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  • 91
    Thermo Fisher anti gata4
    Upstream and downstream regulation of HNF4A in GIAC cells (A) Western Blotting and (B) qRT-PCR assay upon each individual knockdown of indicated MRTFs in Eso26 cells. (C) Co-IP assay of <t>GATA4,</t> GATA6, and HNF4A in Eso26 cells. (D) Pathway enrichment analysis of the differentially expressed genes upon silencing of HNF4A in Eso26 cells. (E) HNF4A ChIP-Seq showing its binding peak at the enhancer of IL4R in Eso26 (EAC), Caco2 (COAD), GP5d (COAD) and LoVo (COAD) cells. (F) qRT-PCR analysis showed that knockdown of HNF4A decreased the expression level of the HNF4A targets of Interleukin signaling. (G) IL4R, LYN, ELK1 and IL6ST was silenced by individual siRNAs in Eso26 cells, and cell proliferation was measured. Mean ± s.d. are shown. *, P
    Anti Gata4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam anti gata4
    Defect of spermatogenesis in Sohlh2 KO testes during early postnatal development. ( A ) A representative image of RT-PCR analysis showing the deficiency of Sohlh2 expression in 2-week-old Sohlh2 KO mice. Total RNA was isolated from WT and Sohlh2 KO testes. RT-PCR was performed using primers for Sohlh1 , Sohlh2 , and Gapdh mRNA. ( B ) Immunohistochemical analysis demonstrating a defect of spermatogenesis in Sohlh2 KO testes during early postnatal development. Sections of WT and Sohlh2 KO testes were stained with hematoxylin or immunostained with antibodies. Anti-SOHLH1, anti-SYCP3, and <t>anti-GATA4</t> antibodies were used to stain spermatogonia, spermatocyte, and sertoli cell, respectively. Testes were prepared from 2-week-old WT and Sohlh2 KO mice. Scale bars represent 25 μm. ( C ) SOHLH2-, SYCP3-, and GATA4-positive cell numbers in 2-week-old Sohlh2 KO versus WT mice. The number of positively stained cells per tubule was determined in the cross section of WT and Sohlh2 KO testes. Each group contained three mice. A minimum of 45 tubules per testis were counted for the cell number analysis. Data are presented as the mean ± SD. * p
    Anti Gata4, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti gata4
    ICM organoids seeded with 50 cells show mutual exclusive expression and sorting. Mouse tet::GATA6 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. ICM organoids were formed with 50 cells and kept undisturbed for 24 ( A ) and 48 h ( B ). ICM organoids show the two first phases: mutual exclusive expression and sorting of GATA6-positive cells. <t>GATA4</t> and SOX17 expression was present at both stages. Microscope: Zeiss LSM880; objective: 40×/1.3 oil differential interference contrast; scale bars, 20 μ m. To see this figure in color, go online.
    Anti Gata4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore gata4
    Dedifferentiation of glioblastoma multiforme (GBM) cell lines into induced glioma stem cells (iGSCs). (A) Shown are microscopic images of GBM cells (top), emerging colonies marked with asterisk (middle) and iGSCs (bottom). Scale bar: 100 μm. (B) Analysis of iGSC2 for pluripotency markers such as Oct4, Nanog and Sox2. Oct4 was detected with immunocytochemistry. DAPI was used for nuclear staining. Nanog and Sox2 expressions were compared using Western blot. Sox2 expression is 2.5-fold higher in iGSCs. Actin was used as control for Western blot. Scale bar: 100 μm. (C) Multilineage differentiation of iGSC2 detected with immunocytochemistry: Tuj-1 for ectoderm, GFAP for neuronal, <t>GATA4</t> for endoderm and SMA for mesoderm. DAPI was used for nuclear staining. Scale bar: 100 μm.
    Gata4, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    R&D Systems gata4
    Endodermal subpopulations emerging after activin A treatment using tfFACS. ( A ) A representative experiment using two-channel FACS analysis of <t>GATA4</t> and SOX17. Compared with the isotype negative control (bottom panels), three distinct cellular populations: SOX17 + GATA4 − , SOX17 + GATA4 + , and SOX17 − GATA4 + are emerging gradually upon differentiation: at day 1, 13% are SOX17 + GATA4 + , increasing to 23% by day 3. Another significant population consists of 18% SOX17 − GATA4 + at day 1 and 25% at day 3. ( B ) After 5 days of differentiation, using three-way multichannel FACS analysis for SOX17, GATA4, and CXCR4, we found that the SOX17 + GATA4 + population dominates the culture (62%) and CXCR4 is expressed in 49% of the cells, most of which are SOX17 + GATA4 + CXCR4 + (41%). There are also approximately 27% GATA4 + CXCR4 − cells, which comprises the population of SOX17 + GATA4 + CXCR4 − cells (21%). ( C ) Post sorting, FACS analysis demonstrated that 97% of day 5 SOX17 + GATA4 + CXCR4 + cells were positive for GATA4, 88% were SOX17 positive, and 95% were CXCR4 positive. This was consistent over 5 separate experiments. ( D ) Expression analysis using RT-qPCR demonstrates that day 5 SOX17 + GATA4 + CXCR4 + and day3 SOX17 + GATA4 + cells have higher level of expression of SOX17, GATA4 and CXCR4 than unsorted fixed cells or day 3 SOX17 − GATA4 − (d3SOX17negGATA4neg) cells.
    Gata4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc gata4
    Pretreatment with curcumin blocks <t>GATA4</t> activation. ( A and B ) Curcumin blocked the acetylation and DNA-binding activity of GATA4 induced by PE infusion ( A ) or AB ( B ). n = 4. Oct-1 DNA-binding activity was used as a control. ( C ) Effect of p300 on the acetylation and DNA-binding activity of GATA4 induced by PE. Cells were infected with Ad-p300, Ad-DN-p300, or Ad-GFP for 24 hours and then treated with 100 μM PE for 24 hours. Extracts were assayed for GATA4 acetylation and DNA-binding activity. ( D ) p300 partially reversed the inhibitory effect of curcumin on the acetylation and DNA-binding activity of GATA4 induced by PE. Cells were infected with Ad-p300 or Ad-GFP for 24 hours, treated with 25 μM curcumin for 60 minutes, and then incubated with 100 μM PE for 24 hours. The results were reproducible in 3 separate experiments.
    Gata4, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GeneTex anti gata4
    The variant of ZFPM2 attenuated the transcriptional activation of <t>GATA4</t> and contributed to the cardiac abnormalities in zebrafish. a Diagram of the human ZFPM2/FOG2 protein domain with the location of its variants identified. The eight zinc-finger motifs (ZNF) are represented by yellow boxes . The nuclear localization signal (NLS) is indicated by a green box . The putative CtBP-binding site (CBS) is represented by a pink box . The N-terminal transcriptional repression domain (TRD) is indicated by a blue box . E1148 K found in B430 is located at the eighth Zinc-finger domain, and E1005G found in B548 is located in the seventh and eighth domains. Sanger sequencing shows the variant in the red frame . Both are conserved in human, mouse, dog and zebrafish. The amino acid residue altered by the mutation is shown in the red box . b Co-immunoprecipitation assays in HEK293T cells revealed that the E1148 K variant significantly damaged the interaction between ZFPM2 and GATA4 on the western blot. The semi-quantitative analysis of the western blot results shows that the E1148 K mutant ZFPM2 protein significantly disrupted the interaction with GATA4 compared to the wild-type ZFPM2 protein. The experiment was repeated three times. (p
    Anti Gata4, supplied by GeneTex, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson gata4
    Celastrol increased HSPs and preserved essential transcription factors. a , b Immunoblot analysis showed an increase in active caspase-3 expression after exposure to DOX and TAA in H9c2 and Huh7 cells, respectively. <t>GATA4</t> in H9c2 and Hnf-1α and
    Gata4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Epitomics anti gata4
    <t>GATA4</t> downregulates the TGFB2 mRNA level through multiple miRNAs. a Schematics of targeting sites on TGFB2 mRNA recognized by 14 miRNAs. b Relative expression level of miRNAs before and after GATA4 overexpression in A549i (through 2 μg/mL of Dox treatment) and A549 cells (through lentivirus infection) ( n = 3 per group). c qPCR analysis of promoter fragment of designated gene from DNA samples prepared from control IgG or anti-FLAG (for pulldown GATA4) DNA samples. Quantity of GATA4 bond DNA was normalized by the value in control IgG-treated samples ( n = 3 per group). d TGFB2 mRNA level in A549 cells overexpressing miR-32, miR-301b, miR-320A, or miR-590, respectively ( n = 3 per group). e β-Galactosidase signal in GATA4 overexpressing A549 cells in the absence or presence of microRNA sponge ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P
    Anti Gata4, supplied by Epitomics, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Selleck Chemicals anti gata4
    The protein expression of <t>GATA4</t> was reduced after AT treatment in mice. (a) Western blot bands of the protein expression of GATA4 and GAPDH and (b) their fold changes in the four groups (NS-SHAM, NS-TAC, AT-SHAM, and AT-TAC). Data are mean ± SEM ( n = 7). * P
    Anti Gata4, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Upstream and downstream regulation of HNF4A in GIAC cells (A) Western Blotting and (B) qRT-PCR assay upon each individual knockdown of indicated MRTFs in Eso26 cells. (C) Co-IP assay of GATA4, GATA6, and HNF4A in Eso26 cells. (D) Pathway enrichment analysis of the differentially expressed genes upon silencing of HNF4A in Eso26 cells. (E) HNF4A ChIP-Seq showing its binding peak at the enhancer of IL4R in Eso26 (EAC), Caco2 (COAD), GP5d (COAD) and LoVo (COAD) cells. (F) qRT-PCR analysis showed that knockdown of HNF4A decreased the expression level of the HNF4A targets of Interleukin signaling. (G) IL4R, LYN, ELK1 and IL6ST was silenced by individual siRNAs in Eso26 cells, and cell proliferation was measured. Mean ± s.d. are shown. *, P

    Journal: bioRxiv

    Article Title: Lineage-Specific Epigenomic and Genomic Activation of the Oncogene HNF4A Promotes Gastrointestinal Adenocarcinomas

    doi: 10.1101/812149

    Figure Lengend Snippet: Upstream and downstream regulation of HNF4A in GIAC cells (A) Western Blotting and (B) qRT-PCR assay upon each individual knockdown of indicated MRTFs in Eso26 cells. (C) Co-IP assay of GATA4, GATA6, and HNF4A in Eso26 cells. (D) Pathway enrichment analysis of the differentially expressed genes upon silencing of HNF4A in Eso26 cells. (E) HNF4A ChIP-Seq showing its binding peak at the enhancer of IL4R in Eso26 (EAC), Caco2 (COAD), GP5d (COAD) and LoVo (COAD) cells. (F) qRT-PCR analysis showed that knockdown of HNF4A decreased the expression level of the HNF4A targets of Interleukin signaling. (G) IL4R, LYN, ELK1 and IL6ST was silenced by individual siRNAs in Eso26 cells, and cell proliferation was measured. Mean ± s.d. are shown. *, P

    Article Snippet: Antibodies and reagents The following antibodies and reagents were used: Anti-HNF4A for ChIP-Seq and WB (Abcam, ab41898), Anti-HNF4A for IHC (a gift from Kenji Daigo and Takao Hamakubo), Anti-ELF3 (Santa Cruz Biotechnology, sc-376055 X), Anti-KLF5 (Santa Cruz Biotechnology, sc-398470 X), Anti-GATA6 (Cell Signaling Technology, 5851), Anti-GATA4 (Thermo Fisher Scientific, MA5-15532), Anti-HNF1A (Cell Signaling Technology,89670), Anti-FLAG (Sigma, F1804), Anti-Actin (Santa Cruz Biotechnology, sc-8432), Anti-H3K27Ac (Abcam, ab4729), Anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 115-035-003), Anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 115-035-144), FITC Annexin V Apoptosis Detection Kit (BD Biosciences, 556547), Lipofectamine RNAiMAX (Thermo Fisher, 13778150), BI-6015 (Cayman, 12032) and siRNAs were purchased from Suzhou GenePharma Co., Ltd. (Sequences are provided in Supplementary Table 1).

    Techniques: Western Blot, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Binding Assay, Expressing

    Sertoli cell number evolution. Counts of GATA4-positive cells in round tubules show that both the number of Sertoli cells per cluster ( A ) and the percentage of seminiferous tubules that contain clusters ( B ) increase with age in Cldn11 −/−

    Journal:

    Article Title: Claudin 11 Deficiency in Mice Results in Loss of the Sertoli Cell Epithelial Phenotype in the Testis 1

    doi: 10.1095/biolreprod.109.078907

    Figure Lengend Snippet: Sertoli cell number evolution. Counts of GATA4-positive cells in round tubules show that both the number of Sertoli cells per cluster ( A ) and the percentage of seminiferous tubules that contain clusters ( B ) increase with age in Cldn11 −/−

    Article Snippet: For double immunolabeling, paraffin sections were prepared as above and incubated overnight with anti-Gata4 antibody (diluted 1:100), followed by sequential incubations with anti-goat immunoglobulin (Ig)-Alexa Fluor 546 secondary antibody (1:1000; Invitrogen) for 2 h at room temperature, anti-BrdU antibody (1:100), and anti-mouse Ig-Alexa Fluor 488 secondary antibody (1:1000; Invitrogen).

    Techniques:

    Nature of cell clusters. Immunolabeling of germ cell markers DDX4 ( A – C ) and calmegin (CLGN), recognized by the TRA369 antibody ( D – F ), and Sertoli cell marker GATA4 ( G – I ) of P28 control ( A , D , and G ) and Cldn11 −/−

    Journal:

    Article Title: Claudin 11 Deficiency in Mice Results in Loss of the Sertoli Cell Epithelial Phenotype in the Testis 1

    doi: 10.1095/biolreprod.109.078907

    Figure Lengend Snippet: Nature of cell clusters. Immunolabeling of germ cell markers DDX4 ( A – C ) and calmegin (CLGN), recognized by the TRA369 antibody ( D – F ), and Sertoli cell marker GATA4 ( G – I ) of P28 control ( A , D , and G ) and Cldn11 −/−

    Article Snippet: For double immunolabeling, paraffin sections were prepared as above and incubated overnight with anti-Gata4 antibody (diluted 1:100), followed by sequential incubations with anti-goat immunoglobulin (Ig)-Alexa Fluor 546 secondary antibody (1:1000; Invitrogen) for 2 h at room temperature, anti-BrdU antibody (1:100), and anti-mouse Ig-Alexa Fluor 488 secondary antibody (1:1000; Invitrogen).

