Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: Primers used for target amplification in real-time PCR.

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques: Amplification

Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: Impaired TCF7L2 induced β-cells dedifferentiation and decreased insulin secretion of MIN6 cells. (A) The expressions of TCF7L2, Hes1 and FoxO1 were measured respectively by western blot after the transfection of TCF7L2 siRNA plasmid. (B) The expression of Hes1 was measured by real-time PCR after the transfection of TCF7L2 siRNA plasmid. (C) The expression of FoxO1 was measured by real-time PCR after the transfection of TCF7L2 siRNA plasmid. (D) The insulin concentration was evaluated by ELISA after the transfection of TCF7L2 siRNA plasmid. (E) The expressions of TCF7L2, Hes1 and FoxO1 were measured respectively by western blot after the transfection of lenti-TCF7L2 plasmid. (F) The expression of Hes1 was measured by real-time PCR after the transfection of lenti-TCF7L2 plasmid. (G) The expression of FoxO1 was measured by real-time PCR after the transfection of lenti-TCF7L2 plasmid. (H) The insulin concentration was evaluated by ELISA after the transfection of lenti-TCF7L2 plasmid. (n = 3 samples per groug, ** P < .01 and * P < .05 as indicated).

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: The effects of TCF7L2 on β-cell dedifferentiation and insulin secretion were mediated by ERK/MAPK signaling pathway. (A) The expressions of total and phosphorylated ERK were evaluated respectively by western blot after TCF7L2 siRNA plasmid transfection. (B) The insulin concentrations in supernatant were measured by ELISA when MIN6 cells were cultured with 10 µM ERK inhibitor U0126 for 30 minutes prior to TCF7L2 siRNA plasmid transfection. (C) The expressions of Hes1 were evaluated by real-time PCR when MIN6 cells were cultured with 10 µM ERK inhibitor U0126 for 30 minutes prior to TCF7L2 siRNA plasmid transfection. (D) The expressions of Foxo1 were evaluated by real-time PCR when MIN6 cells were cultured with 10 µM ERK inhibitor U0126 for 30 minutes prior to TCF7L2 siRNA plasmid transfection. (n = 3 samples per groug, ** P < .01 as indicated).

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques: Western Blot, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: Reciprocal changes of pancreatic TCF7L2 protein during diabetes progression. (A) The body weight of db/db mice (n = 5) was measured after weeks of normal or high-fat diet feeding. (B) Fasting glucose levels in db/db mice (n = 5) were measured after weeks of a normal or high-fat diet. (C) Changes in pancreatic TCF7L2 and FoxO1 levels in db/db mice were measured using western blotting after weeks of normal or high-fat diet feeding. (D) Quantification of TCF7L2 expression in db/db mice after weeks of a normal or high-fat diet. (E) Quantification of FoxO1 expression in db/db mice after weeks of a normal or high-fat diet. (F) Local expression of TCF7L2 in islets was measured by immunohistochemistry after weeks of high-fat diet feeding. (n = 3 samples per groug, ** P < .01 as indicated).

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques: Western Blot, Expressing, Immunohistochemistry

Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: Weekly body weight of TCF7L2 fl/fl and TCF7L2 β−/− mice were recorded. (A) Genotype of the candidate mice were confirmed by PCR using TCF7L2 primer. (Wild type: one band with 476 bp; Heterozygote: two bands with 476 and 586 bp; Homozygote: one band with 586 bp.) WT: Wild type, H20: Negative control. (B) Genotype of the candidate mice were confirmed by PCR using Mip-cre primer. (Target: 300 bp; Control: 200 bp.) WT: Wild type, H20: Negative control. (C) Body weight of TCF7L2 fl/fl (n = 6) and TCF7L2 β−/− (n = 6) mice were measured weekly from 8 to 12 weeks of age, after normal diet feeding. (D) Body weight of TCF7L2 fl/fl (n = 6) and TCF7L2 β−/− (n = 6) mic were measured weekly from 8 to 12 weeks of age, after high-fat diet feeding. All data are represented as mean ± SEM, and statistical analyses were performed using Student’s t -test (** P < .01, * P < .05).

