Structured Review

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.
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1) Product Images from "Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering"

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

Journal: Biophysical Journal

doi: 10.1016/j.bpj.2018.11.011

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Staining, Microscopy

2) Product Images from "A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart"

Article Title: A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky049

GATA4 interacts with β-catenin and fine-tunes the molecular switch driving adult heart disease progression in vivo . ( A ) Spearman's correlation plot of TCF7L2 co-occupancy with GATA4, NKX2.5 and TBX3, highlighting highest correlation with GATA4 (black box). ( B ) Venn diagram of genes bound by TCF7L2 (orange) with upregulated (violet) or downregulated (green) genes with log 2 FC≥0.5, p≤0.05 in β-cat Δex3 ventricles and corresponding motif enrichment of the intersections. ( C ) Venn diagram showing commonly bound genes (319) between TCF7L2 in β-cat Δex3 hearts and GATA4 in normal hearts. ( D ) Immunoblot of GATA4 with β-catenin co-immunoprecipitation in WT, β-cat Δex3 and 6 weeks post-TAC hearts. Input represents the total, sheared chromatin-protein complexes before immunoprecipitation, (*) protein ladder. ( E ) Immunoblot of total β-catenin and active pSer675 β-catenin in the nuclear fractions of control, β-cat Δex3 and 6 weeks post-TAC hearts. TBX5 and GAPDH were used to detect nuclear and cytosolic enrichments respectively. ( F ) IGV binding profiles for TCF7L2 occupancy in β-cat Δex3 hearts along with GATA4 co-occupancy in normal hearts on Hand2 enhancer locus; ChIP-qPCR for GATA4 binding on Hand2 enhancer in normal (WT), 6 weeks post-TAC (WT TAC) and β-cat Δex3 hearts. Relative fold enrichment was calculated to IgG control, normalized to 10% input chromatin (n = 3 hearts/ChIP) ( G ) Profiles of enhancers with GATA4 and TCF7L2 overlapping occupancy ( Hand2 ) and with only TCF7L2 occupancy ( Tbx20 ). Luciferase reporter assay for Hand2 enhancer (-enh) and Tbx20- enh upon β-catenin stabilization, GATA4 overexpression or both normalized to empty vector (EV). (Renilla luciferase was the transfection control, n = 3/independent experiments). Data are mean ± SEM; t -test and ANOVA, Bonferroni's multiple comparison test. ( H ) Spearman's correlation plot depicting high correlations between GATA4 and repressive elements KLF15, H3K27me3 and CTCF in normal hearts specifically on TCF7L2-bound regions in β-cat Δex3 hearts.
Figure Legend Snippet: GATA4 interacts with β-catenin and fine-tunes the molecular switch driving adult heart disease progression in vivo . ( A ) Spearman's correlation plot of TCF7L2 co-occupancy with GATA4, NKX2.5 and TBX3, highlighting highest correlation with GATA4 (black box). ( B ) Venn diagram of genes bound by TCF7L2 (orange) with upregulated (violet) or downregulated (green) genes with log 2 FC≥0.5, p≤0.05 in β-cat Δex3 ventricles and corresponding motif enrichment of the intersections. ( C ) Venn diagram showing commonly bound genes (319) between TCF7L2 in β-cat Δex3 hearts and GATA4 in normal hearts. ( D ) Immunoblot of GATA4 with β-catenin co-immunoprecipitation in WT, β-cat Δex3 and 6 weeks post-TAC hearts. Input represents the total, sheared chromatin-protein complexes before immunoprecipitation, (*) protein ladder. ( E ) Immunoblot of total β-catenin and active pSer675 β-catenin in the nuclear fractions of control, β-cat Δex3 and 6 weeks post-TAC hearts. TBX5 and GAPDH were used to detect nuclear and cytosolic enrichments respectively. ( F ) IGV binding profiles for TCF7L2 occupancy in β-cat Δex3 hearts along with GATA4 co-occupancy in normal hearts on Hand2 enhancer locus; ChIP-qPCR for GATA4 binding on Hand2 enhancer in normal (WT), 6 weeks post-TAC (WT TAC) and β-cat Δex3 hearts. Relative fold enrichment was calculated to IgG control, normalized to 10% input chromatin (n = 3 hearts/ChIP) ( G ) Profiles of enhancers with GATA4 and TCF7L2 overlapping occupancy ( Hand2 ) and with only TCF7L2 occupancy ( Tbx20 ). Luciferase reporter assay for Hand2 enhancer (-enh) and Tbx20- enh upon β-catenin stabilization, GATA4 overexpression or both normalized to empty vector (EV). (Renilla luciferase was the transfection control, n = 3/independent experiments). Data are mean ± SEM; t -test and ANOVA, Bonferroni's multiple comparison test. ( H ) Spearman's correlation plot depicting high correlations between GATA4 and repressive elements KLF15, H3K27me3 and CTCF in normal hearts specifically on TCF7L2-bound regions in β-cat Δex3 hearts.