    Techniques: Immunolabeling, Marker

    Dynamics of Sertoli cell sloughing. Immunolabeling of GATA4 of P13 ( A and B ) and P28 ( C – F ) control ( A and C ) and Cldn11 −/− ( B and D – F ) testes shows the progressive sloughing of Sertoli cells from P13 onward and from the

    Journal:

    Article Title: Claudin 11 Deficiency in Mice Results in Loss of the Sertoli Cell Epithelial Phenotype in the Testis 1

    doi: 10.1095/biolreprod.109.078907

    Figure Lengend Snippet: Dynamics of Sertoli cell sloughing. Immunolabeling of GATA4 of P13 ( A and B ) and P28 ( C – F ) control ( A and C ) and Cldn11 −/− ( B and D – F ) testes shows the progressive sloughing of Sertoli cells from P13 onward and from the

    Article Snippet: For double immunolabeling, paraffin sections were prepared as above and incubated overnight with anti-Gata4 antibody (diluted 1:100), followed by sequential incubations with anti-goat immunoglobulin (Ig)-Alexa Fluor 546 secondary antibody (1:1000; Invitrogen) for 2 h at room temperature, anti-BrdU antibody (1:100), and anti-mouse Ig-Alexa Fluor 488 secondary antibody (1:1000; Invitrogen).

    Techniques: Immunolabeling

    Sertoli cell loss of polarity. Immunolabeling of GATA4 in Sertoli cell nuclei of P28 ( A ) Cldn11 −/− testes shows the progressive change in Sertoli nuclei shape, from round (arrowhead) to fibroblasticlike elongated (arrows). Immunolabeling

    Journal:

    Article Title: Claudin 11 Deficiency in Mice Results in Loss of the Sertoli Cell Epithelial Phenotype in the Testis 1

    doi: 10.1095/biolreprod.109.078907

    Figure Lengend Snippet: Sertoli cell loss of polarity. Immunolabeling of GATA4 in Sertoli cell nuclei of P28 ( A ) Cldn11 −/− testes shows the progressive change in Sertoli nuclei shape, from round (arrowhead) to fibroblasticlike elongated (arrows). Immunolabeling

    Article Snippet: For double immunolabeling, paraffin sections were prepared as above and incubated overnight with anti-Gata4 antibody (diluted 1:100), followed by sequential incubations with anti-goat immunoglobulin (Ig)-Alexa Fluor 546 secondary antibody (1:1000; Invitrogen) for 2 h at room temperature, anti-BrdU antibody (1:100), and anti-mouse Ig-Alexa Fluor 488 secondary antibody (1:1000; Invitrogen).

    Techniques: Immunolabeling

    Gata6 could not replace Gata4 function in the septum transversum mesenchyme, leading to a loss of liver and ventral pancreas. (A) H E sections of E12.5 heterozygous and homozygous knock-in embryos at the level of the abdomen. Remaining liver tissue

    Journal: Developmental biology

    Article Title: Unique functions of Gata4 in mouse liver induction and heart development

    doi: 10.1016/j.ydbio.2015.12.007

    Figure Lengend Snippet: Gata6 could not replace Gata4 function in the septum transversum mesenchyme, leading to a loss of liver and ventral pancreas. (A) H E sections of E12.5 heterozygous and homozygous knock-in embryos at the level of the abdomen. Remaining liver tissue

    Article Snippet: Primary antibodies: GATA4 (eBioscience), MYC (generously provided by Susan Morton/Thomas Jessell), ITGA4 (BD Pharmingen), and MF20 (DSHB) all at 1:100, and BrdU (Abcam) at 1:300.

    Techniques: Knock-In

    Gata6 replaces Gata4 function in extraembryonic tissues. (A) Gross appearance of E12.5 Gata4 Gata6/+ and Gata4 Gata6/Gata6 littermates. A subset of Gata4 Gata6/Gata6 embryos appeared morphologically normal except for the absence of liver (asterisk), while

    Journal: Developmental biology

    Article Title: Unique functions of Gata4 in mouse liver induction and heart development

    doi: 10.1016/j.ydbio.2015.12.007

    Figure Lengend Snippet: Gata6 replaces Gata4 function in extraembryonic tissues. (A) Gross appearance of E12.5 Gata4 Gata6/+ and Gata4 Gata6/Gata6 littermates. A subset of Gata4 Gata6/Gata6 embryos appeared morphologically normal except for the absence of liver (asterisk), while

    Article Snippet: Primary antibodies: GATA4 (eBioscience), MYC (generously provided by Susan Morton/Thomas Jessell), ITGA4 (BD Pharmingen), and MF20 (DSHB) all at 1:100, and BrdU (Abcam) at 1:300.

    Techniques:

    Gata6 was able to replace Gata4 function in extraembryonic tissues

    Journal: Developmental biology

    Article Title: Unique functions of Gata4 in mouse liver induction and heart development

    doi: 10.1016/j.ydbio.2015.12.007

    Figure Lengend Snippet: Gata6 was able to replace Gata4 function in extraembryonic tissues

    Article Snippet: Primary antibodies: GATA4 (eBioscience), MYC (generously provided by Susan Morton/Thomas Jessell), ITGA4 (BD Pharmingen), and MF20 (DSHB) all at 1:100, and BrdU (Abcam) at 1:300.

    Techniques:

    Heart and epicardium development are compromised in Gata4 Gata6/Gata6 mutants. (A) H E staining of E12.5 heterozygous and homozygous knock-in embryos at the level of the heart. Scale bar: 250 um. RA: right atrium, LA: left atrium, RV: right ventricle,

    Journal: Developmental biology

    Article Title: Unique functions of Gata4 in mouse liver induction and heart development

    doi: 10.1016/j.ydbio.2015.12.007

    Figure Lengend Snippet: Heart and epicardium development are compromised in Gata4 Gata6/Gata6 mutants. (A) H E staining of E12.5 heterozygous and homozygous knock-in embryos at the level of the heart. Scale bar: 250 um. RA: right atrium, LA: left atrium, RV: right ventricle,

    Article Snippet: Primary antibodies: GATA4 (eBioscience), MYC (generously provided by Susan Morton/Thomas Jessell), ITGA4 (BD Pharmingen), and MF20 (DSHB) all at 1:100, and BrdU (Abcam) at 1:300.

    Techniques: Staining, Knock-In

    Gata6 was able to replace Gata4 function in extraembryonic tissues

    Journal: Developmental biology

    Article Title: Unique functions of Gata4 in mouse liver induction and heart development

    doi: 10.1016/j.ydbio.2015.12.007

    Figure Lengend Snippet: Gata6 was able to replace Gata4 function in extraembryonic tissues

    Article Snippet: Primary antibodies: GATA4 (eBioscience), MYC (generously provided by Susan Morton/Thomas Jessell), ITGA4 (BD Pharmingen), and MF20 (DSHB) all at 1:100, and BrdU (Abcam) at 1:300.

    Techniques:

    Construction and validation of the Gata4 Gata6 allele. (A) Schematic of targeting strategy. The targeting vector contains Gata6 cDNA with an amino-terminal myc tag, followed by an SV40 polyA tail and a neo cassette flanked by FRT sites, all targeted to

    Journal: Developmental biology

    Article Title: Unique functions of Gata4 in mouse liver induction and heart development

    doi: 10.1016/j.ydbio.2015.12.007

    Figure Lengend Snippet: Construction and validation of the Gata4 Gata6 allele. (A) Schematic of targeting strategy. The targeting vector contains Gata6 cDNA with an amino-terminal myc tag, followed by an SV40 polyA tail and a neo cassette flanked by FRT sites, all targeted to

    Article Snippet: Primary antibodies: GATA4 (eBioscience), MYC (generously provided by Susan Morton/Thomas Jessell), ITGA4 (BD Pharmingen), and MF20 (DSHB) all at 1:100, and BrdU (Abcam) at 1:300.

    Techniques: Plasmid Preparation

    GATA4 downregulates the TGFB2 mRNA level through multiple miRNAs. a Schematics of targeting sites on TGFB2 mRNA recognized by 14 miRNAs. b Relative expression level of miRNAs before and after GATA4 overexpression in A549i (through 2 μg/mL of Dox treatment) and A549 cells (through lentivirus infection) ( n = 3 per group). c qPCR analysis of promoter fragment of designated gene from DNA samples prepared from control IgG or anti-FLAG (for pulldown GATA4) DNA samples. Quantity of GATA4 bond DNA was normalized by the value in control IgG-treated samples ( n = 3 per group). d TGFB2 mRNA level in A549 cells overexpressing miR-32, miR-301b, miR-320A, or miR-590, respectively ( n = 3 per group). e β-Galactosidase signal in GATA4 overexpressing A549 cells in the absence or presence of microRNA sponge ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: GATA4 downregulates the TGFB2 mRNA level through multiple miRNAs. a Schematics of targeting sites on TGFB2 mRNA recognized by 14 miRNAs. b Relative expression level of miRNAs before and after GATA4 overexpression in A549i (through 2 μg/mL of Dox treatment) and A549 cells (through lentivirus infection) ( n = 3 per group). c qPCR analysis of promoter fragment of designated gene from DNA samples prepared from control IgG or anti-FLAG (for pulldown GATA4) DNA samples. Quantity of GATA4 bond DNA was normalized by the value in control IgG-treated samples ( n = 3 per group). d TGFB2 mRNA level in A549 cells overexpressing miR-32, miR-301b, miR-320A, or miR-590, respectively ( n = 3 per group). e β-Galactosidase signal in GATA4 overexpressing A549 cells in the absence or presence of microRNA sponge ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Expressing, Over Expression, Infection, Real-time Polymerase Chain Reaction

    The GATA4-TGF-β2-Wnt-7b signaling axis is clinically relevant. a qRT-PCR analysis of TGFB2 and WNT7B expression level in H226 cell line with GATA4 knockdown ( n = 3 per group). b GATA4 expression level in lung cancer samples and normal lung tissues. c Kaplan–Meier survival curve of GATA4-high and GATA4-low lung cancer patients (Cox log-rank test, p = 0.019). d Reverse correlation of expression level of GATA4 versus TGF-β2 and Wnt-7b in clinical lung cancer samples downloaded from TCGA. e Expression pattern of GATA4 versus TGF-β2 and Wnt-7b in driver mutation positive samples. EGFR mutation positive: Upper panel; Kras mutation positive: middle panel; EML4-ALK mutation positive: lower panel

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: The GATA4-TGF-β2-Wnt-7b signaling axis is clinically relevant. a qRT-PCR analysis of TGFB2 and WNT7B expression level in H226 cell line with GATA4 knockdown ( n = 3 per group). b GATA4 expression level in lung cancer samples and normal lung tissues. c Kaplan–Meier survival curve of GATA4-high and GATA4-low lung cancer patients (Cox log-rank test, p = 0.019). d Reverse correlation of expression level of GATA4 versus TGF-β2 and Wnt-7b in clinical lung cancer samples downloaded from TCGA. e Expression pattern of GATA4 versus TGF-β2 and Wnt-7b in driver mutation positive samples. EGFR mutation positive: Upper panel; Kras mutation positive: middle panel; EML4-ALK mutation positive: lower panel

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Quantitative RT-PCR, Expressing, Mutagenesis

    TGFBR1 is a potential target for GATA4-deficient lung cancer. a and b 5000 cells per well seeded in 96-well plates. Cells were treated with SB525334 (2 μM) and/or trametinib (2 μM). CCK8 value were checked 3 days after drug treatment. a For H23 and b for PC9 ( n = 3 per group). c SB525334 (10 mg/kg/day) synergizes with trametinib (10 mg/kg/day) to shrink lung tumor in KRAS G12D /GATA4-/- mice. MRI image of lung of pre-treatment (left panel) and post-treatment (middle panel) of lung cancer bearing mice (PreRx and PostRx); β-Galactosidase staining of lung section of post-treatment mice (right panel). Scale = 100 μm. d Statistics of lung tumor burdens recorded in MRI ( n = 4 per group). e Statistics of senescence in tumor shown ( c ) ( n = 4 per group). f , g , h and i The tumor sizes and weights of PDX tumors treated with TGFBR inhibitor (SB525334, 10 mg/kg/day), Cisplatin (5 mg/kg, once a week), or combination. SH3166: GATA4-low PDX; SH3a: GATA4-high PDX ( n = 6 per group). j Model of how GATA4 regulate TGF-β2 and Wnt-7b signaling and cellular senescence. See text for details. Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: TGFBR1 is a potential target for GATA4-deficient lung cancer. a and b 5000 cells per well seeded in 96-well plates. Cells were treated with SB525334 (2 μM) and/or trametinib (2 μM). CCK8 value were checked 3 days after drug treatment. a For H23 and b for PC9 ( n = 3 per group). c SB525334 (10 mg/kg/day) synergizes with trametinib (10 mg/kg/day) to shrink lung tumor in KRAS G12D /GATA4-/- mice. MRI image of lung of pre-treatment (left panel) and post-treatment (middle panel) of lung cancer bearing mice (PreRx and PostRx); β-Galactosidase staining of lung section of post-treatment mice (right panel). Scale = 100 μm. d Statistics of lung tumor burdens recorded in MRI ( n = 4 per group). e Statistics of senescence in tumor shown ( c ) ( n = 4 per group). f , g , h and i The tumor sizes and weights of PDX tumors treated with TGFBR inhibitor (SB525334, 10 mg/kg/day), Cisplatin (5 mg/kg, once a week), or combination. SH3166: GATA4-low PDX; SH3a: GATA4-high PDX ( n = 6 per group). j Model of how GATA4 regulate TGF-β2 and Wnt-7b signaling and cellular senescence. See text for details. Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Mouse Assay, Magnetic Resonance Imaging, Staining