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques: Negative Control, Control

Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: TCF7L2 deficiency destroyed glucose tolerance and insulin secretion to some extent. (A) Glucose tolerance tests on TCF7L2 fl/fl (n = 4) and TCF7L2 β −/− (n = 5) mice at 8 weeks. (B) Glucose tolerance tests on TCF7L2 fl/fl (n = 6) and TCF7L2 β−/− (n = 6) mice at 12 weeks, after normal diet feeding. (C) Glucose tolerance tests on TCF7L2 fl/fl (n = 6) and TCF7L2 β−/− (n = 6) mice at 12 weeks, after high-fat diet feeding. (D) Insulin secretion of TCF7L2 fl/fl (n = 4) and TCF7L2 β−/− (n = 5) mice at 8 weeks. (E) Insulin secretion of TCF7L2 fl/fl (n = 6) and TCF7L2 β−/− (n = 6) mice at 12 weeks, after normal diet feeding. (F) Insulin secretion of TCF7L2 fl/fl (n = 6) and TCF7L2 β−/− (n = 6) mice at 12 weeks, after high-fat diet feeding. All data are represented as mean ± SEM, and statistical analyses were performed using Student’s t -test (** P < .01, * P < .05).

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques:

Journal: Clinical Medicine Insights. Endocrinology and Diabetes

Article Title: Inhibitory Effect of TCF7L2 on Pancreatic β-Cell Dedifferentiation via ERK/MAPK Signaling Pathway in Diabetes

doi: 10.1177/11795514241295620

Figure Lengend Snippet: TCF7L2 deficiency induce β-cell dedifferentiation and activation of ERK/MPAK signaling. (A) Genotypes of TCF7L2 fl/fl and TCF7L2 β−/− mice were confirmed by PCR. (B) Expressions of Hes1, FoxO1 and p-ERK in TCF7L2 fl/fl and TCF7L2 β−/− mice were measured respectively by western blotting when the mice were fed with high-fat diet to 12 weeks of age.

Article Snippet: The TCF7L2 antibody (cat. no. H00006934-M06) was purchased from Novus Biologicals Inc. A specific inhibitor of ERK phosphorylation (U0126) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Techniques: Activation Assay, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: TCF7L2 silencing results in altered gene expression patterns accompanied by local genomic reorganization.

doi: 10.1016/j.neo.2020.12.010

Figure Lengend Snippet: Fig. 1. Conceptual approach and gene expression dynamics following TCF7L2 silencing. (A) Schematic summary of the experimental approach. We sought to explore how silencing TCF7L2 impacted the colon cancer gene network in terms of chromatin structure and gene expression. (B) qPCR demonstrated progressive silencing of TCF7L2 over time with a 75% reduction in TCF7L2 transcript levels after 72 hours. Each dot represents a biological replicate, with 3 replicates plotted per time point (n = 3). The green line represents the mean, while the green-shaded ribbon represents the standard deviation. (C) Western blot analysis demonstrated a 70% decrease in TCF4 protein abundance by the last time point with β-actin as loading control. (D) PCA of gene expression (TPM) over the time course, with time points differentiated by color (n = 2). (E) Volcano plots show differential gene expression genome-wide with thresholds set at log 2 FC = 2, and P = 0.05 (equivalent to 1.3 on the –log10 P scale). Genes in red are significantly down-regulated and undergo a > 4-fold decrease in expression, while those in green are significantly up-regulated and experience a > 4-fold increase in expression. The number of genes which fall within these regions are shown. (F) Expression profile of SOX2 , which increases dramatically across the time series. The dots correspond to each biological replicate (n = 2), the shaded ribbon represents the standard deviation. (G) Western blot for active β-catenin over time with β-actin as loading control. No noticeable change in active β-catenin protein levels occurs during the time series. (Color version of figure is available online.)

Article Snippet: Membranes were ncubated with antibodies targeting TCF4 (Origene; TA333645), active -catenin (Millipore; 05-665), or β-actin (CST; 4970S).