Techniques Used: In Vivo, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Over Expression, Plasmid Preparation, Transfection

β-catenin loss of function in CM rescues Wnt-dependent pathological gene regulation in vivo . ( A ) Representative examples of M-mode echocardiograms, and quantification of fractional shortening (FAS) by echocardiographic analysis of 3 weeks TAC-induced Cre pos control and β-cat Δex2–6 mice, n ≥ 7. ( B ) Relative transcript levels of hypertrophic marker Nppb in β-cat Δex2–6 and controls Cre pos ; sham and TAC (n≥8/per group). ( C ) Immunoblot depicting total β-catenin and pSer675-β-catenin upon TAC in β-catenin loss of function (β-cat Δex2–6 ). GAPDH was protein-loading control (n = 2/group). Relative transcript levels of ( D ) classical Wnt target Axin2 and ( E ) newly identified cardiac Wnt targets Tbx20 and Dstn , 6 weeks post-TAC in β-cat Δex2–6 and controls, n≥7. Data are mean ± SEM; ANOVA, Bonferroni's multiple comparison test. Tbp was used for transcript normalization. ( F ) ChIP-qPCR for GATA4 binding on Hand2 enhancer in normal (WT), 6 weeks post-TAC (WT TAC) and β-cat Δex2–6 TAC hearts. Relative fold enrichment was calculated to IgG control, normalized to 10% input chromatin ( n = 3 hearts/ChIP). ( G ) Schematic representation of the findings of this study. In the healthy adult heart, β-catenin/TCF7L2-dependent loci are inactive, inaccessible or bound by transcriptional repressors, resulting in low transcription. On a subset of these loci, GATA4 binds to transcriptionally inactive β-catenin, fine-tuning Wnt-dependent transcription. This chromatin state guaranties normal homeostasis in the adult heart. Pathological stimuli leading to active pSer675-β-catenin accumulation activates Wnt signalling and the epigenetic state switches on to ‘active’, replaced by transcriptionally active pSer675-β-catenin bound to TCF7L2, leading to enriched H3K27ac occupancy and a high Wnt transcriptional activity. This results in the expression of disease-associated genes leading to adverse remodelling and heart failure.
Figure Legend Snippet: β-catenin loss of function in CM rescues Wnt-dependent pathological gene regulation in vivo . ( A ) Representative examples of M-mode echocardiograms, and quantification of fractional shortening (FAS) by echocardiographic analysis of 3 weeks TAC-induced Cre pos control and β-cat Δex2–6 mice, n ≥ 7. ( B ) Relative transcript levels of hypertrophic marker Nppb in β-cat Δex2–6 and controls Cre pos ; sham and TAC (n≥8/per group). ( C ) Immunoblot depicting total β-catenin and pSer675-β-catenin upon TAC in β-catenin loss of function (β-cat Δex2–6 ). GAPDH was protein-loading control (n = 2/group). Relative transcript levels of ( D ) classical Wnt target Axin2 and ( E ) newly identified cardiac Wnt targets Tbx20 and Dstn , 6 weeks post-TAC in β-cat Δex2–6 and controls, n≥7. Data are mean ± SEM; ANOVA, Bonferroni's multiple comparison test. Tbp was used for transcript normalization. ( F ) ChIP-qPCR for GATA4 binding on Hand2 enhancer in normal (WT), 6 weeks post-TAC (WT TAC) and β-cat Δex2–6 TAC hearts. Relative fold enrichment was calculated to IgG control, normalized to 10% input chromatin ( n = 3 hearts/ChIP). ( G ) Schematic representation of the findings of this study. In the healthy adult heart, β-catenin/TCF7L2-dependent loci are inactive, inaccessible or bound by transcriptional repressors, resulting in low transcription. On a subset of these loci, GATA4 binds to transcriptionally inactive β-catenin, fine-tuning Wnt-dependent transcription. This chromatin state guaranties normal homeostasis in the adult heart. Pathological stimuli leading to active pSer675-β-catenin accumulation activates Wnt signalling and the epigenetic state switches on to ‘active’, replaced by transcriptionally active pSer675-β-catenin bound to TCF7L2, leading to enriched H3K27ac occupancy and a high Wnt transcriptional activity. This results in the expression of disease-associated genes leading to adverse remodelling and heart failure.