    GATA4 is an important tumor suppressor in lung cancer. a Scatterplot of relative growth rate against relative expression level. 10,000 H23 cells per well seeded in 96-well plate; siRNA transfected into individual wells. CCK8 value of a well 3 days post siRNA transfection was plotted against relative expression value of the corresponding gene in tumor (one replica). b Western blot analysis of GATA4 expression in cancerous and normal lung cell lines. c Lung cancer cells infected with lentivirus for ectopic expression of GATA4. Soft-agar colony forming ability was compared between lung cancer cell lines (A549, PC-9, and H460) ectopically expressing GATA4 and EGFP. Scale = 500 μm. d Statistics of soft-agar colony result shown in c ( n = 3 per group). e Colony number of 10,000 A549i cells in the absence or presence of Dox checked 14 days after seeding in soft agar plate. Upper panel: statistics of the soft-agar colonies. Lower panel: representative pictures of A549i cells treated with or without 2 μg/mL of Dox ( n = 3 per group, scale = 100 μm) (A549i is an engineered A549 clone harboring Dox inducible GATA4 expression gene element). f Soft-agar colony forming ability of stable H23 with GATA4 knockdown by shRNAs or control shRNA. Upper panel: statistics of soft-agar colony result of H23 knockdown with control or GATA4 targeting shRNAs. Lower panel: Representative pictures ( n = 3 per group, scale = 50 μm). g Lsl-KRAS G12D infected with recombinant lentivirus coexpressing Cre and CRISPR/CAS9 through nasal instillation. Lung tumor formation in KRAS G12D /GATA4-/- mice compared to KRAS G12D /TdTomato-/- mice 10 weeks post-infection. Scale = 200 μm. h Statistics of percentage of tumor area in the lung represented in g (n = 3 per group). i Schematics of intranasal instillation of retrovirus for forced expressing GATA4 in Dox inducible Tet-KRAS G12C /CC10rtTA lung cancer mouse model (referred to as KC). j Pathology of lung tumor formation in KC mice infected with control (mCherry) or GATA4 expressing virus. Scale = 200 μm. k Statistics of tumor area per lung area (n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: GATA4 is an important tumor suppressor in lung cancer. a Scatterplot of relative growth rate against relative expression level. 10,000 H23 cells per well seeded in 96-well plate; siRNA transfected into individual wells. CCK8 value of a well 3 days post siRNA transfection was plotted against relative expression value of the corresponding gene in tumor (one replica). b Western blot analysis of GATA4 expression in cancerous and normal lung cell lines. c Lung cancer cells infected with lentivirus for ectopic expression of GATA4. Soft-agar colony forming ability was compared between lung cancer cell lines (A549, PC-9, and H460) ectopically expressing GATA4 and EGFP. Scale = 500 μm. d Statistics of soft-agar colony result shown in c ( n = 3 per group). e Colony number of 10,000 A549i cells in the absence or presence of Dox checked 14 days after seeding in soft agar plate. Upper panel: statistics of the soft-agar colonies. Lower panel: representative pictures of A549i cells treated with or without 2 μg/mL of Dox ( n = 3 per group, scale = 100 μm) (A549i is an engineered A549 clone harboring Dox inducible GATA4 expression gene element). f Soft-agar colony forming ability of stable H23 with GATA4 knockdown by shRNAs or control shRNA. Upper panel: statistics of soft-agar colony result of H23 knockdown with control or GATA4 targeting shRNAs. Lower panel: Representative pictures ( n = 3 per group, scale = 50 μm). g Lsl-KRAS G12D infected with recombinant lentivirus coexpressing Cre and CRISPR/CAS9 through nasal instillation. Lung tumor formation in KRAS G12D /GATA4-/- mice compared to KRAS G12D /TdTomato-/- mice 10 weeks post-infection. Scale = 200 μm. h Statistics of percentage of tumor area in the lung represented in g (n = 3 per group). i Schematics of intranasal instillation of retrovirus for forced expressing GATA4 in Dox inducible Tet-KRAS G12C /CC10rtTA lung cancer mouse model (referred to as KC). j Pathology of lung tumor formation in KC mice infected with control (mCherry) or GATA4 expressing virus. Scale = 200 μm. k Statistics of tumor area per lung area (n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Expressing, Transfection, Western Blot, Infection, shRNA, Recombinant, CRISPR, Mouse Assay

    Ectopic GATA4 expression induces lung cancer cell senescence. a Microphotograph of A549 cells infected lentivirus overexpressing GATA4. Red arrow-head highlighted the senescent cells. Scale = 100 μm. b β-galactosidase staining of A549 cells infected with GATA4 or EGFP expressing lentivirus. Representative pictures were shown. Scale = 100 μm. c , d Statistics of β-Galactosidase staining of A549 cells ectopically expressing GATA4 through lentivirus infection ( c ) or Doxycycline treatment of A549i cells ( d ) ( n = 3 per group). e Western blot analysis of Doxycycline-inducible GATA4 expression in stable cell lines of H460, EKVX, and Hop62. f β-Galactosidase staining of indicated cell lines before and after GATA4 expression. g Statistics of f ( n = 3 per group). h Western blot analysis of p15INK4b expression in GATA4-expressing A549 cells. i β-galactosidase staining of GATA4-expressing A549 knockdown with control or CDKN2B-targeting shRNAs ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: Ectopic GATA4 expression induces lung cancer cell senescence. a Microphotograph of A549 cells infected lentivirus overexpressing GATA4. Red arrow-head highlighted the senescent cells. Scale = 100 μm. b β-galactosidase staining of A549 cells infected with GATA4 or EGFP expressing lentivirus. Representative pictures were shown. Scale = 100 μm. c , d Statistics of β-Galactosidase staining of A549 cells ectopically expressing GATA4 through lentivirus infection ( c ) or Doxycycline treatment of A549i cells ( d ) ( n = 3 per group). e Western blot analysis of Doxycycline-inducible GATA4 expression in stable cell lines of H460, EKVX, and Hop62. f β-Galactosidase staining of indicated cell lines before and after GATA4 expression. g Statistics of f ( n = 3 per group). h Western blot analysis of p15INK4b expression in GATA4-expressing A549 cells. i β-galactosidase staining of GATA4-expressing A549 knockdown with control or CDKN2B-targeting shRNAs ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Expressing, Infection, Staining, Western Blot, Stable Transfection

    TGF-β2 mediates GATA4-induced senescence upstream of Wnt-7b. a and b qRT-PCR analysis of TGFB2 expression in GATA4 overexpressing A549 cells through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c β-Galactosidase staining of A549 cells with TGFB2 knockdown (middle panel) and rescued by shRNA-resistant TGFB2 . d Statistics of c ( n = 3 per group). e β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting TGFB2 ( n = 3 per group). f β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by TGFB2 expression ( n = 3 per group). g β-Galactosidase staining of A549i cells in the absence or presence of 2 μg/mL of Dox and rescued with recombinant TGF-β protein at indicated concentration ( n = 3 per group). h β-Galactosidase signal in A549 cells knockdown of TGFBR1 and TGFBR2 ( n = 3 per group). i β-Galactosidase signal of A549 cells with SMAD2 knockdown (middle panel) and rescued by shRNA-resistant SMAD2 (right panel). j β-Galactosidase staining of A549 cells with SMAD4 knockdown (middle panel) and rescued by shRNA-resistant SMAD4 (right panel) ( n = 3 per group). k WNT7B expression level in A549 cells with knockdown of TGFBR1 , TGFBR2 , SMAD2 , or SMAD4 , respectively ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: TGF-β2 mediates GATA4-induced senescence upstream of Wnt-7b. a and b qRT-PCR analysis of TGFB2 expression in GATA4 overexpressing A549 cells through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c β-Galactosidase staining of A549 cells with TGFB2 knockdown (middle panel) and rescued by shRNA-resistant TGFB2 . d Statistics of c ( n = 3 per group). e β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting TGFB2 ( n = 3 per group). f β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by TGFB2 expression ( n = 3 per group). g β-Galactosidase staining of A549i cells in the absence or presence of 2 μg/mL of Dox and rescued with recombinant TGF-β protein at indicated concentration ( n = 3 per group). h β-Galactosidase signal in A549 cells knockdown of TGFBR1 and TGFBR2 ( n = 3 per group). i β-Galactosidase signal of A549 cells with SMAD2 knockdown (middle panel) and rescued by shRNA-resistant SMAD2 (right panel). j β-Galactosidase staining of A549 cells with SMAD4 knockdown (middle panel) and rescued by shRNA-resistant SMAD4 (right panel) ( n = 3 per group). k WNT7B expression level in A549 cells with knockdown of TGFBR1 , TGFBR2 , SMAD2 , or SMAD4 , respectively ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Quantitative RT-PCR, Expressing, Infection, Staining, shRNA, Recombinant, Concentration Assay

    GATA4 induces lung cancer cell senescence by downregulating WNT7B. a and b WNT7B expression level in A549 cells ectopically expressing GATA4 through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c TOP-FLASH analysis of GATA4 expressing A549 cells ( n = 6 per group). d Western blot analysis of c-Myc and cyclin D1 in A549i cells in the absence or presence of 2 μg/mL of Dox. e β-Galactosidase staining of A549 cells expressing WNT7B targeting shRNA (middle panel) and rescued by shRNA-resistant WNT7B (right panel) (scale bar 100 μm). f Statistics of e ( n = 3 per group). g β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting WNT7B (A549 tet-shWNT7B) in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). h β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by Wnt-7b expression ( n = 3 per group). i Impact of shRNA targeting CTNNB1 mRNA on induction of senescence in A549 cells ( n = 3 per group). j β-Galactosidase staining of A549 cells treated with ICG-001 and XAV939 at indicated concentration ( n = 3 per group). k qRT-PCR analysis of MMP7 expression of in A549i cells in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). l β-Galactosidase staining of A549 cells knockdown with shRNA targeting MMP7 mRNA ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: GATA4 induces lung cancer cell senescence by downregulating WNT7B. a and b WNT7B expression level in A549 cells ectopically expressing GATA4 through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c TOP-FLASH analysis of GATA4 expressing A549 cells ( n = 6 per group). d Western blot analysis of c-Myc and cyclin D1 in A549i cells in the absence or presence of 2 μg/mL of Dox. e β-Galactosidase staining of A549 cells expressing WNT7B targeting shRNA (middle panel) and rescued by shRNA-resistant WNT7B (right panel) (scale bar 100 μm). f Statistics of e ( n = 3 per group). g β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting WNT7B (A549 tet-shWNT7B) in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). h β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by Wnt-7b expression ( n = 3 per group). i Impact of shRNA targeting CTNNB1 mRNA on induction of senescence in A549 cells ( n = 3 per group). j β-Galactosidase staining of A549 cells treated with ICG-001 and XAV939 at indicated concentration ( n = 3 per group). k qRT-PCR analysis of MMP7 expression of in A549i cells in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). l β-Galactosidase staining of A549 cells knockdown with shRNA targeting MMP7 mRNA ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: The total cell lysate was divided into two aliquots, one was mixed with 40 μL of 1 mg/mL GATA4 antibody (6H10, Thermo Fisher Scientific, Waltham, MA, USA) and 40 μL Protein A/G Agarose beads (20423, Pierce, Thermo Scientific), and the other was mixed with 40 μL 1 mg/mL normal mouse IgG (NI03, Sigma) and 40 μL Agarose beads.

    Techniques: Expressing, Infection, Western Blot, Staining, shRNA, Concentration Assay, Quantitative RT-PCR

    Defect of spermatogenesis in Sohlh2 KO testes during early postnatal development. ( A ) A representative image of RT-PCR analysis showing the deficiency of Sohlh2 expression in 2-week-old Sohlh2 KO mice. Total RNA was isolated from WT and Sohlh2 KO testes. RT-PCR was performed using primers for Sohlh1 , Sohlh2 , and Gapdh mRNA. ( B ) Immunohistochemical analysis demonstrating a defect of spermatogenesis in Sohlh2 KO testes during early postnatal development. Sections of WT and Sohlh2 KO testes were stained with hematoxylin or immunostained with antibodies. Anti-SOHLH1, anti-SYCP3, and anti-GATA4 antibodies were used to stain spermatogonia, spermatocyte, and sertoli cell, respectively. Testes were prepared from 2-week-old WT and Sohlh2 KO mice. Scale bars represent 25 μm. ( C ) SOHLH2-, SYCP3-, and GATA4-positive cell numbers in 2-week-old Sohlh2 KO versus WT mice. The number of positively stained cells per tubule was determined in the cross section of WT and Sohlh2 KO testes. Each group contained three mice. A minimum of 45 tubules per testis were counted for the cell number analysis. Data are presented as the mean ± SD. * p

    Journal: Scientific Reports

    Article Title: SOHLH2 is essential for synaptonemal complex formation during spermatogenesis in early postnatal mouse testes

    doi: 10.1038/srep20980

    Figure Lengend Snippet: Defect of spermatogenesis in Sohlh2 KO testes during early postnatal development. ( A ) A representative image of RT-PCR analysis showing the deficiency of Sohlh2 expression in 2-week-old Sohlh2 KO mice. Total RNA was isolated from WT and Sohlh2 KO testes. RT-PCR was performed using primers for Sohlh1 , Sohlh2 , and Gapdh mRNA. ( B ) Immunohistochemical analysis demonstrating a defect of spermatogenesis in Sohlh2 KO testes during early postnatal development. Sections of WT and Sohlh2 KO testes were stained with hematoxylin or immunostained with antibodies. Anti-SOHLH1, anti-SYCP3, and anti-GATA4 antibodies were used to stain spermatogonia, spermatocyte, and sertoli cell, respectively. Testes were prepared from 2-week-old WT and Sohlh2 KO mice. Scale bars represent 25 μm. ( C ) SOHLH2-, SYCP3-, and GATA4-positive cell numbers in 2-week-old Sohlh2 KO versus WT mice. The number of positively stained cells per tubule was determined in the cross section of WT and Sohlh2 KO testes. Each group contained three mice. A minimum of 45 tubules per testis were counted for the cell number analysis. Data are presented as the mean ± SD. * p

    Article Snippet: The primary antibodies were anti-GATA4 (1:500; Abcam), anti-HORMAD1 (1:500) , anti-SYCP1 (1:750; Abcam), anti-SYCP3 (1:750; Santa Cruz), anti-RAD51, and anti-γH2AX (1:500; Cell Signaling).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Isolation, Immunohistochemistry, Staining

    SIRT6 antagonizes DOX-induced apoptotic pathway in cardiomyocytes via GATA4. ( A ) Quantitative PCR (qPCR) showing the mRNA levels of Bcl-2 , Bcl-xL , Gata4 and Sirt6 in primary mouse cardiomyocytes after the treatment of DOX (1 μM) for 6 h. *** P

