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: Journal of Oncology

Article Title: MiR-522-3p Targets Transcription Factor 4 to Overcome Cisplatin Resistance of Gastric Cells

doi: 10.1155/2022/6082373

Figure Lengend Snippet: miR-522-3p targeted TCF4 in GC cells. (a) RT-qPCR measured TCF4 mRNA level in AGS cells under NC mimics or miR-522-3p mimics transfection. ∗ p < 0.05, miR-522-3p vs. NC mimics group. (b) Western blotting detected miR-522-3p protein level in AGS cells under NC mimics or miR-522-3p mimics transfection. (c) starBase predicted binding fragment of miR-522-3p on TCF4 3′UTR. (d) Luciferase reporter assay assessed relationship of miR-522-3p and TCF4 in GC cells. ∗ p < 0.05, miR-522-3p vs. NC mimics group. (e) RT-qPCR measured TCF4 level in control cells and GC cell lines. ∗∗∗ p < 0.001, AGS, HGC27, MKN-45 vs. GES-1 group. (f) RT-qPCR detected TCF4 level in AGS cells under DDP treatment at different doses.

Article Snippet: Lipofectamine 2000 was from Invitrogen (USA); miR-522-3p mimics and NC mimics from GenePharma (Shanghai, China), transcription factor 4 (TCF4) overexpression vector and empty vector from OriGenl (USA); and TCF4 antibody (ab185736; 1 : 500), GAPDH (ab9485; 1 : 2500), and anti-mouse and anti-rabbit horseradish peroxidaselabeled secondary antibodies from Abcam (Shanghai, China).

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Binding Assay, Luciferase, Reporter Assay, Control

Journal: Journal of Oncology

Article Title: MiR-522-3p Targets Transcription Factor 4 to Overcome Cisplatin Resistance of Gastric Cells

doi: 10.1155/2022/6082373

Figure Lengend Snippet: TCF4 suppressed GC cell resistance to DDP. (a) Colony formation assessed AGS and AGS/DDP cell proliferation under indicated transfection. (b) Flow cytometry evaluated AGS and AGS/DDP cell apoptosis under indicated transfection. (c) Quantification of number of colonies in AGS and AGS/DDP cells under indicated transfection. (d) Quantification of proportion of apoptotic AGS and AGS/DDP cells under indicated transfection. ∗∗ p < 0.01, ∗∗∗ p < 0.001, Over-TCF4 vs. NC group; ## p < 0.01, ### p < 0.001, Over-TCF4 vs. NC group.

Article Snippet: Lipofectamine 2000 was from Invitrogen (USA); miR-522-3p mimics and NC mimics from GenePharma (Shanghai, China), transcription factor 4 (TCF4) overexpression vector and empty vector from OriGenl (USA); and TCF4 antibody (ab185736; 1 : 500), GAPDH (ab9485; 1 : 2500), and anti-mouse and anti-rabbit horseradish peroxidaselabeled secondary antibodies from Abcam (Shanghai, China).

Techniques: Transfection, Flow Cytometry

Journal: Journal of Oncology

Article Title: MiR-522-3p Targets Transcription Factor 4 to Overcome Cisplatin Resistance of Gastric Cells

doi: 10.1155/2022/6082373

Figure Lengend Snippet: MiR-522-3p facilitated GC cell resistance to DDP via targeting TCF4. (a) Colony formation assessed AGS and AGS/DDP cell proliferation after indicated treatment. (b) Flow cytometry evaluated AGS and AGS/DDP cell apoptosis after indicated treatment. ∗∗ p < 0.01, ∗∗∗ p < 0.001, miR-522-3p vs. NC mimics group; ## p < 0.01, ### p < 0.001, miR-522-3+TCF4 vs. miR-522-3p group.

