Journal: Stem Cells International

Article Title: Essentiality of CTNNB1 in Malignant Transformation of Human Embryonic Stem Cells under Long-Term Suboptimal Conditions

doi: 10.1155/2020/5823676

Figure Lengend Snippet: CTNNB1 expression is upregulated during the malignant transformation of hESCs. MS/MS spectra of iTRAQ-labeled peptides from CTNNB1. (a) The relative peak heights of iTRAQ labels. (b) A representative tandem mass spectrum of a peptide in each protein. Reporter ions in (a) demonstrate examples of equal, low, and high protein levels in normal and aberrant ch HES-3 cells. Normal, SIMP, COMP, and NTERA-2 cells are labeled as 114, 115, 116, and 117, respectively. The x -axis of a mass spectrum represents the correlation between the mass of a given ion and the number of elementary charges. This is represented as the IUPAC standard m/z to denote the quantity generated by dividing the mass of an ion by the unified atomic mass unit and its charge number (positive absolute value). Data are represented in Dalton (Da) as the unit of mass. The y -axis of a mass spectrum represents the signal intensity of ions.

Article Snippet: CTNNB1 shRNAs plasmid, pLKO.1-puro-shCTNNB1 (pLKO.1 puro shRNA beta-catenin, Plasmid #18803) were from Addgene, with empty vector (pLKO.1 puro, Plasmid, #8453) as control.

Techniques: Expressing, Transformation Assay, Tandem Mass Spectroscopy, Multiplex sample analysis, Labeling, Generated

Journal: Stem Cells International

Article Title: Essentiality of CTNNB1 in Malignant Transformation of Human Embryonic Stem Cells under Long-Term Suboptimal Conditions

doi: 10.1155/2020/5823676

Figure Lengend Snippet: shRNA induces the downregulation of CTNNB1 expression in hESCs. (a) Western blot analysis of CTNNB1 protein in the shCTNNB1 group, empty vector group (MOCK), and uninfected control group (WT) hESCs. (b) Immunofluorescence assay of CTNNB1 and OCT4 in shRNAs, MOCK, and WT hESCs (scale bar = 100 μ m). (c) Semiquantitative RT-PCR validation of mRNA expression levels of pluripotency genes in shCTNNB1, MOCK, and WT hESCs. Error bars denote mean ± SD from three independent experiments ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, relative to WT hESCs).

Article Snippet: CTNNB1 shRNAs plasmid, pLKO.1-puro-shCTNNB1 (pLKO.1 puro shRNA beta-catenin, Plasmid #18803) were from Addgene, with empty vector (pLKO.1 puro, Plasmid, #8453) as control.

Techniques: shRNA, Expressing, Western Blot, Plasmid Preparation, Control, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery

Journal: Stem Cells International

Article Title: Essentiality of CTNNB1 in Malignant Transformation of Human Embryonic Stem Cells under Long-Term Suboptimal Conditions

doi: 10.1155/2020/5823676

Figure Lengend Snippet: shCTNNB1 hESCs exhibit low cell proliferation and decreased telomere length as compared to WT cells. (a) Cell growth curves of shCTNNB1, MOCK, and WT hESCs. The data are represented as an average of two independent experiments ( n = 2). (b) Cell cycle analysis of shCTNNB1, MOCK, and WT hESCs. (c) Wound-healing assay. Wounds were created by manual scraping the cell clones, and images were captured as the reference point. The image of the healing state was acquired after 18 h (scale bar = 200 μ m). (d) Decreased telomerase activity in shCTNNB1 cells. Heated HSF and WT cell lysates were used as negative controls. (e) Telomere length in shCTNNB1 hESCs. Metaphase cells from the indicated groups were analyzed by the quantitative telomere-FISH assay. Strand-specific telomeric DNA probes (red) show telomere repeats; the nuclei were stained with DAPI (blue). Telomere length is represented in arbitrary units of fluorescence (a.u.) (scale bar = 10 μ m). The histograms show the frequency of different telomere lengths in CTNNB1 -silenced hESCs. The results are represented as an average of two or more independent experiments. Error bars indicate mean ± SD from three independent experiments ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01, relative to WT hESCs).

Article Snippet: CTNNB1 shRNAs plasmid, pLKO.1-puro-shCTNNB1 (pLKO.1 puro shRNA beta-catenin, Plasmid #18803) were from Addgene, with empty vector (pLKO.1 puro, Plasmid, #8453) as control.