Techniques Used: In Vivo, Mouse Assay, Marker, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Activity Assay, Expressing

TCF7L2 cooperates with cardiac-TFs to enable heart-specific gene regulation. ( A ) Comparison of TCF7L2-bound regions in in vivo models of β-catenin stabilization (β-cat Δex3 ) in CMs and hepatocytes. GO biological processes for unique regions in CM (magenta) and hepatocytes (blue). ( B ) TCF7L2 occupancy profiles on identified Hand2, Tbx20, Rock2 and Dstn enhancers and common Wnt targets Axin2 and Lef1 in β-catenin stabilized CM (magenta) and hepatocytes (blue). ( C ) Table enlisting the transcription factors (TFs) enriched on TCF7L2-bound regions in β-cat Δex3 ventricles by de-novo motif search using MEME-SpaMo. ( D ) Heatmap depicting the occupancy of cardiac-TFs GATA4 and NKX2.5 in the normal heart on regions occupied by TCF7L2 in β-cat Δex3 hearts. Regions ± 5 kb are shown. ( E ) Average profiles of GATA4 and NKX2.5 occupancy on TCF7L2-bound liver and heart-specific regions. Data are mean ± SEM; t -test.
Figure Legend Snippet: TCF7L2 cooperates with cardiac-TFs to enable heart-specific gene regulation. ( A ) Comparison of TCF7L2-bound regions in in vivo models of β-catenin stabilization (β-cat Δex3 ) in CMs and hepatocytes. GO biological processes for unique regions in CM (magenta) and hepatocytes (blue). ( B ) TCF7L2 occupancy profiles on identified Hand2, Tbx20, Rock2 and Dstn enhancers and common Wnt targets Axin2 and Lef1 in β-catenin stabilized CM (magenta) and hepatocytes (blue). ( C ) Table enlisting the transcription factors (TFs) enriched on TCF7L2-bound regions in β-cat Δex3 ventricles by de-novo motif search using MEME-SpaMo. ( D ) Heatmap depicting the occupancy of cardiac-TFs GATA4 and NKX2.5 in the normal heart on regions occupied by TCF7L2 in β-cat Δex3 hearts. Regions ± 5 kb are shown. ( E ) Average profiles of GATA4 and NKX2.5 occupancy on TCF7L2-bound liver and heart-specific regions. Data are mean ± SEM; t -test.

Techniques Used: In Vivo

3) Product Images from "Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells"

Article Title: Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085645

Cardiac-specific markers in TiPSC-CMs. (A) RT-PCR analyses of beating EBs derived from T05 and T07 for the pluripotent marker genes OCT3/4 , NANOG , and REX1 , the cardiac-related transcription factors GATA4 and MEF2C , and the cardiac-specific marker genes ANP , αMHC , βMHC , MLC2A , MLC2V , cTNNI , and SCN5A . (B) Immunofluorescence staining for cardiac-specific markers in cardiomyocytes derived from T07. Scale bar shows 50 µm.
Figure Legend Snippet: Cardiac-specific markers in TiPSC-CMs. (A) RT-PCR analyses of beating EBs derived from T05 and T07 for the pluripotent marker genes OCT3/4 , NANOG , and REX1 , the cardiac-related transcription factors GATA4 and MEF2C , and the cardiac-specific marker genes ANP , αMHC , βMHC , MLC2A , MLC2V , cTNNI , and SCN5A . (B) Immunofluorescence staining for cardiac-specific markers in cardiomyocytes derived from T07. Scale bar shows 50 µm.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Marker, Aqueous Normal-phase Chromatography, Immunofluorescence, Staining