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: SIRT6 antagonizes DOX-induced apoptotic pathway in cardiomyocytes via GATA4. ( A ) Quantitative PCR (qPCR) showing the mRNA levels of Bcl-2 , Bcl-xL , Gata4 and Sirt6 in primary mouse cardiomyocytes after the treatment of DOX (1 μM) for 6 h. *** P

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Real-time Polymerase Chain Reaction

    SIRT6 recruits TIP60 for GATA4 acetylation at Lysine 328/330. ( A ) Immunoblots of anti-HA immunoprecipitates showing the acetylation level of HA-GATA4 in HEK293 cells overexpressing ectopic HA-GATA4 treated with indicated histone acetyltransferase (HAT) inhibitors, i.e. Cpth2 (20 μM), Mg149 (20 μM) and C646 (10 μM), for 6 h. Lower, fold change of the acetylation level of HA-GATA4 relative to HA-GATA4 only control, determined by ImageJ. n = 3. *** P

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: SIRT6 recruits TIP60 for GATA4 acetylation at Lysine 328/330. ( A ) Immunoblots of anti-HA immunoprecipitates showing the acetylation level of HA-GATA4 in HEK293 cells overexpressing ectopic HA-GATA4 treated with indicated histone acetyltransferase (HAT) inhibitors, i.e. Cpth2 (20 μM), Mg149 (20 μM) and C646 (10 μM), for 6 h. Lower, fold change of the acetylation level of HA-GATA4 relative to HA-GATA4 only control, determined by ImageJ. n = 3. *** P

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Western Blot, HAT Assay

    Hypoacetylation of GATA4 diminishes cardio-protection against DOX. ( A ) Western blotting analysis of the levels of FLAG-SIRT6 protein and HA-GATA4 acetylation in the precipitated pool of HA-GATA4 with DOX treatment at 0, 1, 2 μM for 6 h (left). Percent level of acetylated GATA4 relative to vehicle/no FLAG-SIRT6 control (middle) and percent FLAG-SIRT6 bound to HA-GATA4 relative to vehicle control (right). Quantification was performed by ImageJ. n = 3. * P

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: Hypoacetylation of GATA4 diminishes cardio-protection against DOX. ( A ) Western blotting analysis of the levels of FLAG-SIRT6 protein and HA-GATA4 acetylation in the precipitated pool of HA-GATA4 with DOX treatment at 0, 1, 2 μM for 6 h (left). Percent level of acetylated GATA4 relative to vehicle/no FLAG-SIRT6 control (middle) and percent FLAG-SIRT6 bound to HA-GATA4 relative to vehicle control (right). Quantification was performed by ImageJ. n = 3. * P

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Western Blot

    SIRT6 enhances GATA4 acetylation and protects against DOX-induced myocyte apoptosis independently of its deacylase activity. ( A ) Western blotting showing the acetylation level of GATA4 immunoprecipitated from HEK293 cells overexpressing of FLAG-SIRT6 and HA-GATA4. Acetylation level was determined with anti-acetyl lysine (AcK) antibodies. Lower, fold change of the acetylation level of GATA4 relative to HA-GATA4 only control, determined by ImageJ. n = 6. *** P

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: SIRT6 enhances GATA4 acetylation and protects against DOX-induced myocyte apoptosis independently of its deacylase activity. ( A ) Western blotting showing the acetylation level of GATA4 immunoprecipitated from HEK293 cells overexpressing of FLAG-SIRT6 and HA-GATA4. Acetylation level was determined with anti-acetyl lysine (AcK) antibodies. Lower, fold change of the acetylation level of GATA4 relative to HA-GATA4 only control, determined by ImageJ. n = 6. *** P

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Activity Assay, Western Blot, Immunoprecipitation

    SIRT6 physically interacts with GATA4. ( A ) Co-immunoprecipitation using anti-HA agarose was performed in HEK293 cells transfected with indicated constructs. The level of FLAG-GATA4 was detected by western blotting in purified pool of HA-SIRT6. ( B ) Co-immunoprecipitation using anti-FLAG agarose was performed in HEK293 cells transfected with indicated constructs. The level of HA-SIRT6 was detected by western blotting in purified pool of FLAG-GATA4. ( C, D ) Endogenous interaction of GATA4 and SIRT6 in H9C2 cells was evaluated by co-immunoprecipitation (Co-IP) and immunostaining with anti-GATA4 and anti-SIRT6 antibodies. Scale bar, 50 μm. ( E ) GST pull-down assay was performed to test the in vitro binding of purified His-GATA4 and GST-fused SIRT6 from E. coli . Coomassie blue staining indicates the loading levels of GST and GST-SIRT6. ( F ) Diagram of full-length (F-L) and fragmented human GATA4 with one or two zinc finger DNA-binding domains. ( G ) Domain-based truncations of HA-GATA4 were co-expressed with FLAG-SIRT6 in HEK293 cells. HA-GATA4 protein was precipitated and FLAG-SIRT6 was analyzed by western blotting.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: SIRT6 physically interacts with GATA4. ( A ) Co-immunoprecipitation using anti-HA agarose was performed in HEK293 cells transfected with indicated constructs. The level of FLAG-GATA4 was detected by western blotting in purified pool of HA-SIRT6. ( B ) Co-immunoprecipitation using anti-FLAG agarose was performed in HEK293 cells transfected with indicated constructs. The level of HA-SIRT6 was detected by western blotting in purified pool of FLAG-GATA4. ( C, D ) Endogenous interaction of GATA4 and SIRT6 in H9C2 cells was evaluated by co-immunoprecipitation (Co-IP) and immunostaining with anti-GATA4 and anti-SIRT6 antibodies. Scale bar, 50 μm. ( E ) GST pull-down assay was performed to test the in vitro binding of purified His-GATA4 and GST-fused SIRT6 from E. coli . Coomassie blue staining indicates the loading levels of GST and GST-SIRT6. ( F ) Diagram of full-length (F-L) and fragmented human GATA4 with one or two zinc finger DNA-binding domains. ( G ) Domain-based truncations of HA-GATA4 were co-expressed with FLAG-SIRT6 in HEK293 cells. HA-GATA4 protein was precipitated and FLAG-SIRT6 was analyzed by western blotting.

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Immunoprecipitation, Transfection, Construct, Western Blot, Purification, Co-Immunoprecipitation Assay, Immunostaining, Pull Down Assay, In Vitro, Binding Assay, Staining

    A schematic model: a SIRT6–TIP60–GATA4 axis couples gene transcription and epigenetic activation. SIRT6 initially occupies the promoter region via a nucleosome-binding property and recruits TIP60 to balance dynamic acetylation of local histones. When GATA4 comes by recognizing the GATA sequence and simultaneously interacts and blocks SIRT6 deacetylase activity, TIP60 acetylates GATA4 to enhance its transcription activity. The inhibition of SIRT6 disrupts local histone acetylation balance, thus ensuring an open chromatin structure for transcription.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: A schematic model: a SIRT6–TIP60–GATA4 axis couples gene transcription and epigenetic activation. SIRT6 initially occupies the promoter region via a nucleosome-binding property and recruits TIP60 to balance dynamic acetylation of local histones. When GATA4 comes by recognizing the GATA sequence and simultaneously interacts and blocks SIRT6 deacetylase activity, TIP60 acetylates GATA4 to enhance its transcription activity. The inhibition of SIRT6 disrupts local histone acetylation balance, thus ensuring an open chromatin structure for transcription.

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Activation Assay, Binding Assay, Sequencing, Histone Deacetylase Assay, Activity Assay, Inhibition

    The SIRT6–TIP60–GATA4 axis facilitates chromatin accessibility. ( A, B ) ChIP assay was performed using anti-TIP60 or anti-H3K9ac antibody in WT and SIRT6 KO HEK293 cells with or without overexpression of FLAG-TIP60. The enrichment of TIP60 and H3K9ac on Bcl-2 promoter was analyzed by qPCR. ** P

    Journal: Nucleic Acids Research

    Article Title: Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis

    doi: 10.1093/nar/gkaa214

    Figure Lengend Snippet: The SIRT6–TIP60–GATA4 axis facilitates chromatin accessibility. ( A, B ) ChIP assay was performed using anti-TIP60 or anti-H3K9ac antibody in WT and SIRT6 KO HEK293 cells with or without overexpression of FLAG-TIP60. The enrichment of TIP60 and H3K9ac on Bcl-2 promoter was analyzed by qPCR. ** P

    Article Snippet: Anti-GATA4 (ab84593), anti-SIRT6 (ab62739) and anti-H3 (ab1791) antibodies were purchased from Abcam (UK).

    Techniques: Chromatin Immunoprecipitation, Over Expression, Real-time Polymerase Chain Reaction

    GATA4 accumulates during mouse aging, human aging, and mouse IR-induced senescence

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

    Article Title: The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

    doi: 10.1126/science.aaa5612

    Figure Lengend Snippet: GATA4 accumulates during mouse aging, human aging, and mouse IR-induced senescence

    Article Snippet: The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R & D systems); p16 (rabbit, Abcam); p16 (mouse, Thermo Scientific); p16 (rabbit, Bethyl); GFAP (goat, Abcam).

    Techniques:

    GATA4 regulates cellular senescence

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

    Article Title: The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

    doi: 10.1126/science.aaa5612

    Figure Lengend Snippet: GATA4 regulates cellular senescence

    Article Snippet: The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R & D systems); p16 (rabbit, Abcam); p16 (mouse, Thermo Scientific); p16 (rabbit, Bethyl); GFAP (goat, Abcam).

    Techniques:

    Selective autophagy degrades GATA4 in a p62-dependent manner to prevent senescence

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

    Article Title: The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

    doi: 10.1126/science.aaa5612

    Figure Lengend Snippet: Selective autophagy degrades GATA4 in a p62-dependent manner to prevent senescence

    Article Snippet: The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R & D systems); p16 (rabbit, Abcam); p16 (mouse, Thermo Scientific); p16 (rabbit, Bethyl); GFAP (goat, Abcam).

    Techniques:

    Gene expression profiling reveals that GATA4 controls the SASP

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

    Article Title: The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

    doi: 10.1126/science.aaa5612

    Figure Lengend Snippet: Gene expression profiling reveals that GATA4 controls the SASP

    Article Snippet: The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R & D systems); p16 (rabbit, Abcam); p16 (mouse, Thermo Scientific); p16 (rabbit, Bethyl); GFAP (goat, Abcam).

    Techniques: Expressing

    GATA4 regulates NF-κB

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

    Article Title: The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

    doi: 10.1126/science.aaa5612

    Figure Lengend Snippet: GATA4 regulates NF-κB

    Article Snippet: The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R & D systems); p16 (rabbit, Abcam); p16 (mouse, Thermo Scientific); p16 (rabbit, Bethyl); GFAP (goat, Abcam).

    Techniques:

    The GATA4 pathway functions independently of the p53 and p16 pathways and is regulated by the DDR kinases ATM and ATR

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

    Article Title: The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

    doi: 10.1126/science.aaa5612

    Figure Lengend Snippet: The GATA4 pathway functions independently of the p53 and p16 pathways and is regulated by the DDR kinases ATM and ATR

    Article Snippet: The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R & D systems); p16 (rabbit, Abcam); p16 (mouse, Thermo Scientific); p16 (rabbit, Bethyl); GFAP (goat, Abcam).

    Techniques:

    Tsc22d3-2 knockout males are sterile. A, Representative pictures from 2- and 6-month-old control ( top ) and knockout ( bottom ) adult testes; B, H E-stained paraffin sections; C, immunohistochemistry of Sertoli and Leydig cells counterstained with H E-stained paraffin sections from control and knockout testes. In B and C, note absence of mature spermatozoa ( white arrowhead ) and apparent hyperproliferation of Leydig cells ( black arrow ) in the knockouts. Panel C is a higher-magnification image (rabbit anti-GATA4 antibody) with counterstaining with hematoxylin (blue nuclei); elongated spermatids ( white arrowhead ) are seen in controls. D, Corticosterone (cort), FSH, LH, and testosterone (testo) hormone levels in plasma (n ≥ 10 animals per group, 3–4 months old). E, Total number of cells per seminiferous tubule (n ≥ 20 tubules per group; left ), ratio of Ki-67-positive cells divided by total cell number, middle ), and ratio of TUNEL-positive cells divided by total cell number per seminiferous tubule, n = 18 tubules, right ). F, Quantification of Sertoli cells per tubule (F) (n ≥ 25 tubules) and Leydig cells in the intertubular space (G) (n ≥ 27) at 2 months (n = 2) and 6 months (n = 4 animals). H, qRT-PCR of Fshr , Gata4 , and Mvh control ( black bars ) and knockout ( white bars ) animals. I, qRT-PCR of Ppar γ 2 , Tsc22d1 , Gr , Ar , Klf5 , and Klf15 in testis from control ( black bars ) and knockout ( white bars ) animals. Scale bar , 1 mm (A), 100 μm (B), and 50 μm (C). *, P

    Journal: Molecular Endocrinology

    Article Title: The Glucocorticoid-Induced Leucine Zipper (Gilz/Tsc22d3-2) Gene Locus Plays a Crucial Role in Male Fertility

    doi: 10.1210/me.2011-1249

    Figure Lengend Snippet: Tsc22d3-2 knockout males are sterile. A, Representative pictures from 2- and 6-month-old control ( top ) and knockout ( bottom ) adult testes; B, H E-stained paraffin sections; C, immunohistochemistry of Sertoli and Leydig cells counterstained with H E-stained paraffin sections from control and knockout testes. In B and C, note absence of mature spermatozoa ( white arrowhead ) and apparent hyperproliferation of Leydig cells ( black arrow ) in the knockouts. Panel C is a higher-magnification image (rabbit anti-GATA4 antibody) with counterstaining with hematoxylin (blue nuclei); elongated spermatids ( white arrowhead ) are seen in controls. D, Corticosterone (cort), FSH, LH, and testosterone (testo) hormone levels in plasma (n ≥ 10 animals per group, 3–4 months old). E, Total number of cells per seminiferous tubule (n ≥ 20 tubules per group; left ), ratio of Ki-67-positive cells divided by total cell number, middle ), and ratio of TUNEL-positive cells divided by total cell number per seminiferous tubule, n = 18 tubules, right ). F, Quantification of Sertoli cells per tubule (F) (n ≥ 25 tubules) and Leydig cells in the intertubular space (G) (n ≥ 27) at 2 months (n = 2) and 6 months (n = 4 animals). H, qRT-PCR of Fshr , Gata4 , and Mvh control ( black bars ) and knockout ( white bars ) animals. I, qRT-PCR of Ppar γ 2 , Tsc22d1 , Gr , Ar , Klf5 , and Klf15 in testis from control ( black bars ) and knockout ( white bars ) animals. Scale bar , 1 mm (A), 100 μm (B), and 50 μm (C). *, P

    Article Snippet: Sertoli and Leydig cells were labeled with the rabbit anti-GATA4 (GATA-binding protein 4) (ab84593; Abcam plc, Cambridge, UK; dilution 1:140) with EnVision (DakoCytomation, Glostrup, Denmark) used as secondary antibody ( ).