Techniques: Gene Expression, Standard Deviation, Western Blot, Quantitative Proteomics, Control, Genome Wide, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: TCF7L2 silencing results in altered gene expression patterns accompanied by local genomic reorganization.

doi: 10.1016/j.neo.2020.12.010

Figure Lengend Snippet: Fig. 2. Changes in global and local Hi-C partitioning over time. (A) Algebraic difference between genome-wide Hi-C contact maps at 0 and 72 hours demonstrates an overall increase in contact frequency over time. (B) Global partitioning (A/B compartmentalization) dynamics revealed that 1091 100-kb genomic bins switched compartments at one or more time points genome-wide. The Fiedler number (see Methods) of each loci are k-means clustered into 8 groups (denoted to the left) exhibiting different switching dynamics. The number of genomic bins in each cluster is specified on the right. A 95% change in Fiedler number from one time point to another was set as a threshold to remove inherent noise. (C) Local partitioning (TAD organization) of the region on Chromosome 19 (34.6 - 45.2 Mb) containing two CEACAM gene groups. Hi-C contact maps are shown at 100kb resolution with TAD domains at 0h denoted by solid, black lines and at 72h denoted by dashed, black lines. The CEACAM gene groups are denoted by blue lines in the expression array. ChIPseq data for TCF4 and SP1 binding demonstrated that TCF4 and SP1 may bind within the TAD boundary region. (D) qPCR was performed on TCF7L2 silenced cells at the 72-hour time point to determine the expression of CEACAM1 in various colon cancer cell lines. Expression of TCF7L2 and CEACAM1 was normalized to the negative control (Time 0 - not plotted), which was uniformly set to 1 for each cell line. All cell lines tested demonstrated an up-regulation of CEACAM1 , however the weakest response was observed in COLO201. (E) Diagram illustrating coupled chromosome structure and gene expression between two conditions as well as a possible explanation for this coupling, i.e., condition specific transcription factor activity. (F) Transcription factor enrichment analysis for the 64 genes found in the differentially conformed region of chromosome 19 (see 2C) showed enrichment for SP1, KLF4, ZFX, and MZF1 when compared against a background of 24,752 genes.

Article Snippet: Membranes were ncubated with antibodies targeting TCF4 (Origene; TA333645), active -catenin (Millipore; 05-665), or β-actin (CST; 4970S).

Techniques: Hi-C, Genome Wide, Expressing, Binding Assay, Negative Control, Gene Expression, Activity Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Chemokine receptor 7 mediates miRNA ‐182 to regulate cerebral ischemia/reperfusion injury in rats

doi: 10.1111/cns.14056

Figure Lengend Snippet: The CXCR7/miR‐182 axis affects ischemic/reperfusion (I/R) progression mediated through TCF7L2. (A) All downregulated miRNA gene regulatory networks. (B) Putative miR‐182 binding sequence in the 3′ untranslated region (UTR) of TCF7L2 mRNA. A mutation was generated in the TCF7L2 3′UTR sequence at the complementary site of the seed region in miR‐182. A TCF7L2 3′UTR fragment with a wild‐type (TCF7L2‐Wt) or a mutant (TCF7L2‐Mut) miR‐182‐binding sequence was cloned downstream of the luciferase reporter gene. (C) The TCF7L2‐Wt or TCF7L2‐Mut reporter vector cotransfected into C6 cells with vehicle (control), miR‐182 mimic, or negative control (miRNA‐Scr). The normalized luciferase activity in the control group was set as the relative luciferase activity. The data are presented as the mean ± standard error of the mean ( n = 3). (D) Representative western blot bands showing TCF7L2 expression in the miRNA‐Scr, miR‐182 mimic, anti‐miRNA‐Scr, and anti‐miR‐182 groups of C6 cells ( n = 3). (E) Densitometric quantification of TCF7L2/β‐actin. (F) Representative western blot bands showing TCF7L2 expression in the shRNA‐Scr, shRNA‐CXCR7, shRNA‐CXCR7 + miRNA‐Scr, and shRNA‐CXCR7 + miR‐182 mimic groups of C6 cells. (G) Densitometric quantification of TCF7L2/β‐actin. * p < 0.05 versus the miRNA‐Scr group