4) Product Images from "Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation"

Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

Journal: Stem Cells International

doi: 10.1155/2014/379678

Nanog colocalizes with Gata4 and FoxA2 in definitive endoderm cells. Cells were differentiated according to protocol described in Section 2 and analysed by confocal immunofluorescence (a) for Nanog, Gata4, and FoxA2 at day 8 of differentiation. (b) Analysis showing the percentage of cells coexpressing Nanog-Gata4 and Nanog-FoxA2 in D3 differentiated cells. Data are mean ± ESM from 7–9 fields.
Figure Legend Snippet: Nanog colocalizes with Gata4 and FoxA2 in definitive endoderm cells. Cells were differentiated according to protocol described in Section 2 and analysed by confocal immunofluorescence (a) for Nanog, Gata4, and FoxA2 at day 8 of differentiation. (b) Analysis showing the percentage of cells coexpressing Nanog-Gata4 and Nanog-FoxA2 in D3 differentiated cells. Data are mean ± ESM from 7–9 fields.

Techniques Used: Immunofluorescence

5) Product Images from "A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation"

Article Title: A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-9-44

The proximal WT1 binding site in mouse Sry promoter is functional . A. A DNA-binding assay was performed with in vitro produced WT1 proteins and a 32 P-labeled oligonucleotide probe corresponding to the consensus WT1 binding site of the human AMH promoter. WT1 binding (+ or - KTS isoforms) to the labeled probe was blocked by 10-fold excess of unlabeled probe (self) and unlabeled oligo corresponding to the proximal WT1 site (site 1) of the mouse Sry promoter. B. Western blot analysis of nuclear extracts (10 μg) from PGR 9E11 cells (a pig genital ridge cell line) using antisera against WT1 and GATA4. Nuclear extracts from HeLa cells overexpressing WT1(-KTS) and MA-10 Leydig cells were used as positive controls for WT1 and GATA4 expression, respectively. C. Transfection studies performed in homologous PGR cells confirm the importance of the proximal WT1 site for basal Sry promoter activity. PGR cells were transfected with the deleted or mutated mouse Sry promoter constructs (500 ng) as indicated. Results are shown as % activity relative to the intact -340 bp construct ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05). D. GATA4 is associated with the SRY promoter in PGR genital ridge cells. PGR cell lysates were prepared and interaction of GATA4 with the endogenous pig SRY promoter was studied by ChIP. An aliquot of chromatin preparation before immunoprecipitation (20% input) was used as positive control. Chromatin was precipitated with a GATA4 antiserum (αGATA4) or incubated with goat-IgG (IgG) which served as a negative control. A 320-bp DNA fragment spanning a portion of the SRY promoter containing the proximal WT1 binding site (ChIP targeting region 2) was amplified by PCR as indicated by the arrow. A more distal SRY promoter fragment lacking this WT1 site (ChIP targeting region 1) was not amplified.
Figure Legend Snippet: The proximal WT1 binding site in mouse Sry promoter is functional . A. A DNA-binding assay was performed with in vitro produced WT1 proteins and a 32 P-labeled oligonucleotide probe corresponding to the consensus WT1 binding site of the human AMH promoter. WT1 binding (+ or - KTS isoforms) to the labeled probe was blocked by 10-fold excess of unlabeled probe (self) and unlabeled oligo corresponding to the proximal WT1 site (site 1) of the mouse Sry promoter. B. Western blot analysis of nuclear extracts (10 μg) from PGR 9E11 cells (a pig genital ridge cell line) using antisera against WT1 and GATA4. Nuclear extracts from HeLa cells overexpressing WT1(-KTS) and MA-10 Leydig cells were used as positive controls for WT1 and GATA4 expression, respectively. C. Transfection studies performed in homologous PGR cells confirm the importance of the proximal WT1 site for basal Sry promoter activity. PGR cells were transfected with the deleted or mutated mouse Sry promoter constructs (500 ng) as indicated. Results are shown as % activity relative to the intact -340 bp construct ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05). D. GATA4 is associated with the SRY promoter in PGR genital ridge cells. PGR cell lysates were prepared and interaction of GATA4 with the endogenous pig SRY promoter was studied by ChIP. An aliquot of chromatin preparation before immunoprecipitation (20% input) was used as positive control. Chromatin was precipitated with a GATA4 antiserum (αGATA4) or incubated with goat-IgG (IgG) which served as a negative control. A 320-bp DNA fragment spanning a portion of the SRY promoter containing the proximal WT1 binding site (ChIP targeting region 2) was amplified by PCR as indicated by the arrow. A more distal SRY promoter fragment lacking this WT1 site (ChIP targeting region 1) was not amplified.