Article Snippet: Lipofectamine 2000 was from Invitrogen (USA); miR-522-3p mimics and NC mimics from GenePharma (Shanghai, China), transcription factor 4 (TCF4) overexpression vector and empty vector from OriGenl (USA); and TCF4 antibody (ab185736; 1 : 500), GAPDH (ab9485; 1 : 2500), and anti-mouse and anti-rabbit horseradish peroxidaselabeled secondary antibodies from Abcam (Shanghai, China).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: TCF4 mediates NED in human prostate cancer cell lines. A. Unsupervised comparison of transcriptome of LNCaP cultured in FBS vs charcoal-stripped FBS (cFBS) was carried out. B. Comparison of transcriptome between LNCaP-cFBS and LNCaP-Enz (resistant to enzalutamide). Markers of NED (ChgA, NSE, and PTHrP) were significantly increased in LNCaP-EnzR. C . Human prostate cancer cell lines LNCaP and 22Rv1 were treated with increasing concentrations of enzalutamide (0-10 μM) under an androgen-deprived condition (RPMI-1640/10% cFBS) for 48 hours. Immunoblot demonstrated that NED markers (ChgA, NSE, and PTHrP) were induced by enzalutamide. D. QPCR demonstrated that NED marker (ChgA, NSE, and PTHrP) mRNA expression levels increased also after treatment with enzalutamide at the indicated concentrations (0-10 μM) for 48 hours.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Cell Culture, Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: TCF4 mediated enzalutamide resistance in human prostate cancer cell lines. A. TCF4 cDNA was transiently transfected into LNCaP, 22Rv1, and VCaP using lipofectamine. Cells were analyzed 48 hours after transfection. The results demonstrated that the overexpression of TCF4 induced the mRNA expression levels of LNCaP, 22Rv1, and VCaP. As control, parental lines transfected with the plasmid backbone was used. B . In addition to increasing NED, overexpression of TCF4 increased the cellular proliferation rate of LNCaP, VCaP, and 22Rv1. The result shows cell counts at 72 hours after transfection. C. LNCaP and 22Rv1 were treated with enzalutamide for 48 hours. Where indicated, TCF4 or the control POU2F2 expression was silenced using shRNA approach. Increased protein levels of neuroendocrine markers following enzalutamide (enz) treatment was blocked by TCF4 shRNA in LNCaP and 22Rv1. POU2F2 was used as a negative control because it is a transcription factor whose consensus binding element was also found commonly in the promoter regions of the neuroendocrine markers. D. LNCaP was treated with 10 μM enzalutamide and cells were harvested at the indicated time. Kinetics of NED markers and TCF4 transcription following enzalutamide treatment was analyzed using QPCR. The results demonstrated increased mRNA levels of TCF4 in 3 hours while the expression levels of NED markers (ChgA, NSE, and PTHrP) was induced at 24 hours. This observation suggests that TCF4 signals upstream of NED markers.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Transfection, Over Expression, Expressing, Plasmid Preparation, shRNA, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: Effect of blocking TCF4/β-catenin (PKF118-310) or PTHrP (PTHrP (7-34) ) on enzalutamide resistant prostate cancer cells. A. LNCaP, 22Rv1, and VCaP were treated with enzalutamide (10 μM) and/or XAV939 (10 μM), β-catenin degradation activator for 48 hours. XAV939 treatment was carried out 5 min prior to the addition of enzalutamide. PTHrP mRNA induction after 48 hours of treatment with 10 μM enzalutamide was completely blocked by XAV939, β-catenin inhibitor in LNCaP, 22Rv1 and VCaP. B. LNCaP, 22Rv1, and VCaP were treated with 10 uM enzalutamide for 48 hours. The PTHrP receptor, PTH1R, mRNA level significantly increased after enzalutamide treatment in all three cell lines. C. Three enzalutamide-resistant human prostate cancer cell lines (LNCaP-EnzR, 22Rv1-EnzR, and VCaP-EnzR) were treated with 10 μM enzalutamide and increasing concentrations of PKF118-310 and PTHrP (7-34) as indicated. After 48 hours, viable cells were counted. In the presence of 10 μM Enz, PKF118-310 or PTHrP (7-34) increased enzalutamide sensitivity in the enzalutamide resistant prostate cancer cell lines, LNCaP-EnzR, VCaP-EnzR, and 22Rv1-EnzR. D. Enzalutamide-resistant human prostate cancer cell lines were treated with a fixed concentration of PKF118-310 (5 μM) or PTHrP (7-34) (10 μM) and varying concentrations of enzalutamide (0-10 μM). After 48 hours, viable cells were counted. In the presence of 5 μM of PKF118-310 or 10 μM of PTHrP (7-34) , enzalutamide inhibited cellular proliferation in a concentration-dependent manner.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Blocking Assay, Concentration Assay

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: Human CRPC tissue microarray (TMA) analysis. TMA was obtained from the rapid autopsy program at the University of Michigan. This array contains 51 CRPC samples as well as 16 benign prostate tissues and 12 localized prostate cancer tissues for controls. A. Immunofluorescence microscopy demonstrated a consistent co-localization of TCF4 (red) and PTHrP (green) in CRPC tissues. B. There was a correlation between PTHrP and TCF4 expression. C. Protein expression levels of TCF4 and D. PTHrP in patients with localized CaP (Local CaP) and metastatic CaP (Meta CaP). TCF4 and PTHrP protein levels were higher in CaP when compared with benign and increased even further in metastatic group when compared with localized CaP group. Benign n=16, Local CaP n=12, meta CaP n=51.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Microarray, Immunofluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: TCF4 induces enzalutamide resistance in the human prostate cancer cell line, LNCaP. LNCaP transfected with tetracycline-inducible TCF4 plasmid (LNCaP-TCF4) was injected into the flanks of twenty Rag2-/-, γ c -/- immunodeficient mice. When tumors reached an average size of 3 mm, the mice were divided into four groups of five each. Where indicated, 10 mg/kg enzalutamide was delivered orally daily. In the designated groups, TCF4 was delivering doxycycline via the drinking water. At the end of the indicated duration, all tumors were harvested and analyzed for protein and mRNA expression. A. When TCF4 expression was induced with doxycycline (Doxy), tumor growth rate increased when compared to the control group. In the absence of TCF4 induction, enzalutamide treatment slowed tumor growth rate. However, enzalutamide treatment had no demonstrable inhibitory effect in TCF4-induced doxycycline group. Con = LNCaP-TCF4 without doxycycline. Enz = enzalutamide. B. H&E staining. There was no difference among all groups. C. Immunofluorescence staining for PTHrP (green), TCF4 (red) with DAPI (blue) staining. Increased TCF4 and PTHrP protein levels were observed following the induction of TCF4 with doxycycline. Directly supporting the tissue culture data, enzalutamide treatment also increased protein levels of TCF4 and PTHrP. D and E . Effect of enzalutamide and TCF4 overexpression on TCF4 and PTHrP by western blot analysis ( D) and QPCR ( E ). Immunoblot and QPCR both demonstrated increased expression of TCF4 and PTHrP following TCF4 induction (Doxy). A more modest increase in TCF4 and PTHrP expression was observed with enzalutamide (enz) treatment. Error bars indicate average ± SE and * p-value<0.05.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Transfection, Plasmid Preparation, Injection, Expressing, Staining, Immunofluorescence, Over Expression, Western Blot

