myc tcf  (Addgene inc)

Journal: Metabolism: clinical and experimental

Article Title: TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes.

doi: 10.1016/j.metabol.2022.155240

Figure Lengend Snippet: Fig. 4. TCF7L2 dose-dependently modulates WNT/β-catenin signaling. (A) AXIN2 mRNA levels (one-way ANOVA followed by Tukey's multiple comparisons test), (B) TOPFlash promoter activity following 6 h treatment with vehicle (Veh) or 50 ng/ml WNT3A (n = 12 wells/group) (two-way ANOVA, aaaTreatment * Genotype p < 0.001, bbbTreatment P < 0.001, cccGenotype P < 0.001 followed by ˇSíd´ak's multiple comparisons test), (C) whole cell lysate active β-catenin protein levels (one-way ANOVA followed by Tukey's multiple comparisons test), and (D) TCF7 mRNA levels following TCF7L2 KD in DFAT abdominal APs and primary abdominal APs (n = 2 subjects [0F]; mean age = 45.5. Mean BMI = 29.12; rs7903146 genotype [CC]). (One-way ANOVA followed by Tukey's multiple comparisons test.) (E) TCF7 protein levels in TCF7L2 KD DFAT abdominal APs (one-way ANOVA followed by Tukey's multiple comparisons test). shCN = scrambled control, sh897 = moderate and sh843 = high TCF7L2-KD DFAT abdominal APs. Actin was used as a loading control for western blots. qRT-PCR data were normalized to 18S rRNA levels. Histograms are means ± SEM. Data obtained from 3 independent experiments. ###P < 0.001, ##P < 0.01, #P < 0.05 (adjusted for multiple comparisons).

Article Snippet: The TCF7L2 sequence (from TCF4E pcDNA3, a gift from Frank McCormick, Addgene #32738) [31] was cloned into the pCW57.1 lentiviral vector (gift from David Root, Addgene plasmid #41393) and oligonucleotides for sh843 were cloned into tet-pLKO-puro doxycyclineinducible expression lentiviral vector (gift from Dmitri Wiederschain, Addgene #21915) [32] for inducible TCF7L2 over-expression and KD respectively.

Techniques: Activity Assay, Control, Western Blot, Quantitative RT-PCR

Journal: Metabolism: clinical and experimental

Article Title: TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes.

doi: 10.1016/j.metabol.2022.155240

Figure Lengend Snippet: Fig. 5. Global transcriptional profiling reveals that TCF7L2 regulates multiple aspects of AP biology. (A) Principal component analysis (PCA) of the global tran scriptomic profile of control (scrambled), moderate (sh897) and high-efficiency (sh843) TCF7L2-KD DFAT abdominal APs over 3 independent passages. (B) Venn diagram showing the overlap between differentially regulated genes from paired comparisons (FDR < 0.05). (C and D) Volcano plots showing the genes differentially regulated between control and (C) sh843 (TCF7 was one of top 30 differentially regulated genes highlighted in yellow); or (D) sh897, TCF7L2-KD DFAT abdominal APs. Highlighted are the top 30 differentially regulated genes. (E) Overrepresentation analyses of genes differentially regulated in sh843 and sh897 DFAT abdominal APs, vs. controls in gene ontology biological processes gene sets. P-value was adjusted using the FDR (Benjamini-Hochberg) procedure. Gene ratio represents the percentage of total DEGs in the given GO term. (F) Dimensional reduction of transcription factors similarity by co-regulation of targets (see Supplementary methods). Colors represent high dimensional clustering by affinity propagation. (G) Dendrogram of transcription factors Euclidean distance. (H) JUB, MEF2A and TEAD1 expression levels in high- and low-efficiency TCF7L2-KD cells (vs. scramble control) are shown. Padj is annotated for RNAseq log2 fold-change measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The TCF7L2 sequence (from TCF4E pcDNA3, a gift from Frank McCormick, Addgene #32738) [31] was cloned into the pCW57.1 lentiviral vector (gift from David Root, Addgene plasmid #41393) and oligonucleotides for sh843 were cloned into tet-pLKO-puro doxycyclineinducible expression lentiviral vector (gift from Dmitri Wiederschain, Addgene #21915) [32] for inducible TCF7L2 over-expression and KD respectively.

Techniques: Control, Expressing

Journal: Metabolism: clinical and experimental

Article Title: TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes.

doi: 10.1016/j.metabol.2022.155240

Figure Lengend Snippet: Fig. 7. Schematic summarizing the role of TCF7L2 and impact of rs7903146 genotype on human AP biology. TCF7L2 has dose-dependent effects on adipo genesis with moderate and high-efficiency knockdown leading to enhanced and impaired adipogenesis respectively in vitro. In fractionated adipose tissue expression of TCF7L2 in APs correlates positively with donor BMI whilst the T2D risk variant (T) at rs7903146 is associated with reduced AP TCF7L2 expression. Based on these findings we speculate that lean homozygous T2D risk variant carriers would have the lowest AP TCF7L2 expression and conse quently impaired adipogenesis, larger adipocytes, increased adipo-IR and higher susceptibility to T2D compared with obese T2D risk variant carriers.

Article Snippet: The TCF7L2 sequence (from TCF4E pcDNA3, a gift from Frank McCormick, Addgene #32738) [31] was cloned into the pCW57.1 lentiviral vector (gift from David Root, Addgene plasmid #41393) and oligonucleotides for sh843 were cloned into tet-pLKO-puro doxycyclineinducible expression lentiviral vector (gift from Dmitri Wiederschain, Addgene #21915) [32] for inducible TCF7L2 over-expression and KD respectively.

Techniques: Knockdown, In Vitro, Expressing, Variant Assay