Article Snippet: The membrane was blocked at room temperature for 2 h with 5% skim milk or bovine serum albumin and then incubated overnight at 4°C with the following primary antibodies: anti‐CXCR7 (1:100, rabbit, NBP2‐24779, Novus Biologicals, Littleton, CO, USA), anti‐TCF7L2 (1:500, rabbit, A19548, ABclonal, Wuhan, China), anti‐phospho‐TAZ resistant (Ser89) (1:1000, rabbit, 59971 S, Cell Signaling Technology, Danvers, MA, USA), anti‐phospho‐YAP (Ser127) (1:1000, rabbit, 13,008 T, phospho‐Yap (Ser127), Cell Signaling Technology, Danvers, MA, USA), anti‐β‐actin (1:500, rabbit, AF5006, Beyotime, Shanghai, China), and anti‐GAPDH (1:2000, AF1186, Beyotime, Shanghai, China).

Techniques: Binding Assay, Sequencing, Mutagenesis, Generated, Clone Assay, Luciferase, Plasmid Preparation, Negative Control, Activity Assay, Western Blot, Expressing, shRNA

Journal: CNS Neuroscience & Therapeutics

Article Title: Chemokine receptor 7 mediates miRNA ‐182 to regulate cerebral ischemia/reperfusion injury in rats

doi: 10.1111/cns.14056

Figure Lengend Snippet: CXCR7 plays a protective role in the brain‐mediated Hippo signaling pathway. (A) The 10 most significantly enriched ischemia/reperfusion (I/R)‐associated pathways with downregulated CXCR7 expression. (B) Representative protein expression levels of TCF7L2, p‐YAP, and p‐TAZ detected at 24 h following surgery in the sham, MCAO, and shRNA‐CXCR7 + MCAO groups ( n = 3). (C) Densitometric quantification of TCF7L2/β‐actin. (D) Densitometric quantification of p‐YAP/β‐actin. (E) Densitometric quantification of p‐TAZ/β‐actin. * p < 0.05 versus the sham group, # p < 0.05 versus the MCAO group

Article Snippet: The membrane was blocked at room temperature for 2 h with 5% skim milk or bovine serum albumin and then incubated overnight at 4°C with the following primary antibodies: anti‐CXCR7 (1:100, rabbit, NBP2‐24779, Novus Biologicals, Littleton, CO, USA), anti‐TCF7L2 (1:500, rabbit, A19548, ABclonal, Wuhan, China), anti‐phospho‐TAZ resistant (Ser89) (1:1000, rabbit, 59971 S, Cell Signaling Technology, Danvers, MA, USA), anti‐phospho‐YAP (Ser127) (1:1000, rabbit, 13,008 T, phospho‐Yap (Ser127), Cell Signaling Technology, Danvers, MA, USA), anti‐β‐actin (1:500, rabbit, AF5006, Beyotime, Shanghai, China), and anti‐GAPDH (1:2000, AF1186, Beyotime, Shanghai, China).

Techniques: Expressing, shRNA

Journal: CNS Neuroscience & Therapeutics

Article Title: Chemokine receptor 7 mediates miRNA ‐182 to regulate cerebral ischemia/reperfusion injury in rats

doi: 10.1111/cns.14056

Figure Lengend Snippet: Schematic illustration of CXCR7 ameliorating ischemia/reperfusion (I/R) injury in acute ischemic stroke. (A) CXCR7 is upregulated in MCAO rats, and miR‐182 promotes pathological process and plays a protective role in the brain by forming an endogenous competitive RNA network with TCF7L2/Hippo. (B) Decreased expression of CXCR7 disrupts the miR‐182 expression, reducing the inhibitory effect of downstream target genes, thereby affecting the activation of Hippo pathway and subsequently aggravating cerebral ischemia reperfusion injury

Article Snippet: The membrane was blocked at room temperature for 2 h with 5% skim milk or bovine serum albumin and then incubated overnight at 4°C with the following primary antibodies: anti‐CXCR7 (1:100, rabbit, NBP2‐24779, Novus Biologicals, Littleton, CO, USA), anti‐TCF7L2 (1:500, rabbit, A19548, ABclonal, Wuhan, China), anti‐phospho‐TAZ resistant (Ser89) (1:1000, rabbit, 59971 S, Cell Signaling Technology, Danvers, MA, USA), anti‐phospho‐YAP (Ser127) (1:1000, rabbit, 13,008 T, phospho‐Yap (Ser127), Cell Signaling Technology, Danvers, MA, USA), anti‐β‐actin (1:500, rabbit, AF5006, Beyotime, Shanghai, China), and anti‐GAPDH (1:2000, AF1186, Beyotime, Shanghai, China).