Techniques: Cell Cycle Assay, Wound Healing Assay, Clone Assay, Activity Assay, Staining, Fluorescence

Journal: Theranostics

Article Title: A tumor suppressor enhancing module orchestrated by GATA4 denotes a therapeutic opportunity for GATA4 deficient HCC patients

doi: 10.7150/thno.38060

Figure Lengend Snippet: GATA4 assembled a tumor suppressor enhancing module between itself and β-catenin. (a) b-catenin enhances GATA4 transcriptional activity in a dose-dependent manner. HEK 293 cells (1×10 5 ) were transfected with the GATA4 reporter plasmid (0.1 mg) and increased amounts of b-catenin expression plasmid (0.05, 0.1, 0.2 mg) together w/o GATA4 expression plasmid(0.2 mg). Luciferase was monitored 24 hours later. (b) ChIP-seq result of DNA fragments enriched with antibody targeting b-catenin from HepG2i cells cultured w/o DOX. (c) b-catenin was recruited to GATA4 chromosome binding site. HepG2 cells (1.6×10 8 ) were treated w/o DOX (1 mg/mL) treatment. ChIP-Seq was conducted on DNA samples enriched with indicated antibodies from indicated cells. (d) b-catenin bound promoter region of DCN and SORSBI genes in the presence of GATA4. 1: input, 2: b- catenin enriched DNA elements in HepG2i cells in absence of DOX, 3: b-catenin enriched DNA elements in HepG2i cells in the presence of DOX, 4: GATA4 enriched DNA elements in HepG2i cells in the presence of DOX. (e) β-catenin promoted GATA4 to transcribe DCN and SORBS1 expression. CTNNB1 , DCN and SORBS1 mRNA level in DOX treated HepG2i cells after transfected with shRNA-luc or shRNA-CTNNB1. (f) DCN and SORSBI inhibited cell proliferation. HepG2-Teton-DCN and HepG2-Teton-SORBS1 cells were treated w/o DOX (1 mg/mL) for indicated time points followed by monitoring viability with CCK-8 reagents ( Left ) and statistics of colony formation ( Right ). Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote SD. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Following shRNAs were purchased from the Sigma Mission shRNA Library: shGATA4 (TRCN0000020424), shCTNNB1 (TRCN0000314920), and Luciferase shRNA (shLuc, SHC007). psPAX2, pMD2.G, pCAG-IRES-Neo and pLKO.1-puro were purchased from Addgene.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, ChIP-sequencing, Cell Culture, Binding Assay, shRNA, CCK-8 Assay

Journal: Theranostics

Article Title: A tumor suppressor enhancing module orchestrated by GATA4 denotes a therapeutic opportunity for GATA4 deficient HCC patients

doi: 10.7150/thno.38060

Figure Lengend Snippet: GATA4 inhibited canonical Wnt signaling by blocking β-catenin's recruitment of essential co-transcription factors. (a) Heatmap representation of the mRNA expression level between HepG2i cells treated with or without DOX. (b) GATA4 expression inhibited canonical Wnt signaling pathway. RNA was extracted from HepG2i treated w/o DOX (1 mg/mL) for 48 hours. Expression of the indicated genes was quantified through qPCR ( left ) or immunoblot analysis ( right ). (c) GATA4 inhibited transcriptional activity of b-catenin. Left: HEK 293 cells (1×10 5 ) were transfected with the Top-Flash reporter plasmid (0.1 mg) and b-catenin expression plasmid(0.1 mg )together with increased amounts of GATA4 expression plasmid (0.05, 0.1, 0.2 mg), followed by monitoring luciferase 24 hours later. Right: HepG2 cells (1×10 5 ) were transfected with the Top-Flash reporter plasmid and GATA4 expression plasmid. Cells were then left treated w/o GSK3b-Inhibitor for 12 hours before luciferase assays were performed. (d) Upper: Senescence-associated β-galactosidase staining of ICG001 and Vehicle treated HepG2 cells. HepG2 cells were treated w/o b-catenin inhibitor, ICG001 (0.1 mg/mL) for 48 hours. Lower: Senescence-associated β-galactosidase staining of HepG2 cells which harboring shLuciferase (shLuc), shCTNNB1 or HepG2 cells harboring shCTNNB1 transfected with PC3.1-CTNNB1 plasmid (Re-shCTNNB1), Scale bars:100mm. (e) GATA4 suppressed the interaction between b-catenin and LEF1/TCF1. HepG2i cells (5×10 7 ) were treated w/o DOX for 48 hours followed by immunoprecipitation with anti-b-catenin antibody. The immunoprecipitants and whole cell lysate were analyzed by immunoblots with indicated antibodies. (f) Bimolecular fluorescence complementation assay determining the impact of GATA4 on interaction between b-catenin and LEF1 or TCF1. HEK 293 cells (1×10 5 ) were transfected with the b-catenin-N-luciferase plasmid (b-catenin, 0.1 mg) and LEF1-C-luciferase (LEF1, 0.1 mg) or TCF1-C-luciferase (TCF1, 0.1 mg). Luciferase assays were performed 24 hours after transfection. Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote SD. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Following shRNAs were purchased from the Sigma Mission shRNA Library: shGATA4 (TRCN0000020424), shCTNNB1 (TRCN0000314920), and Luciferase shRNA (shLuc, SHC007). psPAX2, pMD2.G, pCAG-IRES-Neo and pLKO.1-puro were purchased from Addgene.

Techniques: Blocking Assay, Expressing, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Staining, Immunoprecipitation, Bimolecular Fluorescence Complementation Assay