    Techniques: Knock-Out, Staining, Immunohistochemistry, TUNEL Assay, Quantitative RT-PCR

    Cardiomyogenic competency of BM-derived cells. BM cells were cultured in the absence or presence of 8 μM BIX01294, 50 ng/mL Wnt3a, or 50 nM TSA for 48 h before the addition of fresh media by itself or supplemented with Wnt11. Seven days later, cultures were harvested for RNA, and assayed by real-time qPCR. (A, B) PCR analyses of Nkx2.5 and GATA4 expression, respectively. Expression of these cardiac genes by BM cells was minimal in nontreated and Wnt3a or TSA-treated cultures, regardless of whether the cultures were subsequently exposed to the cardiogenic stimulating factor Wnt11. In contrast, BIX01294 pretreatment did yield small increases of expression over basal levels. However, when BM cells were pretreated with BIX01294, subsequent exposure to Wnt11 enhanced expression of Nkx2.5 and GATA4 by > 132-fold and 82-fold, respectively. BIX, BIX01294; W3a, Wnt3a * P

    Journal: Stem Cells and Development

    Article Title: The Histone Methyltransferase Inhibitor BIX01294 Enhances the Cardiac Potential of Bone Marrow Cells

    doi: 10.1089/scd.2012.0181

    Figure Lengend Snippet: Cardiomyogenic competency of BM-derived cells. BM cells were cultured in the absence or presence of 8 μM BIX01294, 50 ng/mL Wnt3a, or 50 nM TSA for 48 h before the addition of fresh media by itself or supplemented with Wnt11. Seven days later, cultures were harvested for RNA, and assayed by real-time qPCR. (A, B) PCR analyses of Nkx2.5 and GATA4 expression, respectively. Expression of these cardiac genes by BM cells was minimal in nontreated and Wnt3a or TSA-treated cultures, regardless of whether the cultures were subsequently exposed to the cardiogenic stimulating factor Wnt11. In contrast, BIX01294 pretreatment did yield small increases of expression over basal levels. However, when BM cells were pretreated with BIX01294, subsequent exposure to Wnt11 enhanced expression of Nkx2.5 and GATA4 by > 132-fold and 82-fold, respectively. BIX, BIX01294; W3a, Wnt3a * P

    Article Snippet: The next day, the rabbit anti-GATA4 antibody (ab61170; Abcam) was diluted with a 1:1,000 blocking solution and applied to the slides overnight at 4°C.

    Techniques: Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing

    Immunofluorescent analysis of cardiac differentiation of BM-derived cells. Cardiac proteins were identified using protein-specific antibodies labeled with DyLight 488 ( green ). In addition, cells were counterstained with 4′,6-diamidino-2-phenyindole (DAPI, blue ) and rhodamine-coupled wheat germ agglutinin ( red ), respectively, to identify nuclei and visualize individual cells. BM cells were exposed to BIX01294 for 48 h before their culture for 7 days in the (A) absence or (B) presence of Wnt11, and subsequently immunostained for GATA4. In the absence of Wnt11, very few cells were observed that exhibited positive staining for GATA4 ( arrow ). In contrast, Wnt11 provoked greater numbers of BIX01294-pretreated cells to exhibit GATA4 reactivity, as indicated by the green , yellow fluorescent dots within individual nuclei ( arrows ). BM cultures immunostained against titin after being cultured in the (C) absence or (D, E) presence of BIX01294, and then subsequently exposed to Wnt11 for 7 days. Both (F) sarcomeric α-actin and (H) desmin displayed positive immunofluorescent staining following the 2-step BIX01294/Wnt11 treatment. Sarcomeric α-actinin immunostained BM cells cultured with (H) Wnt11 only or (I) Wnt11 following pretreatment with BIX01294. Arrows in (D, F, I) identify individual cells that exhibit the myofibrillar proteins in a preorganized pattern within the cytoplasm. For (A, B, H, I) , both the BIX01294-pretreatment and subsequent 7-day culture were carried out in the same dish, before cytospinning trypsinized cells onto glass slides for immunostaining. For (C–G) , cells were harvested following the 48-h pretreatment and replated into chamber slides for subsequent Wnt11 culture and immunostaining. Scale bars=20 μm.

    Journal: Stem Cells and Development

    Article Title: The Histone Methyltransferase Inhibitor BIX01294 Enhances the Cardiac Potential of Bone Marrow Cells

    doi: 10.1089/scd.2012.0181

    Figure Lengend Snippet: Immunofluorescent analysis of cardiac differentiation of BM-derived cells. Cardiac proteins were identified using protein-specific antibodies labeled with DyLight 488 ( green ). In addition, cells were counterstained with 4′,6-diamidino-2-phenyindole (DAPI, blue ) and rhodamine-coupled wheat germ agglutinin ( red ), respectively, to identify nuclei and visualize individual cells. BM cells were exposed to BIX01294 for 48 h before their culture for 7 days in the (A) absence or (B) presence of Wnt11, and subsequently immunostained for GATA4. In the absence of Wnt11, very few cells were observed that exhibited positive staining for GATA4 ( arrow ). In contrast, Wnt11 provoked greater numbers of BIX01294-pretreated cells to exhibit GATA4 reactivity, as indicated by the green , yellow fluorescent dots within individual nuclei ( arrows ). BM cultures immunostained against titin after being cultured in the (C) absence or (D, E) presence of BIX01294, and then subsequently exposed to Wnt11 for 7 days. Both (F) sarcomeric α-actin and (H) desmin displayed positive immunofluorescent staining following the 2-step BIX01294/Wnt11 treatment. Sarcomeric α-actinin immunostained BM cells cultured with (H) Wnt11 only or (I) Wnt11 following pretreatment with BIX01294. Arrows in (D, F, I) identify individual cells that exhibit the myofibrillar proteins in a preorganized pattern within the cytoplasm. For (A, B, H, I) , both the BIX01294-pretreatment and subsequent 7-day culture were carried out in the same dish, before cytospinning trypsinized cells onto glass slides for immunostaining. For (C–G) , cells were harvested following the 48-h pretreatment and replated into chamber slides for subsequent Wnt11 culture and immunostaining. Scale bars=20 μm.

    Article Snippet: The next day, the rabbit anti-GATA4 antibody (ab61170; Abcam) was diluted with a 1:1,000 blocking solution and applied to the slides overnight at 4°C.

    Techniques: Derivative Assay, Labeling, Staining, Cell Culture, Immunostaining

    ICM organoids seeded with 50 cells show mutual exclusive expression and sorting. Mouse tet::GATA6 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. ICM organoids were formed with 50 cells and kept undisturbed for 24 ( A ) and 48 h ( B ). ICM organoids show the two first phases: mutual exclusive expression and sorting of GATA6-positive cells. GATA4 and SOX17 expression was present at both stages. Microscope: Zeiss LSM880; objective: 40×/1.3 oil differential interference contrast; scale bars, 20 μ m. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: ICM organoids seeded with 50 cells show mutual exclusive expression and sorting. Mouse tet::GATA6 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. ICM organoids were formed with 50 cells and kept undisturbed for 24 ( A ) and 48 h ( B ). ICM organoids show the two first phases: mutual exclusive expression and sorting of GATA6-positive cells. GATA4 and SOX17 expression was present at both stages. Microscope: Zeiss LSM880; objective: 40×/1.3 oil differential interference contrast; scale bars, 20 μ m. To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Expressing, Microscopy

    ICM organoids show secretion of basement membrane component laminin. Mouse tet::GATA4 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. Cells that were not stimulated with dox (− dox) served as controls. ( A ) Heterogeneous laminin distribution in 1-day-old ICM organoids at stage of mutual exclusive expression. ( B ) After 48 h of ICM organoid formation, secretion of laminin is restricted to the outer layer. ( C ) After 72 h, at the stage of NANOG downregulation, laminin layers of different thicknesses were detected between the PrE layer and inner Epi core ( red arrows and red boxes , second and third rows ). Different structures could be observed and indicate differentiation toward visceral endoderm (VE) and parietal endoderm (PE): aligned columnar cell structure ( green box , first row ), aligned cuboidal cell morphology ( white arrows , third row , first column ), and vacuoles ( yellow arrows and box , third row ). Characteristics for VE were columnar- or cuboidal-shaped cells and low laminin expression; for PE, they were smaller-sized cells and loosely connected to the Epi core and high laminin expression. For more details, please also see text. Images show a single slice from the ICM organoids’ center. Microscope: Zeiss LSM780; objective: 63×/1.40 oil; scale bars, 20 μ m. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: ICM organoids show secretion of basement membrane component laminin. Mouse tet::GATA4 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. Cells that were not stimulated with dox (− dox) served as controls. ( A ) Heterogeneous laminin distribution in 1-day-old ICM organoids at stage of mutual exclusive expression. ( B ) After 48 h of ICM organoid formation, secretion of laminin is restricted to the outer layer. ( C ) After 72 h, at the stage of NANOG downregulation, laminin layers of different thicknesses were detected between the PrE layer and inner Epi core ( red arrows and red boxes , second and third rows ). Different structures could be observed and indicate differentiation toward visceral endoderm (VE) and parietal endoderm (PE): aligned columnar cell structure ( green box , first row ), aligned cuboidal cell morphology ( white arrows , third row , first column ), and vacuoles ( yellow arrows and box , third row ). Characteristics for VE were columnar- or cuboidal-shaped cells and low laminin expression; for PE, they were smaller-sized cells and loosely connected to the Epi core and high laminin expression. For more details, please also see text. Images show a single slice from the ICM organoids’ center. Microscope: Zeiss LSM780; objective: 63×/1.40 oil; scale bars, 20 μ m. To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Expressing, Microscopy

    ICM organoids mimic the mouse ICM. Mouse tet::GATA4 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. Cells that were not stimulated with dox (− dox) served as control. ( A ) After removal of dox, GATA6 and NANOG were coexpressed in cells of the two-dimensional monolayer of mESCs (see box for a magnified section of a coexpressing cell). ( B ) 24 h after ICM organoid formation, GATA6 and NANOG were mutually exclusively expressed within the ICM organoids. ( C ) After 48 h, GATA6-positive cells arranged at the rim of the ICM organoids. ( D ) After 72 h, NANOG was downregulated. Images show a single slice from the aggregate’s center. Microscope: Zeiss LSM780; objective: 63×/1.40 oil; scale bars, 20 μ m. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: ICM organoids mimic the mouse ICM. Mouse tet::GATA4 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. Cells that were not stimulated with dox (− dox) served as control. ( A ) After removal of dox, GATA6 and NANOG were coexpressed in cells of the two-dimensional monolayer of mESCs (see box for a magnified section of a coexpressing cell). ( B ) 24 h after ICM organoid formation, GATA6 and NANOG were mutually exclusively expressed within the ICM organoids. ( C ) After 48 h, GATA6-positive cells arranged at the rim of the ICM organoids. ( D ) After 72 h, NANOG was downregulated. Images show a single slice from the aggregate’s center. Microscope: Zeiss LSM780; objective: 63×/1.40 oil; scale bars, 20 μ m. To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Microscopy

    Lineage composition and spatial distribution of GATA6 and NANOG in ICM organoids resemble those of mid- and late mouse blastocysts. ( A ) Three-dimensional imaging and three-dimensional image analysis form the basis for a quantitative comparison between ICM organoids of mouse tet::GATA4 ES cells and blastocysts. Quantitative data of early, mid-, and late blastocysts were taken from Saiz et al. ( 22 ) ( B ) Fluorescence intensity levels of GATA6 and NANOG of individual cells in ICM organoids after 24 and 48 h. The data points are colored by cell population. ( C and D ) Lineage composition is shown as percentage of the total number of cells in ICM organoids after 24 and 48 h and in the ICM of early, mid-, and late blastocysts. ( E and F ) Lineage composition of neighboring cells is shown as percentage of the total of neighboring cells in ICM organoids after 24 and 48 h and the ICM of mid- and late blastocysts. The error bars indicate the standard error of the mean. The number of independent experiments for ICM organoids or blastocysts is, respectively, 76, 147. DN: double negative (NANOG−/GATA6−), DP: double positive (NANOG+/GATA6+), N+/G− (NANOG+/GATA6−), N−/G+ (NANOG−/GATA6+). To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: Lineage composition and spatial distribution of GATA6 and NANOG in ICM organoids resemble those of mid- and late mouse blastocysts. ( A ) Three-dimensional imaging and three-dimensional image analysis form the basis for a quantitative comparison between ICM organoids of mouse tet::GATA4 ES cells and blastocysts. Quantitative data of early, mid-, and late blastocysts were taken from Saiz et al. ( 22 ) ( B ) Fluorescence intensity levels of GATA6 and NANOG of individual cells in ICM organoids after 24 and 48 h. The data points are colored by cell population. ( C and D ) Lineage composition is shown as percentage of the total number of cells in ICM organoids after 24 and 48 h and in the ICM of early, mid-, and late blastocysts. ( E and F ) Lineage composition of neighboring cells is shown as percentage of the total of neighboring cells in ICM organoids after 24 and 48 h and the ICM of mid- and late blastocysts. The error bars indicate the standard error of the mean. The number of independent experiments for ICM organoids or blastocysts is, respectively, 76, 147. DN: double negative (NANOG−/GATA6−), DP: double positive (NANOG+/GATA6+), N+/G− (NANOG+/GATA6−), N−/G+ (NANOG−/GATA6+). To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Imaging, Fluorescence

