Journal: Cell reports

Article Title: Mesenchymal Stromal Cells Are Required for Regeneration and Homeostatic Maintenance of Skeletal Muscle

doi: 10.1016/j.celrep.2019.04.074

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TCF4 polyclonal antibody , Novus , CatNBP1-88633 RRID:AB_11021504.

Techniques: Control, Recombinant, RNA Sequencing, Software

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 2. TCF4/TCF7L2 mediates the upregulation of TDO2 in APC-mutated colorectal cancer (CRC) cells. A, Immunoblots for TDO2 and β-catenin in colorectal cancer cell lines RKO (human) and MC38 (mouse) with their isogenic APC-KO counterparts. At least three independent experiments were performed. B, RT-qPCR showed APC-deleted RKO and MC38 cell lines exhibit increased Tdo2 mRNA expression. At least three independent experiments were performed. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. FC, fold change. C, DNA sequence binding motif for the transcription factor TCF4/TCF7L2. Pro- moter regions of human and mouse TDO2 genes harbor TCF4 binding motifs near the transcription start site. The motif sequence is conserved in human and mouse genes. D, ChIP-seq in APC-WT and APC-KO MC38 cells showed binding peaks for TCF4 on the promoters of the TDO2 gene. E, ChIP-PCR using the TCF4 antibody showed enriched binding to the promoter regions of the TDO2 gene in DLD-1 cells. GAPDH as a negative control; MYC and AXIN2 as positive controls. F, Luciferase activity of the human TDO2 (hTDO2) promoter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) when cotransfected with dominant-negative (DN) TCF4. ***, P < 0.001. Two independent experiments were performed. G, Luciferase activity of the hTDO2 pro- moter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) and dominant-negative TCF4. **, P < 0.01. H, Luciferase activity of the TCF4 binding motif–mutated hTDO2 promoter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) and dominant-negative TCF4. n.s., P > 0.05. I, Immunoblots for TDO2 and TCF4 in APC-KO MC38 cell lysates after transfecting with siControl (siCon) or three different siTCF4s.

Article Snippet: The pcDNA/Myc DeltaN TCF4 expression vector (RRID:Addgene_16513) was transfected to express dominant-negative TCF4.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Sequencing, Binding Assay, ChIP-sequencing, Negative Control, Luciferase, Activity Assay, Dominant Negative Mutation

Journal: Cellular & Molecular Biology Letters

Article Title: The promoting roles of GLP1R and GIPR in stemness maintenance and multiple lineage-specific differentiation of PDLSCs

doi: 10.1186/s11658-026-00867-2

Figure Lengend Snippet: Enhanced cell growth of PDLSCs facilitated by GIPRA/MAPK/ERK axis. a Western blot assay showing the expression of Ki-67, PCNA, and CCND3 affected by GIPRA as well as inhibition of cAMP/PKA/CREB, MAPK/ERK, Wnt/β-catenin, or JAK/STAT3 signaling pathways. The interpretation of bubble colors in the line chart is illustrated in Fig. a. b CCK-8 assay indicating the cell growth enhanced by GIPRA, but suppressed by ERK Inh ( n = 5 in each group). The interpretation of line colors in the line chart is illustrated in Fig. b. c Venn diagram showing the intersection of differentially binding genes by p-CREB, p-ERK1/2, TCF4, and p-STAT3 (log FC > 1, P < 0.05) compared between GIPRA and NC group. d The bubble plot displays the top ten functions of cell division and cell cycle (GO biological process) associated with the 5994 (red color highlighted) specifically binding genes by p-ERK1/2 in c . e – g IGV showing the binding peaks of p-ERK1/2 on MKI67 ( e ), PCNA ( f ), and CCND3 genes ( g ). The differential binding peaks of p-ERK1/2 on these gene promoters are highlighted by blue boxes. h – k Line plot illustrates the distribution of ChIP-seq signal intensity of p-ERK1/2 ( h ), p-CREB ( i ), TCF4 ( j ), and p-STAT3 ( k ) across the transcription start sites (±3 kb) of differentially binding genes in PDLSCs treated with GIPRA as well as GIPRA plus ERK Inh. NC, normal PDLSCs as negative control; Inh., inhibitor

Article Snippet: Immunoprecipitation was carried out overnight at 4 °C using 1 μg IP grade antibodies against p-CREB1 (cat. no. 81871-1-RR, Proteintech), p-ERK1/2 (Thr202/Tyr204), p-STAT3 (Ser727) (same with Western blot), and TCF4 (cat. no. 22337-1-AP, Proteintech).

Techniques: Western Blot, Expressing, Inhibition, Protein-Protein interactions, CCK-8 Assay, Binding Assay, ChIP-sequencing, Negative Control

Journal: Biochimica et biophysica acta

Article Title: Sex-determining region Y-box 9 acts downstream of NADPH oxidase to influence the effect of leptin on PPARγ1 expression in hepatic stellate cells.

doi: 10.1016/j.bbadis.2016.09.001

Figure Lengend Snippet: Fig. 5. Crosstalk between NADPH oxidase pathway and β-catenin pathway or DLk1 pathway. (A) Western blot analysis of β-catenin protein levels or luciferase assay of β-catenin trans- activation activity (n = 3). HSCs (for western blot analysis) or HSCs transfected with 1.6 μg of pGL3-OT or pGL3-OF (for evaluating β-catenin trans-activation activity) were pretreated with or without 1 μM DPI for 1 h before addition of leptin for another 24 h. Western blot analysis or luciferase assay was performed. Luciferase activities denoted the ratio of the signals from pGL3-OT and pGL3-OF after normalizing by the internal control. ⁎P b 0.05. (B) Detection of DLK1 expression at the levels of mRNA, protein, and promoter activity (n = 3). HSCs (for analysis by real-time PCR and western blot) or HSCs transfected with 1.6 μg pDLK1Luc were pretreated with or without 1 μM DPI for 1 h before addition of leptin for another 24 h. The levels of DLK1 mRNA, DLK1 protein, and DLK1 promoter activity were assessed, respectively, by real-time PCR, western blot analysis, and luciferase assay. ⁎P b 0.05. (C) Western blot analysis of the protein levels of β-catenin and DLK1 (n = 3). HSCs in six-well plastic plate were transfected with 1 μg p47phox siRNA (p47phox siR) or the control siRNA (control siR) and then incubated with leptin for 48 h. Western bolt analysis was performed.

Article Snippet: We also transfected HSCs with pGL3-OT or pGL3-OF (Addgene, used for evaluating β-catenin trans-activation activity) or HSCs were transfectedwith pDLK1Luc (a plasmid harboringmouse DLK1 promoter, a gift from Dr.

Techniques: Western Blot, Luciferase, Activation Assay, Activity Assay, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, Incubation