Techniques: Expressing, Activation Assay

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: siRNA-mediated knockdown of TCF72 in mouse islets and clonal β-cell lines leads to defective glucose-stimulated insulin secretion. Mouse islets ( A , top left ), MIN6 ( B , top left ), and INS-1(832/13) ( C , top left ) β-cells were transfected as described in research design and methods . Immunoblot analysis was carried out using 50 μg of protein extract. Anti-TCF7L2 antibody (Abnova, Taiwan) was used at 1:1,000 and revealed by chemiluminescence after blotting with anti-mouse–horseradish peroxidase (HRP) conjugated antibody. Band intensity was quantified using ImageJ software. Real-time PCR analysis of RNA extracts from mouse islets ( A , bottom left ), MIN6 ( B , bottom left ), and INS-1(832/13) ( C , bottom left ) β-cells revealed that siRNA treatment led to 75 ± 3.57%, 71.0 ± 0.7%, and 75.2 ± 19.8% decrease, respectively, in TCF7L2 message levels. Mouse islets ( A , right ), MIN6 ( B , right ), and INS-1(832/13) ( C , right ) β-cells were treated with siRNA as described above prior to insulin secretion assays as described in research design and methods . Released and total insulin were assayed using an insulin radioimmunoassay kit from Linco. Data are means ± SEM, n = 3. A : □, control siRNA; ■, TCF7L2 siRNA. B : □, 3.0 mmol/l glucose; ▨, 10 mmol/l glucose; ■, 30 mmol/l glucose. C : □, 3.0 mmol/l glucose; ▨, 10 mmol/l glucose; ■, 30 mmol/l glucose; ▤, 3.0 mmol/l glucose + KCl.

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Knockdown, Transfection, Western Blot, Software, Real-time Polymerase Chain Reaction, RIA Assay, Control

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: TCF7L2 knockdown in mouse pancreatic islets and INS-1(832/13) β-cells does not alter glucose-stimulated changes in intracellular free [Ca 2+ ] but alters total cellular ATP and ADP levels. Mouse islets were treated and intracellular adenine nucleotide measured as described in research design and methods ( A ). Dissociated mouse islet cells ( B and C ) and INS-1(832/13) β-cells ( D and E ) were treated with 10 nmol/l fluorescein-labeled siRNA ( B and C ) or pEGFP-TCF7L2 shRNA ( D and E ) and incubated with FURA-Red (200 nmol/l; B–E ) for [Ca 2+ ] i measurement. Typical traces are shown ( C and D ). Glucose-induced (3.0 versus 11 mmol/l) [Ca 2+ ] i changes in individual glucose-responsive dissociated mouse islet cells in which TCF7L2 was silenced ( B ) and the number of INS-1(832/13) cells within the cell population that responded to (20 mmol/l) glucose ( E ) are shown. Data are means ± SEM, n = 3, unless otherwise stated in the research design and methods . AUC, area under the curve; FITC, fluorescein isothiocyanate.

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Knockdown, Labeling, shRNA, Incubation

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: A : Overexpression of TCF7L2-EGFP has no effect on glucose-stimulated insulin secretion. Insulin secretion from mouse islets overexpressing TCF7L2-EGFP was assessed as described in research design and methods . □, 3.0 mmol/l glucose; ■, 15 mmol/l glucose; ▧, 3.0 mmol/l glucose + KCl. B : Overexpression and subcellular localization of TCF7L2-EGFP in INS-1(832/13) cells was assessed by confocal microscopy 48 h after plasmid transfection. Scale bar = 15 μm. [Ca 2+ ] i imaging was performed as described in research design and methods . C and E : Representative traces of [Ca 2+ ] i responses to glucose (3 versus 20 mmol/l) stimulation are shown for EGFP-N1 vector ( C ) and TCF7L2-EGFP ( E ). The proportion of cells that displayed glucose– or GLP-1–induced [Ca 2+ ] i changes are shown in D and F , respectively (□, EGFP-N1; ■, TCF7L2-EGFP). Data are means ± SEM, n = 3. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Over Expression, Confocal Microscopy, Plasmid Preparation, Transfection, Imaging

