oncotarget

Article Title: Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling

doi: 10.18632/oncotarget.9967

Figure Lengend Snippet: BEAS-2B cells were exposed to increasing concentrations (0–5 μm) of Cr(VI) for 24 h. ( A ) The relative miR-21 level was determined by Taqman real-time PCR. ( B ) Immunoblot analysis of PDCD4 protein levels after acute Cr(VI) treatment. ( C ) Representative images of fluorescence immunostaining of PDCD4 ( D ) Cr(VI) increases the binding of miR-21 to the 3′-UTR of PDCD4. BEAS-2B cells were transfected with renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), and pGL3-promoters and treated with 5 μM Cr(VI) for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods. The results are expressed as a relative activity (relative luminescence units (RLU)) normalized to the luciferase activity in the vector control cells without treatment. ( E ) Immunoblot analysis demonstrates that acute treatment of Cr(VI) decreases E-cadherin levels associated with an increase in β-catenin and TCF4 protein levels in BEAS-2B cells. Data presented in the bar graphs are the mean ± SD of three independent experiments. *indicates a statistically significant difference from control cells with p < 0.05.

Article Snippet: Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Immunostaining, Binding Assay, Transfection, Construct, Negative Control, Luciferase, Activity Assay, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling

doi: 10.18632/oncotarget.9967

Figure Lengend Snippet: BEAS-2B cells were maintained in a medium containing various concentrations of Cr(VI) (0.125, 0.25 and 0.5 μM) for 6 months. ( A ) Total cell lysates were prepared for Western blot analysis using specific antibodies against E-Cadherin, Active-β-catenin, β-catenin, TCF4 and GAPDH. Representative images of fluorescence immunostaining of ( B ) E-cadherin ( C ) β- catenin and ( D ) TCF4.

Article Snippet: Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Western Blot, Fluorescence, Immunostaining

Journal: Oncotarget

Article Title: Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling

doi: 10.18632/oncotarget.9967

Figure Lengend Snippet: BEAS-2B cells were exposed to various concentrations of Cr(VI) (0.125, 0.25 and 0.5 μM) for 6 months. ( A ) Expression levels for uPAR and c-Myc were analyzed by Western blot. To evaluate invasive capacity ( B – C ) 1 × 10 5 cells from each group were seeded on the top chamber of 24-well plate culture inserts coated with 20 μl of matrigel in duplicate. Cells were cultured for additional 48 h. Invaded cells on the bottom of insert were stained and quantified. ( D – E ) For the ChIP assay, TCF4 binding regions on uPAR and c-Myc promoters were identified. Chromatin isolated from chronic Cr(VI) exposed BEAS-2B cells were immunoprecipitated with an anti-TCF4 antibody or control mouse IgG. The TCF4 binding to the uPAR and c-Myc promoters was analyzed by RT PCR with specific primers. Data presented in the bar graphs are the mean ± SD of three independent experiments. *indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Cell Culture, Staining, Binding Assay, Isolation, Immunoprecipitation, Control, Reverse Transcription Polymerase Chain Reaction

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: 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