    Mouse ICM organoids express the PrE markers GATA4 and SOX17. Mouse tet::GATA6 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. After the removal of dox, 200 cells formed ICM organoids and were cultured for 24 h or 48 h. ( A ) GATA4 and SOX17 expression was found on the edge of the ICM organoids 24 h after formation. NANOG expression was found within the ICM organoids. ( B ) GATA4 and SOX17 expression was maintained 48 h after ICM organoid formation. Microscope: Zeiss LSM880; objective: 40×/1.3 oil differential interference contrast; scale bars, 20 μ m. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: Mouse ICM organoids express the PrE markers GATA4 and SOX17. Mouse tet::GATA6 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. After the removal of dox, 200 cells formed ICM organoids and were cultured for 24 h or 48 h. ( A ) GATA4 and SOX17 expression was found on the edge of the ICM organoids 24 h after formation. NANOG expression was found within the ICM organoids. ( B ) GATA4 and SOX17 expression was maintained 48 h after ICM organoid formation. Microscope: Zeiss LSM880; objective: 40×/1.3 oil differential interference contrast; scale bars, 20 μ m. To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Cell Culture, Expressing, Microscopy

    Generation and imaging pipeline for aggregates of mESCs (ICM organoids). Mouse tet::GATA4 ESCs were pre-cultured for 3 days in medium containing serum and LIF (S + L) and PD0325901 (PD03). At day 3, cells were stimulated with dox for 6 h to induce PrE differentiation. After dox removal, 200 cells were seeded in microwell plates coated with 1% low melt agarose. Aggregates were formed in medium containing S + L and were kept undisturbed. After 24, 48, and 72 h, aggregates were collected, stained, mounted, and imaged. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: Generation and imaging pipeline for aggregates of mESCs (ICM organoids). Mouse tet::GATA4 ESCs were pre-cultured for 3 days in medium containing serum and LIF (S + L) and PD0325901 (PD03). At day 3, cells were stimulated with dox for 6 h to induce PrE differentiation. After dox removal, 200 cells were seeded in microwell plates coated with 1% low melt agarose. Aggregates were formed in medium containing S + L and were kept undisturbed. After 24, 48, and 72 h, aggregates were collected, stained, mounted, and imaged. To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Imaging, Cell Culture, Staining

    ICM organoids show characteristics of epithelisation. Mouse tet::GATA4 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. Cells that were not stimulated with dox (− dox) served as controls. Aggregates were formed and kept undisturbed for 72 h. ICM organoids show punctate patterns of ZO-1 at the outer cell layer (see zoomed regions in boxes and arrowheads ). Control aggregates show continuous ZO-1 staining at the junctions (see zoomed regions in boxes and arrowheads ). Images show single slices from the ICM organoid’s center. Microscope: Zeiss LSM780; objective: 63×/1.40 oil; scale bars, 20 μ m. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering

    doi: 10.1016/j.bpj.2018.11.011

    Figure Lengend Snippet: ICM organoids show characteristics of epithelisation. Mouse tet::GATA4 ESCs were stimulated for 6 h with doxycycline (+ dox) to induce PrE differentiation. Cells that were not stimulated with dox (− dox) served as controls. Aggregates were formed and kept undisturbed for 72 h. ICM organoids show punctate patterns of ZO-1 at the outer cell layer (see zoomed regions in boxes and arrowheads ). Control aggregates show continuous ZO-1 staining at the junctions (see zoomed regions in boxes and arrowheads ). Images show single slices from the ICM organoid’s center. Microscope: Zeiss LSM780; objective: 63×/1.40 oil; scale bars, 20 μ m. To see this figure in color, go online.

    Article Snippet: The primary antibodies were anti-GATA4 (1:200; Santa Cruz, Dallas, TX) and anti-SOX17 (1:200; R & D systems).

    Techniques: Staining, Microscopy

    Expression profile of HIF2α during cardiac differentiation. (A) Immunostaining for cardiomyocyte markers Gata4, Myosin and Troponin T in mouse ESC-derived EBs at day 10. Scale bar, 100 μm. (B) qRT-PCR analysis of Oct4 , Nanog and HIF2α in mouse ESCs and different days of ESCs-derived EBs. Data represent the mean ± s.d. of three biological replicates. *p

    Journal: Journal of Translational Medicine

    Article Title: HIF2α induces cardiomyogenesis via Wnt/β-catenin signaling in mouse embryonic stem cells

    doi: 10.1186/s12967-015-0447-7

    Figure Lengend Snippet: Expression profile of HIF2α during cardiac differentiation. (A) Immunostaining for cardiomyocyte markers Gata4, Myosin and Troponin T in mouse ESC-derived EBs at day 10. Scale bar, 100 μm. (B) qRT-PCR analysis of Oct4 , Nanog and HIF2α in mouse ESCs and different days of ESCs-derived EBs. Data represent the mean ± s.d. of three biological replicates. *p

    Article Snippet: The primary antibodies were Gata4 (G4; Santa Cruz, 1:100), Myosin (MF-20; DSHB, 1:50) and Troponin T (1:1000, Abcam).

    Techniques: Expressing, Immunostaining, Derivative Assay, Quantitative RT-PCR

    Dedifferentiation of glioblastoma multiforme (GBM) cell lines into induced glioma stem cells (iGSCs). (A) Shown are microscopic images of GBM cells (top), emerging colonies marked with asterisk (middle) and iGSCs (bottom). Scale bar: 100 μm. (B) Analysis of iGSC2 for pluripotency markers such as Oct4, Nanog and Sox2. Oct4 was detected with immunocytochemistry. DAPI was used for nuclear staining. Nanog and Sox2 expressions were compared using Western blot. Sox2 expression is 2.5-fold higher in iGSCs. Actin was used as control for Western blot. Scale bar: 100 μm. (C) Multilineage differentiation of iGSC2 detected with immunocytochemistry: Tuj-1 for ectoderm, GFAP for neuronal, GATA4 for endoderm and SMA for mesoderm. DAPI was used for nuclear staining. Scale bar: 100 μm.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Dedifferentiation of patient-derived glioblastoma multiforme cell lines results in a cancer stem cell-like state with mitogen-independent growth

    doi: 10.1111/jcmm.12479

    Figure Lengend Snippet: Dedifferentiation of glioblastoma multiforme (GBM) cell lines into induced glioma stem cells (iGSCs). (A) Shown are microscopic images of GBM cells (top), emerging colonies marked with asterisk (middle) and iGSCs (bottom). Scale bar: 100 μm. (B) Analysis of iGSC2 for pluripotency markers such as Oct4, Nanog and Sox2. Oct4 was detected with immunocytochemistry. DAPI was used for nuclear staining. Nanog and Sox2 expressions were compared using Western blot. Sox2 expression is 2.5-fold higher in iGSCs. Actin was used as control for Western blot. Scale bar: 100 μm. (C) Multilineage differentiation of iGSC2 detected with immunocytochemistry: Tuj-1 for ectoderm, GFAP for neuronal, GATA4 for endoderm and SMA for mesoderm. DAPI was used for nuclear staining. Scale bar: 100 μm.

    Article Snippet: The following primary antibodies were used: Oct-4, GFAP (1:200, Cell Signaling), ALDH1A1 (1:200), smooth muscle actin (1:50), GATA4 (1:200; Sigma-Aldrich), CD44 (1:400, Abcam) and β-III tubulin (1:200, Millipore).

    Techniques: Immunocytochemistry, Staining, Western Blot, Expressing

    Endodermal subpopulations emerging after activin A treatment using tfFACS. ( A ) A representative experiment using two-channel FACS analysis of GATA4 and SOX17. Compared with the isotype negative control (bottom panels), three distinct cellular populations: SOX17 + GATA4 − , SOX17 + GATA4 + , and SOX17 − GATA4 + are emerging gradually upon differentiation: at day 1, 13% are SOX17 + GATA4 + , increasing to 23% by day 3. Another significant population consists of 18% SOX17 − GATA4 + at day 1 and 25% at day 3. ( B ) After 5 days of differentiation, using three-way multichannel FACS analysis for SOX17, GATA4, and CXCR4, we found that the SOX17 + GATA4 + population dominates the culture (62%) and CXCR4 is expressed in 49% of the cells, most of which are SOX17 + GATA4 + CXCR4 + (41%). There are also approximately 27% GATA4 + CXCR4 − cells, which comprises the population of SOX17 + GATA4 + CXCR4 − cells (21%). ( C ) Post sorting, FACS analysis demonstrated that 97% of day 5 SOX17 + GATA4 + CXCR4 + cells were positive for GATA4, 88% were SOX17 positive, and 95% were CXCR4 positive. This was consistent over 5 separate experiments. ( D ) Expression analysis using RT-qPCR demonstrates that day 5 SOX17 + GATA4 + CXCR4 + and day3 SOX17 + GATA4 + cells have higher level of expression of SOX17, GATA4 and CXCR4 than unsorted fixed cells or day 3 SOX17 − GATA4 − (d3SOX17negGATA4neg) cells.

    Journal: PLoS ONE

    Article Title: A New FACS Approach Isolates hESC Derived Endoderm Using Transcription Factors

    doi: 10.1371/journal.pone.0017536

    Figure Lengend Snippet: Endodermal subpopulations emerging after activin A treatment using tfFACS. ( A ) A representative experiment using two-channel FACS analysis of GATA4 and SOX17. Compared with the isotype negative control (bottom panels), three distinct cellular populations: SOX17 + GATA4 − , SOX17 + GATA4 + , and SOX17 − GATA4 + are emerging gradually upon differentiation: at day 1, 13% are SOX17 + GATA4 + , increasing to 23% by day 3. Another significant population consists of 18% SOX17 − GATA4 + at day 1 and 25% at day 3. ( B ) After 5 days of differentiation, using three-way multichannel FACS analysis for SOX17, GATA4, and CXCR4, we found that the SOX17 + GATA4 + population dominates the culture (62%) and CXCR4 is expressed in 49% of the cells, most of which are SOX17 + GATA4 + CXCR4 + (41%). There are also approximately 27% GATA4 + CXCR4 − cells, which comprises the population of SOX17 + GATA4 + CXCR4 − cells (21%). ( C ) Post sorting, FACS analysis demonstrated that 97% of day 5 SOX17 + GATA4 + CXCR4 + cells were positive for GATA4, 88% were SOX17 positive, and 95% were CXCR4 positive. This was consistent over 5 separate experiments. ( D ) Expression analysis using RT-qPCR demonstrates that day 5 SOX17 + GATA4 + CXCR4 + and day3 SOX17 + GATA4 + cells have higher level of expression of SOX17, GATA4 and CXCR4 than unsorted fixed cells or day 3 SOX17 − GATA4 − (d3SOX17negGATA4neg) cells.

    Article Snippet: Goat anti-human SOX17 and GATA4 (both from R & D systems Inc.) were used, but the common serotype of these primary antibodies meant that secondary fluorescent antibodies would not distinguish between them.

    Techniques: FACS, Negative Control, Expressing, Quantitative RT-PCR

    GSEA analysis of the definitive endoderm (DE) gene sets for the day 5 SOX17 + GATA4 + CXCR4 + group. As shown in ( A–C ), the MGI gene set is highly enriched in the d5 SOX17 + GATA4 + CXCR4 + cells in multiple comparisons. d5SOX17 + GATA4 + CXCR4 + vs. hESC (unfixed hESCs+fixed hESCs): P

    Journal: PLoS ONE

    Article Title: A New FACS Approach Isolates hESC Derived Endoderm Using Transcription Factors

    doi: 10.1371/journal.pone.0017536

    Figure Lengend Snippet: GSEA analysis of the definitive endoderm (DE) gene sets for the day 5 SOX17 + GATA4 + CXCR4 + group. As shown in ( A–C ), the MGI gene set is highly enriched in the d5 SOX17 + GATA4 + CXCR4 + cells in multiple comparisons. d5SOX17 + GATA4 + CXCR4 + vs. hESC (unfixed hESCs+fixed hESCs): P

    Article Snippet: Goat anti-human SOX17 and GATA4 (both from R & D systems Inc.) were used, but the common serotype of these primary antibodies meant that secondary fluorescent antibodies would not distinguish between them.

    Techniques:

    Pretreatment with curcumin blocks GATA4 activation. ( A and B ) Curcumin blocked the acetylation and DNA-binding activity of GATA4 induced by PE infusion ( A ) or AB ( B ). n = 4. Oct-1 DNA-binding activity was used as a control. ( C ) Effect of p300 on the acetylation and DNA-binding activity of GATA4 induced by PE. Cells were infected with Ad-p300, Ad-DN-p300, or Ad-GFP for 24 hours and then treated with 100 μM PE for 24 hours. Extracts were assayed for GATA4 acetylation and DNA-binding activity. ( D ) p300 partially reversed the inhibitory effect of curcumin on the acetylation and DNA-binding activity of GATA4 induced by PE. Cells were infected with Ad-p300 or Ad-GFP for 24 hours, treated with 25 μM curcumin for 60 minutes, and then incubated with 100 μM PE for 24 hours. The results were reproducible in 3 separate experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: Curcumin prevents and reverses murine cardiac hypertrophy

    doi: 10.1172/JCI32865

    Figure Lengend Snippet: Pretreatment with curcumin blocks GATA4 activation. ( A and B ) Curcumin blocked the acetylation and DNA-binding activity of GATA4 induced by PE infusion ( A ) or AB ( B ). n = 4. Oct-1 DNA-binding activity was used as a control. ( C ) Effect of p300 on the acetylation and DNA-binding activity of GATA4 induced by PE. Cells were infected with Ad-p300, Ad-DN-p300, or Ad-GFP for 24 hours and then treated with 100 μM PE for 24 hours. Extracts were assayed for GATA4 acetylation and DNA-binding activity. ( D ) p300 partially reversed the inhibitory effect of curcumin on the acetylation and DNA-binding activity of GATA4 induced by PE. Cells were infected with Ad-p300 or Ad-GFP for 24 hours, treated with 25 μM curcumin for 60 minutes, and then incubated with 100 μM PE for 24 hours. The results were reproducible in 3 separate experiments.

    Article Snippet: The antibodies used to recognize histones H3 and H4 at their N-terminal lysine residues and GATA4 were purchased from Upstate Biotechnology.