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: Silencing and overexpression of TCF7L2 in mouse pancreatic islets lead to changes in expression of islet genes. Mouse pancreatic islets were cultured continuously in the presence of control (□), TCF7L2 siRNA (▨), or control and TCF7L2-EGFP (■) virus for 48 h. Total RNA was isolated as described in research design and methods , and islet gene expression was assessed by real-time PCR. Genes are grouped according to function: transcription factors ( A ), exocytosis ( B ), hormones, glucose sensing, and membrane transport ( C ), and signaling ( D ). Data are means ± SEM, n = 3. * P < 0.05 versus control.

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Over Expression, Expressing, Cell Culture, Control, Virus, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Membrane

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: Syntaxin 1A and Munc18-1 are regulated by TCF7L2. Mouse pancreatic islets were treated with TCF7L2 siRNA as described in research design and methods , and protein content was assessed by immunoblot analysis. A : Typical blots are shown. B : Quantification of immunoblots from three separate sets of samples using ImageJ is shown (□, control siRNA; ■, TCF7L2 siRNA). TIRF microscopy of MIN6 cells treated with TCF7L2 shRNA and quantification of granule number at the cell surface was as described in research design and methods . Images shown are averages of 1-s movies acquired at 50 Hz and 20-ms exposure at 100 nm/pixel. Data are means ± SEM, n = 3 (30–35 cells per condition) ( C ). Scale bar = 2 μm. Example graphs ( D ) of granule movement analysis from a representative control cell at different times and representative tracks ( E ) of the different categories of granule movement observed after the addition of 30 mmol/l glucose. All tracks are 30 s or longer (1 = beginning of track, 2 = end), and comparison ( F ) of the proportion of moving granules in TCF7L2 silenced and control β-cells before and during application of high glucose ( n = 5; 15–25 separate cells per condition; □, scrambled; ■, TCF7L2 shRNA). (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Western Blot, Control, Microscopy, shRNA, Comparison

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: TCF7L2 expression does not affect the morphologically docked granule pool. A : Mouse islets were transfected with siRNA as described. The total number of granules, morphologically docked, and within 200 nm of the membrane pools were analyzed as described in research design and methods ; n = 14–16 cells per condition. B and C : Representative β-cells with examples of docked granules and mitochondria (scrambled siRNA [ B ] and TCF7L2 siRNA [ C ]). Scale bars = 2 μm.

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Expressing, Transfection, Membrane

Journal: Diabetes

Article Title: TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

doi: 10.2337/db08-1187

Figure Lengend Snippet: TCF7L2 depletion affects membrane capacitance or voltage-gated Ca 2+ currents. Changes in β-cell membrane capacitance (C m ) in response to a train of 10 pulses of 200-, 500-, or 2,500-msec depolarizations from −70 mV to 0 mV ( n = 16, scrambled; n = 18, TCF7L2). A : Absolute change in membrane capacitance. The number of data points shown is reduced compared with those acquired by 200× for clarity. To filter the data, a threshold for capacitance (7 pF bottom , 13 pF top ) was set considering the smaller/larger cells outliers. The pipette solution contained (mmol/l): 125 Cs-glutamate, 10 NaCl, 10 CsCl, 1 MgCl 2 , 3 MgATP, 0.1 cAMP, 5 HEPES (pH 7.15 with CsOH). The extracellular (bath) solution contained (mmol/l): 118 NaCl, 20 TEA-Cl, 5.6 KCl, 5 glucose, 2.6 CaCl 2 , 1.2 MgCl 2 , 5 HEPES (pH 7.4 with NaOH). B : Voltage-gated Ca 2+ currents ( n = 22, scrambled; n = 27, TCF7L2). ■, TCF7L2 siRNA; □, scrambled siRNA.

Article Snippet: Mouse monoclonal antibody for TCF7L2 was purchased from Abnova (Taiwan).

Techniques: Membrane, Transferring