Techniques Used: Binding Assay, Functional Assay, DNA Binding Assay, In Vitro, Produced, Labeling, Western Blot, Expressing, Transfection, Activity Assay, Construct, Chromatin Immunoprecipitation, Immunoprecipitation, Positive Control, Incubation, Negative Control, Amplification, Polymerase Chain Reaction

A mutated form of WT1 (WT1 R394W) that causes retention of Müllerian ducts in humans fails to synergize with GATA4 . A. HeLa cells were co-transfected with the mouse -180 bp Amh promoter and either an empty expression vector (control) or expression vectors for GATA4 (100 ng), WT1(-KTS) wild-type (500 ng) or WT1(-KTS) R394W DDS mutant (500 ng), used alone or in combination as indicated. All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
Figure Legend Snippet: A mutated form of WT1 (WT1 R394W) that causes retention of Müllerian ducts in humans fails to synergize with GATA4 . A. HeLa cells were co-transfected with the mouse -180 bp Amh promoter and either an empty expression vector (control) or expression vectors for GATA4 (100 ng), WT1(-KTS) wild-type (500 ng) or WT1(-KTS) R394W DDS mutant (500 ng), used alone or in combination as indicated. All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).

Techniques Used: Transfection, Expressing, Plasmid Preparation, Mutagenesis, Activation Assay

Transcriptional synergism between GATA4 and WT1 on the Amh promoter requires GATA4 and WT1 binding to their respective regulatory elements . A. The native -180 bp Amh promoter containing the GATA and WT1 elements in their natural context. B. A synthetic reporter containing two consensus GATA motifs upstream of the minimal Amh promoter with its WT1 binding site mutated. C. A synthetic reporter containing two consensus GATA motifs upstream of the minimal Amh promoter with an intact WT1 binding site. D. The minimal Amh promoter containing only a WT1 binding site. In all experiments, HeLa cells were co-transfected with the different promoter constructs (500 ng) along with an empty vector or expression vectors for the WT1(-KTS) (500 ng) and GATA4 (100 ng) used alone or in combination. All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
Figure Legend Snippet: Transcriptional synergism between GATA4 and WT1 on the Amh promoter requires GATA4 and WT1 binding to their respective regulatory elements . A. The native -180 bp Amh promoter containing the GATA and WT1 elements in their natural context. B. A synthetic reporter containing two consensus GATA motifs upstream of the minimal Amh promoter with its WT1 binding site mutated. C. A synthetic reporter containing two consensus GATA motifs upstream of the minimal Amh promoter with an intact WT1 binding site. D. The minimal Amh promoter containing only a WT1 binding site. In all experiments, HeLa cells were co-transfected with the different promoter constructs (500 ng) along with an empty vector or expression vectors for the WT1(-KTS) (500 ng) and GATA4 (100 ng) used alone or in combination. All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).