    Techniques: Activation Assay, Binding Assay, Activity Assay, Infection, Incubation

    The variant of ZFPM2 attenuated the transcriptional activation of GATA4 and contributed to the cardiac abnormalities in zebrafish. a Diagram of the human ZFPM2/FOG2 protein domain with the location of its variants identified. The eight zinc-finger motifs (ZNF) are represented by yellow boxes . The nuclear localization signal (NLS) is indicated by a green box . The putative CtBP-binding site (CBS) is represented by a pink box . The N-terminal transcriptional repression domain (TRD) is indicated by a blue box . E1148 K found in B430 is located at the eighth Zinc-finger domain, and E1005G found in B548 is located in the seventh and eighth domains. Sanger sequencing shows the variant in the red frame . Both are conserved in human, mouse, dog and zebrafish. The amino acid residue altered by the mutation is shown in the red box . b Co-immunoprecipitation assays in HEK293T cells revealed that the E1148 K variant significantly damaged the interaction between ZFPM2 and GATA4 on the western blot. The semi-quantitative analysis of the western blot results shows that the E1148 K mutant ZFPM2 protein significantly disrupted the interaction with GATA4 compared to the wild-type ZFPM2 protein. The experiment was repeated three times. (p

    Journal: Journal of Translational Medicine

    Article Title: Multiple gene variations contributed to congenital heart disease via GATA family transcriptional regulation

    doi: 10.1186/s12967-017-1173-0

    Figure Lengend Snippet: The variant of ZFPM2 attenuated the transcriptional activation of GATA4 and contributed to the cardiac abnormalities in zebrafish. a Diagram of the human ZFPM2/FOG2 protein domain with the location of its variants identified. The eight zinc-finger motifs (ZNF) are represented by yellow boxes . The nuclear localization signal (NLS) is indicated by a green box . The putative CtBP-binding site (CBS) is represented by a pink box . The N-terminal transcriptional repression domain (TRD) is indicated by a blue box . E1148 K found in B430 is located at the eighth Zinc-finger domain, and E1005G found in B548 is located in the seventh and eighth domains. Sanger sequencing shows the variant in the red frame . Both are conserved in human, mouse, dog and zebrafish. The amino acid residue altered by the mutation is shown in the red box . b Co-immunoprecipitation assays in HEK293T cells revealed that the E1148 K variant significantly damaged the interaction between ZFPM2 and GATA4 on the western blot. The semi-quantitative analysis of the western blot results shows that the E1148 K mutant ZFPM2 protein significantly disrupted the interaction with GATA4 compared to the wild-type ZFPM2 protein. The experiment was repeated three times. (p

    Article Snippet: The antibodies used for the western blotting were anti-GATA4 (Genetex, USA), anti-FOG2 (Santa Cruz, USA), anti-Flag (Abmart, China), and anti-HA (Abmart, China).

    Techniques: Variant Assay, Activation Assay, Binding Assay, Sequencing, Mutagenesis, Immunoprecipitation, Western Blot

    Two possible genetic etiologies of CHD. a Monogenic mutations in a key cardiac regulator may be the cause of CHD. A pathogenic E1148 K variant of ZFPM2 in patient B430 decreased the binding of ZFPM2 and GATA4 and attenuated the transcriptional activation of GATA4 on the promoter of ANF. b The majority of the variants in the GATA family member genes and JAG1 occurred sporadically in the patients. However, patients B294 and B393 carried two variants in two genes. B393 carried one variant in JAG1 and one variant in GATA6 variant, shown in a blue color , and B294 carried one variant in JAG1 and one variant in GATA5, shown in a green color . The results support the notion that rare, moderate-effect gene variants occur simultaneously in patients and may increase the susceptibility to cardiac malformations. The interaction between NOTCH signaling and the GATA family members is indispensable for cardiac development

    Journal: Journal of Translational Medicine

    Article Title: Multiple gene variations contributed to congenital heart disease via GATA family transcriptional regulation

    doi: 10.1186/s12967-017-1173-0

    Figure Lengend Snippet: Two possible genetic etiologies of CHD. a Monogenic mutations in a key cardiac regulator may be the cause of CHD. A pathogenic E1148 K variant of ZFPM2 in patient B430 decreased the binding of ZFPM2 and GATA4 and attenuated the transcriptional activation of GATA4 on the promoter of ANF. b The majority of the variants in the GATA family member genes and JAG1 occurred sporadically in the patients. However, patients B294 and B393 carried two variants in two genes. B393 carried one variant in JAG1 and one variant in GATA6 variant, shown in a blue color , and B294 carried one variant in JAG1 and one variant in GATA5, shown in a green color . The results support the notion that rare, moderate-effect gene variants occur simultaneously in patients and may increase the susceptibility to cardiac malformations. The interaction between NOTCH signaling and the GATA family members is indispensable for cardiac development

    Article Snippet: The antibodies used for the western blotting were anti-GATA4 (Genetex, USA), anti-FOG2 (Santa Cruz, USA), anti-Flag (Abmart, China), and anti-HA (Abmart, China).

    Techniques: Variant Assay, Binding Assay, Activation Assay

    Variants in the GATA family members. Blue boxes represent the transcription activation domains (TADs), and red boxes indicate the Zinc finger domains (ZFN). Sanger sequencing data are displayed, and the variants are shown in the red frames . Alignment of amino acid residues adjacent to the variants shows a conservation among different species, including human, mouse, dog and zebrafish. a Human GATA4 protein domain with the variants identified in the TOF patients. The GATA4 protein is composed of four functional domains. V380 M is located in the second TAD, and P407Q is located near the second TAD. Sanger sequencing data are displayed, and the variants are shown in the red frames . Amino acid in 407 is conserved in human, mouse and dog but not in zebrafish. b Human GATA5 protein domain with the variants identified in the TOF patients. GATA5 protein also has four functional domains, and the two novel variants are not located in the functional domain. Sanger sequencing data are displayed, and the variants are shown in the red frames . c Human GATA6 protein domain with the variants identified in the TOF patients. The GATA6 protein has five functional domains, including two TADs, two ZNFs and one nuclear location signal (NLS), which is presented by the yellow box . The variants D111 N and H324Q are localized at the TAD1 and TAD2, respectively. The amino acid in 111 is highly conserved in human, mouse, dog and zebrafish

    Journal: Journal of Translational Medicine

    Article Title: Multiple gene variations contributed to congenital heart disease via GATA family transcriptional regulation

    doi: 10.1186/s12967-017-1173-0

    Figure Lengend Snippet: Variants in the GATA family members. Blue boxes represent the transcription activation domains (TADs), and red boxes indicate the Zinc finger domains (ZFN). Sanger sequencing data are displayed, and the variants are shown in the red frames . Alignment of amino acid residues adjacent to the variants shows a conservation among different species, including human, mouse, dog and zebrafish. a Human GATA4 protein domain with the variants identified in the TOF patients. The GATA4 protein is composed of four functional domains. V380 M is located in the second TAD, and P407Q is located near the second TAD. Sanger sequencing data are displayed, and the variants are shown in the red frames . Amino acid in 407 is conserved in human, mouse and dog but not in zebrafish. b Human GATA5 protein domain with the variants identified in the TOF patients. GATA5 protein also has four functional domains, and the two novel variants are not located in the functional domain. Sanger sequencing data are displayed, and the variants are shown in the red frames . c Human GATA6 protein domain with the variants identified in the TOF patients. The GATA6 protein has five functional domains, including two TADs, two ZNFs and one nuclear location signal (NLS), which is presented by the yellow box . The variants D111 N and H324Q are localized at the TAD1 and TAD2, respectively. The amino acid in 111 is highly conserved in human, mouse, dog and zebrafish

    Article Snippet: The antibodies used for the western blotting were anti-GATA4 (Genetex, USA), anti-FOG2 (Santa Cruz, USA), anti-Flag (Abmart, China), and anti-HA (Abmart, China).

    Techniques: Activation Assay, Sequencing, Functional Assay

    Celastrol increased HSPs and preserved essential transcription factors. a , b Immunoblot analysis showed an increase in active caspase-3 expression after exposure to DOX and TAA in H9c2 and Huh7 cells, respectively. GATA4 in H9c2 and Hnf-1α and

    Journal: Cell Stress & Chaperones

    Article Title: Celastrol, an oral heat shock activator, ameliorates multiple animal disease models of cell death

    doi: 10.1007/s12192-014-0536-1

    Figure Lengend Snippet: Celastrol increased HSPs and preserved essential transcription factors. a , b Immunoblot analysis showed an increase in active caspase-3 expression after exposure to DOX and TAA in H9c2 and Huh7 cells, respectively. GATA4 in H9c2 and Hnf-1α and

    Article Snippet: In vitro MTT-based toxicology assay kit (TOX-1), doxorubicin, thioacetamide (TAA), triptolide (TTD), and acetaminophen (APAP) were purchased from Sigma, Inc. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Millipore, Inc. For Western blot analysis, hsf1, hsp90, hsp70, hsp40, hsp27 (Enzo Life Sciences), GAPDH, GATA4 (BD Biosciences), cleaved caspase-3 (Cell Signaling), and CD68 (Abcam, Cambridge, USA) primary antibodies were used.

    Techniques: Expressing

    GATA4 downregulates the TGFB2 mRNA level through multiple miRNAs. a Schematics of targeting sites on TGFB2 mRNA recognized by 14 miRNAs. b Relative expression level of miRNAs before and after GATA4 overexpression in A549i (through 2 μg/mL of Dox treatment) and A549 cells (through lentivirus infection) ( n = 3 per group). c qPCR analysis of promoter fragment of designated gene from DNA samples prepared from control IgG or anti-FLAG (for pulldown GATA4) DNA samples. Quantity of GATA4 bond DNA was normalized by the value in control IgG-treated samples ( n = 3 per group). d TGFB2 mRNA level in A549 cells overexpressing miR-32, miR-301b, miR-320A, or miR-590, respectively ( n = 3 per group). e β-Galactosidase signal in GATA4 overexpressing A549 cells in the absence or presence of microRNA sponge ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: GATA4 downregulates the TGFB2 mRNA level through multiple miRNAs. a Schematics of targeting sites on TGFB2 mRNA recognized by 14 miRNAs. b Relative expression level of miRNAs before and after GATA4 overexpression in A549i (through 2 μg/mL of Dox treatment) and A549 cells (through lentivirus infection) ( n = 3 per group). c qPCR analysis of promoter fragment of designated gene from DNA samples prepared from control IgG or anti-FLAG (for pulldown GATA4) DNA samples. Quantity of GATA4 bond DNA was normalized by the value in control IgG-treated samples ( n = 3 per group). d TGFB2 mRNA level in A549 cells overexpressing miR-32, miR-301b, miR-320A, or miR-590, respectively ( n = 3 per group). e β-Galactosidase signal in GATA4 overexpressing A549 cells in the absence or presence of microRNA sponge ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Expressing, Over Expression, Infection, Real-time Polymerase Chain Reaction

    The GATA4-TGF-β2-Wnt-7b signaling axis is clinically relevant. a qRT-PCR analysis of TGFB2 and WNT7B expression level in H226 cell line with GATA4 knockdown ( n = 3 per group). b GATA4 expression level in lung cancer samples and normal lung tissues. c Kaplan–Meier survival curve of GATA4-high and GATA4-low lung cancer patients (Cox log-rank test, p = 0.019). d Reverse correlation of expression level of GATA4 versus TGF-β2 and Wnt-7b in clinical lung cancer samples downloaded from TCGA. e Expression pattern of GATA4 versus TGF-β2 and Wnt-7b in driver mutation positive samples. EGFR mutation positive: Upper panel; Kras mutation positive: middle panel; EML4-ALK mutation positive: lower panel

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: The GATA4-TGF-β2-Wnt-7b signaling axis is clinically relevant. a qRT-PCR analysis of TGFB2 and WNT7B expression level in H226 cell line with GATA4 knockdown ( n = 3 per group). b GATA4 expression level in lung cancer samples and normal lung tissues. c Kaplan–Meier survival curve of GATA4-high and GATA4-low lung cancer patients (Cox log-rank test, p = 0.019). d Reverse correlation of expression level of GATA4 versus TGF-β2 and Wnt-7b in clinical lung cancer samples downloaded from TCGA. e Expression pattern of GATA4 versus TGF-β2 and Wnt-7b in driver mutation positive samples. EGFR mutation positive: Upper panel; Kras mutation positive: middle panel; EML4-ALK mutation positive: lower panel

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Quantitative RT-PCR, Expressing, Mutagenesis

    TGFBR1 is a potential target for GATA4-deficient lung cancer. a and b 5000 cells per well seeded in 96-well plates. Cells were treated with SB525334 (2 μM) and/or trametinib (2 μM). CCK8 value were checked 3 days after drug treatment. a For H23 and b for PC9 ( n = 3 per group). c SB525334 (10 mg/kg/day) synergizes with trametinib (10 mg/kg/day) to shrink lung tumor in KRAS G12D /GATA4-/- mice. MRI image of lung of pre-treatment (left panel) and post-treatment (middle panel) of lung cancer bearing mice (PreRx and PostRx); β-Galactosidase staining of lung section of post-treatment mice (right panel). Scale = 100 μm. d Statistics of lung tumor burdens recorded in MRI ( n = 4 per group). e Statistics of senescence in tumor shown ( c ) ( n = 4 per group). f , g , h and i The tumor sizes and weights of PDX tumors treated with TGFBR inhibitor (SB525334, 10 mg/kg/day), Cisplatin (5 mg/kg, once a week), or combination. SH3166: GATA4-low PDX; SH3a: GATA4-high PDX ( n = 6 per group). j Model of how GATA4 regulate TGF-β2 and Wnt-7b signaling and cellular senescence. See text for details. Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: TGFBR1 is a potential target for GATA4-deficient lung cancer. a and b 5000 cells per well seeded in 96-well plates. Cells were treated with SB525334 (2 μM) and/or trametinib (2 μM). CCK8 value were checked 3 days after drug treatment. a For H23 and b for PC9 ( n = 3 per group). c SB525334 (10 mg/kg/day) synergizes with trametinib (10 mg/kg/day) to shrink lung tumor in KRAS G12D /GATA4-/- mice. MRI image of lung of pre-treatment (left panel) and post-treatment (middle panel) of lung cancer bearing mice (PreRx and PostRx); β-Galactosidase staining of lung section of post-treatment mice (right panel). Scale = 100 μm. d Statistics of lung tumor burdens recorded in MRI ( n = 4 per group). e Statistics of senescence in tumor shown ( c ) ( n = 4 per group). f , g , h and i The tumor sizes and weights of PDX tumors treated with TGFBR inhibitor (SB525334, 10 mg/kg/day), Cisplatin (5 mg/kg, once a week), or combination. SH3166: GATA4-low PDX; SH3a: GATA4-high PDX ( n = 6 per group). j Model of how GATA4 regulate TGF-β2 and Wnt-7b signaling and cellular senescence. See text for details. Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Mouse Assay, Magnetic Resonance Imaging, Staining