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: Effect of TCF4/β-catenin inhibitor (PKF118-310) and PTHrP antagonist (PTHrP (7-34) ) in enzalutamide-resistant prostate cancer. After injection of LNCaP-EnzR into the flanks of forty Rag2-/-, γ c -/- immunodeficient mice, all mice were surgically castrated divided into four groups of ten each. Animals in predesignated groups were treated daily with PKF118-310 (0.85 mg/kg intraperitoneal) and/or PTHrP (7-34) (0.2 mg/kg subcutaneous). All mice were administered daily 10 mg/kg enzalutamide orally. A. Treatment of PKF118-310 and/or PTHrP (7-34) with 10 mg/kg enzalutamide decreased tumor growth compare with vehicle treatment control group (con). B. H&E staining. There was no difference among all groups. C. Immunofluorescence staining for TCF4 (green), PTHrP (red) with DAPI (blue) staining. Consistent with its mechanism of action, PFK118-310 treatment decreased PTHrP protein levels. However, there was no effect on TCF4 levels. In contrast, PTHrP (7-34) had no demonstrable effect on the protein levels of both TCF4 and PTHrP. Treatment of PKF118-310 decreased PTHrP protein ( D ) and mRNA expression ( E ). Error bars indicate average ± SE and * p-value<0.05.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Injection, Staining, Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Positive Feedback Regulation between Phospholipase D and Wnt Signaling Promotes Wnt-Driven Anchorage-Independent Growth of Colorectal Cancer Cells

doi: 10.1371/journal.pone.0012109

Figure Lengend Snippet: (A) Deletion analysis of pGL4-PLD2 in HCT116 cells. A schematic representation of pGL4-PLD2 reporter constructs is shown. Cells were cotransfected with pGL4-PLD2 and the indicated expression vectors, followed by determination of luciferase activity. (B) Diagrammatic representation of the −2180 to +491 region of the human PLD2 promoter. Numbers above the lines refer to the transcription start site of the PLD2 gene (+1). Two putative binding sites for TCF-4 are indicated on the sequence (the arrows indicate the direction). (C) HCT 116 cells were transfected with the luciferase reporter plasmid containing the wild type (wt) PLD2 promoter, one or double TBE mutant forms (mt) of PLD2 promoter, and treated with Wnt3a (150 ng/ml) or BIO (1 µM). Luciferase activities were measured. * P <0.05 versus wtTBE/Wnt3a; † P <0.01 versus wtTBE/BIO. (D) Cells were co-transfected with the indicated expression vectors, along with the wt or TBE mutant forms of the PLD2 promoter. Luciferase activities were measured. * P <0.05 versus transfected with wtTBE/β-catenin; † P <0.05 versus wtTBE/TCF4; ** P <0.05 versus transfected with wtTBE/β-catenin/TCF4. (E) Arrows indicate position of primers used in the ChIP experiment. The ChIP assay was performed using preimmune IgG, anti-β-catenin, or anti-HDAC1 antibody and analyzed by Q-RT-PCR. As a positive control, ChIP analysis of the NOS2 promoter containing TBE was performed. * P <0.05 versus β-catenin/vehicle; † P <0.05 versus HDAC1/vehicle. Data are representative of four independent experiments. (F) Schematic diagram for comparison of TBEs on PLD2 promoter regions from various species. TCF-4 binding elements in 5′ flanking regions of the human PLD2 transcriptional start site (TSS) were compared with those of the genomes from 4 different species. A core motif , CTTTG(A/T)(A/T) [or the complementary sequence (A/T)(A/T)CAAAG] of TCF binding sequences on PLD2 promoter is highly conserved across species.

Article Snippet: Constitutive active mutant of β-catenin (S37A β-catenin) and wild type TCF-4 expression vectors were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University). shRNA for β-catenin was kindly provided by Dr. H. Clevers (Utrecht, The.

Techniques: Construct, Expressing, Luciferase, Activity Assay, Binding Assay, Sequencing, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Comparison