    GATA4 is an important tumor suppressor in lung cancer. a Scatterplot of relative growth rate against relative expression level. 10,000 H23 cells per well seeded in 96-well plate; siRNA transfected into individual wells. CCK8 value of a well 3 days post siRNA transfection was plotted against relative expression value of the corresponding gene in tumor (one replica). b Western blot analysis of GATA4 expression in cancerous and normal lung cell lines. c Lung cancer cells infected with lentivirus for ectopic expression of GATA4. Soft-agar colony forming ability was compared between lung cancer cell lines (A549, PC-9, and H460) ectopically expressing GATA4 and EGFP. Scale = 500 μm. d Statistics of soft-agar colony result shown in c ( n = 3 per group). e Colony number of 10,000 A549i cells in the absence or presence of Dox checked 14 days after seeding in soft agar plate. Upper panel: statistics of the soft-agar colonies. Lower panel: representative pictures of A549i cells treated with or without 2 μg/mL of Dox ( n = 3 per group, scale = 100 μm) (A549i is an engineered A549 clone harboring Dox inducible GATA4 expression gene element). f Soft-agar colony forming ability of stable H23 with GATA4 knockdown by shRNAs or control shRNA. Upper panel: statistics of soft-agar colony result of H23 knockdown with control or GATA4 targeting shRNAs. Lower panel: Representative pictures ( n = 3 per group, scale = 50 μm). g Lsl-KRAS G12D infected with recombinant lentivirus coexpressing Cre and CRISPR/CAS9 through nasal instillation. Lung tumor formation in KRAS G12D /GATA4-/- mice compared to KRAS G12D /TdTomato-/- mice 10 weeks post-infection. Scale = 200 μm. h Statistics of percentage of tumor area in the lung represented in g (n = 3 per group). i Schematics of intranasal instillation of retrovirus for forced expressing GATA4 in Dox inducible Tet-KRAS G12C /CC10rtTA lung cancer mouse model (referred to as KC). j Pathology of lung tumor formation in KC mice infected with control (mCherry) or GATA4 expressing virus. Scale = 200 μm. k Statistics of tumor area per lung area (n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: GATA4 is an important tumor suppressor in lung cancer. a Scatterplot of relative growth rate against relative expression level. 10,000 H23 cells per well seeded in 96-well plate; siRNA transfected into individual wells. CCK8 value of a well 3 days post siRNA transfection was plotted against relative expression value of the corresponding gene in tumor (one replica). b Western blot analysis of GATA4 expression in cancerous and normal lung cell lines. c Lung cancer cells infected with lentivirus for ectopic expression of GATA4. Soft-agar colony forming ability was compared between lung cancer cell lines (A549, PC-9, and H460) ectopically expressing GATA4 and EGFP. Scale = 500 μm. d Statistics of soft-agar colony result shown in c ( n = 3 per group). e Colony number of 10,000 A549i cells in the absence or presence of Dox checked 14 days after seeding in soft agar plate. Upper panel: statistics of the soft-agar colonies. Lower panel: representative pictures of A549i cells treated with or without 2 μg/mL of Dox ( n = 3 per group, scale = 100 μm) (A549i is an engineered A549 clone harboring Dox inducible GATA4 expression gene element). f Soft-agar colony forming ability of stable H23 with GATA4 knockdown by shRNAs or control shRNA. Upper panel: statistics of soft-agar colony result of H23 knockdown with control or GATA4 targeting shRNAs. Lower panel: Representative pictures ( n = 3 per group, scale = 50 μm). g Lsl-KRAS G12D infected with recombinant lentivirus coexpressing Cre and CRISPR/CAS9 through nasal instillation. Lung tumor formation in KRAS G12D /GATA4-/- mice compared to KRAS G12D /TdTomato-/- mice 10 weeks post-infection. Scale = 200 μm. h Statistics of percentage of tumor area in the lung represented in g (n = 3 per group). i Schematics of intranasal instillation of retrovirus for forced expressing GATA4 in Dox inducible Tet-KRAS G12C /CC10rtTA lung cancer mouse model (referred to as KC). j Pathology of lung tumor formation in KC mice infected with control (mCherry) or GATA4 expressing virus. Scale = 200 μm. k Statistics of tumor area per lung area (n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Expressing, Transfection, Western Blot, Infection, shRNA, Recombinant, CRISPR, Mouse Assay

    Ectopic GATA4 expression induces lung cancer cell senescence. a Microphotograph of A549 cells infected lentivirus overexpressing GATA4. Red arrow-head highlighted the senescent cells. Scale = 100 μm. b β-galactosidase staining of A549 cells infected with GATA4 or EGFP expressing lentivirus. Representative pictures were shown. Scale = 100 μm. c , d Statistics of β-Galactosidase staining of A549 cells ectopically expressing GATA4 through lentivirus infection ( c ) or Doxycycline treatment of A549i cells ( d ) ( n = 3 per group). e Western blot analysis of Doxycycline-inducible GATA4 expression in stable cell lines of H460, EKVX, and Hop62. f β-Galactosidase staining of indicated cell lines before and after GATA4 expression. g Statistics of f ( n = 3 per group). h Western blot analysis of p15INK4b expression in GATA4-expressing A549 cells. i β-galactosidase staining of GATA4-expressing A549 knockdown with control or CDKN2B-targeting shRNAs ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: Ectopic GATA4 expression induces lung cancer cell senescence. a Microphotograph of A549 cells infected lentivirus overexpressing GATA4. Red arrow-head highlighted the senescent cells. Scale = 100 μm. b β-galactosidase staining of A549 cells infected with GATA4 or EGFP expressing lentivirus. Representative pictures were shown. Scale = 100 μm. c , d Statistics of β-Galactosidase staining of A549 cells ectopically expressing GATA4 through lentivirus infection ( c ) or Doxycycline treatment of A549i cells ( d ) ( n = 3 per group). e Western blot analysis of Doxycycline-inducible GATA4 expression in stable cell lines of H460, EKVX, and Hop62. f β-Galactosidase staining of indicated cell lines before and after GATA4 expression. g Statistics of f ( n = 3 per group). h Western blot analysis of p15INK4b expression in GATA4-expressing A549 cells. i β-galactosidase staining of GATA4-expressing A549 knockdown with control or CDKN2B-targeting shRNAs ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Expressing, Infection, Staining, Western Blot, Stable Transfection

    TGF-β2 mediates GATA4-induced senescence upstream of Wnt-7b. a and b qRT-PCR analysis of TGFB2 expression in GATA4 overexpressing A549 cells through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c β-Galactosidase staining of A549 cells with TGFB2 knockdown (middle panel) and rescued by shRNA-resistant TGFB2 . d Statistics of c ( n = 3 per group). e β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting TGFB2 ( n = 3 per group). f β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by TGFB2 expression ( n = 3 per group). g β-Galactosidase staining of A549i cells in the absence or presence of 2 μg/mL of Dox and rescued with recombinant TGF-β protein at indicated concentration ( n = 3 per group). h β-Galactosidase signal in A549 cells knockdown of TGFBR1 and TGFBR2 ( n = 3 per group). i β-Galactosidase signal of A549 cells with SMAD2 knockdown (middle panel) and rescued by shRNA-resistant SMAD2 (right panel). j β-Galactosidase staining of A549 cells with SMAD4 knockdown (middle panel) and rescued by shRNA-resistant SMAD4 (right panel) ( n = 3 per group). k WNT7B expression level in A549 cells with knockdown of TGFBR1 , TGFBR2 , SMAD2 , or SMAD4 , respectively ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: TGF-β2 mediates GATA4-induced senescence upstream of Wnt-7b. a and b qRT-PCR analysis of TGFB2 expression in GATA4 overexpressing A549 cells through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c β-Galactosidase staining of A549 cells with TGFB2 knockdown (middle panel) and rescued by shRNA-resistant TGFB2 . d Statistics of c ( n = 3 per group). e β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting TGFB2 ( n = 3 per group). f β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by TGFB2 expression ( n = 3 per group). g β-Galactosidase staining of A549i cells in the absence or presence of 2 μg/mL of Dox and rescued with recombinant TGF-β protein at indicated concentration ( n = 3 per group). h β-Galactosidase signal in A549 cells knockdown of TGFBR1 and TGFBR2 ( n = 3 per group). i β-Galactosidase signal of A549 cells with SMAD2 knockdown (middle panel) and rescued by shRNA-resistant SMAD2 (right panel). j β-Galactosidase staining of A549 cells with SMAD4 knockdown (middle panel) and rescued by shRNA-resistant SMAD4 (right panel) ( n = 3 per group). k WNT7B expression level in A549 cells with knockdown of TGFBR1 , TGFBR2 , SMAD2 , or SMAD4 , respectively ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Quantitative RT-PCR, Expressing, Infection, Staining, shRNA, Recombinant, Concentration Assay

    GATA4 induces lung cancer cell senescence by downregulating WNT7B. a and b WNT7B expression level in A549 cells ectopically expressing GATA4 through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c TOP-FLASH analysis of GATA4 expressing A549 cells ( n = 6 per group). d Western blot analysis of c-Myc and cyclin D1 in A549i cells in the absence or presence of 2 μg/mL of Dox. e β-Galactosidase staining of A549 cells expressing WNT7B targeting shRNA (middle panel) and rescued by shRNA-resistant WNT7B (right panel) (scale bar 100 μm). f Statistics of e ( n = 3 per group). g β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting WNT7B (A549 tet-shWNT7B) in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). h β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by Wnt-7b expression ( n = 3 per group). i Impact of shRNA targeting CTNNB1 mRNA on induction of senescence in A549 cells ( n = 3 per group). j β-Galactosidase staining of A549 cells treated with ICG-001 and XAV939 at indicated concentration ( n = 3 per group). k qRT-PCR analysis of MMP7 expression of in A549i cells in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). l β-Galactosidase staining of A549 cells knockdown with shRNA targeting MMP7 mRNA ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Journal: Nature Communications

    Article Title: Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

    doi: 10.1038/s41467-019-09295-7

    Figure Lengend Snippet: GATA4 induces lung cancer cell senescence by downregulating WNT7B. a and b WNT7B expression level in A549 cells ectopically expressing GATA4 through lentivirus infection ( a ) or Doxycycline treatment of A549i cells ( b ) ( n = 3 per group). c TOP-FLASH analysis of GATA4 expressing A549 cells ( n = 6 per group). d Western blot analysis of c-Myc and cyclin D1 in A549i cells in the absence or presence of 2 μg/mL of Dox. e β-Galactosidase staining of A549 cells expressing WNT7B targeting shRNA (middle panel) and rescued by shRNA-resistant WNT7B (right panel) (scale bar 100 μm). f Statistics of e ( n = 3 per group). g β-Galactosidase staining of A549 expressing Dox-inducible shRNA targeting WNT7B (A549 tet-shWNT7B) in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). h β-Galactosidase staining of A549i in the absence or presence of 2 μg/mL of Dox and rescued by Wnt-7b expression ( n = 3 per group). i Impact of shRNA targeting CTNNB1 mRNA on induction of senescence in A549 cells ( n = 3 per group). j β-Galactosidase staining of A549 cells treated with ICG-001 and XAV939 at indicated concentration ( n = 3 per group). k qRT-PCR analysis of MMP7 expression of in A549i cells in the absence or presence of 2 μg/mL of Dox ( n = 3 per group). l β-Galactosidase staining of A549 cells knockdown with shRNA targeting MMP7 mRNA ( n = 3 per group). Bars are represented as mean ± SEM of the indicated number ( n ) of repeats. *P

    Article Snippet: Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody.

    Techniques: Expressing, Infection, Western Blot, Staining, shRNA, Concentration Assay, Quantitative RT-PCR

    The protein expression of GATA4 was reduced after AT treatment in mice. (a) Western blot bands of the protein expression of GATA4 and GAPDH and (b) their fold changes in the four groups (NS-SHAM, NS-TAC, AT-SHAM, and AT-TAC). Data are mean ± SEM ( n = 7). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Apocynum Tablet Protects against Cardiac Hypertrophy via Inhibiting AKT and ERK1/2 Phosphorylation after Pressure Overload

    doi: 10.1155/2014/769515

    Figure Lengend Snippet: The protein expression of GATA4 was reduced after AT treatment in mice. (a) Western blot bands of the protein expression of GATA4 and GAPDH and (b) their fold changes in the four groups (NS-SHAM, NS-TAC, AT-SHAM, and AT-TAC). Data are mean ± SEM ( n = 7). * P

    Article Snippet: Following antibodies were used in this study: anti-phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-PKB (Ser473, Cell Signaling Technology), anti-ERK1/2 (Santa Cruz Technology, Delaware, CA, USA), and anti-GATA4 (Selleckchem Technology, Houston, TX, USA).

    Techniques: Expressing, Mouse Assay, Western Blot

    A model of pathways in the cardioprotection of AT in response to pressure stress overload. TAC (stress overload) could activate phosphorylation of the protein kinases of ERK1/2 and AKT, enhance the expression of GATA4, promote the transcription of hypertrophic gene, and result in cardiac hypertrophy and cardiac fibrosis. AT could inhibit the phosphorylation of ERK1/2 and AKT, reduce GATA4, and inhibit pathological development of cardiac hypertrophy. ⨂ denotes inhibition of protein kinase by AT treatment.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Apocynum Tablet Protects against Cardiac Hypertrophy via Inhibiting AKT and ERK1/2 Phosphorylation after Pressure Overload

    doi: 10.1155/2014/769515

    Figure Lengend Snippet: A model of pathways in the cardioprotection of AT in response to pressure stress overload. TAC (stress overload) could activate phosphorylation of the protein kinases of ERK1/2 and AKT, enhance the expression of GATA4, promote the transcription of hypertrophic gene, and result in cardiac hypertrophy and cardiac fibrosis. AT could inhibit the phosphorylation of ERK1/2 and AKT, reduce GATA4, and inhibit pathological development of cardiac hypertrophy. ⨂ denotes inhibition of protein kinase by AT treatment.

    Article Snippet: Following antibodies were used in this study: anti-phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-PKB (Ser473, Cell Signaling Technology), anti-ERK1/2 (Santa Cruz Technology, Delaware, CA, USA), and anti-GATA4 (Selleckchem Technology, Houston, TX, USA).

    Techniques: Expressing, Inhibition