Techniques Used: Binding Assay, Transfection, Construct, Plasmid Preparation, Expressing, Activation Assay

SRY promoter GATA motifs bind GATA4 . An EMSA was performed with recombinant GATA4 protein and a 32 P-labeled oligonucleotide probe corresponding to the first consensus GATA motif (GATA site 1) of the pig SRY promoter. Competition with an oligonucleotide containing a mutated GATA motif (GATA to GGTA) was used to confirm the specificity of GATA4 binding (GATA mut site 1). GATA4 binding to the more distal GATA motifs (GATA sites 2–4) was then assessed by competition experiments using the indicated oligonucleotides.
Figure Legend Snippet: SRY promoter GATA motifs bind GATA4 . An EMSA was performed with recombinant GATA4 protein and a 32 P-labeled oligonucleotide probe corresponding to the first consensus GATA motif (GATA site 1) of the pig SRY promoter. Competition with an oligonucleotide containing a mutated GATA motif (GATA to GGTA) was used to confirm the specificity of GATA4 binding (GATA mut site 1). GATA4 binding to the more distal GATA motifs (GATA sites 2–4) was then assessed by competition experiments using the indicated oligonucleotides.

Techniques Used: Recombinant, Labeling, Binding Assay

GATA4 and WT1 transcriptionally cooperate to activate the SRY promoter . A. In addition to multiple GATA motifs, two potential WT1 binding sites (indicated by gray circles) are present in the first 2 kilobases of the mouse, human, and pig SRY promoters. B. Nucleotide sequence of the potential WT1 binding sites in the mouse, pig, and human SRY promoters. C. Western blot analysis of HeLa cells extracts (10 μg) overexpressing GATA4 and/or WT1 (+/- KTS) isoforms. D. WT1 and GATA4 transcriptionally cooperate. HeLa cells were co-transfected with either a -1090 bp mouse, -1400 bp pig, or -1250 bp human SRY -luciferase promoter construct (500 ng) along with an empty vector or expression vectors (500 ng) for WT1(-KTS) or WT1(+KTS) in the absence (-) or presence (+) of GATA4 (50 ng). All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
Figure Legend Snippet: GATA4 and WT1 transcriptionally cooperate to activate the SRY promoter . A. In addition to multiple GATA motifs, two potential WT1 binding sites (indicated by gray circles) are present in the first 2 kilobases of the mouse, human, and pig SRY promoters. B. Nucleotide sequence of the potential WT1 binding sites in the mouse, pig, and human SRY promoters. C. Western blot analysis of HeLa cells extracts (10 μg) overexpressing GATA4 and/or WT1 (+/- KTS) isoforms. D. WT1 and GATA4 transcriptionally cooperate. HeLa cells were co-transfected with either a -1090 bp mouse, -1400 bp pig, or -1250 bp human SRY -luciferase promoter construct (500 ng) along with an empty vector or expression vectors (500 ng) for WT1(-KTS) or WT1(+KTS) in the absence (-) or presence (+) of GATA4 (50 ng). All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).

Techniques Used: Binding Assay, Sequencing, Western Blot, Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Activation Assay

The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1 . A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a 32 P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
Figure Legend Snippet: The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1 . A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a 32 P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).

Techniques Used: Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Recombinant, Labeling, Functional Assay, Construct, Activation Assay

The zinc finger domains and C-terminal region of the GATA4 protein are required for the transcriptional cooperation and direct interaction with WT1 . A. The domain of the GATA4 protein involved in the transcriptional cooperation with WT1 was identified using the full-length GATA protein and four different GATA4 deletion mutants as indicated. HeLa cells were co-transfected with the -340 bp mouse Sry promoter (500 ng) along with an empty vector or expression vectors for the different GATA4 constructs in the absence (-) or presence (+) of a WT1(+KTS) expression vector (500 ng). All promoter activities are reported as fold activation over control ± S.E.M. The dotted line indicates the activation induced by WT1(+KTS) alone. *, Significantly different from the activation elicited by WT1 alone (P
Figure Legend Snippet: The zinc finger domains and C-terminal region of the GATA4 protein are required for the transcriptional cooperation and direct interaction with WT1 . A. The domain of the GATA4 protein involved in the transcriptional cooperation with WT1 was identified using the full-length GATA protein and four different GATA4 deletion mutants as indicated. HeLa cells were co-transfected with the -340 bp mouse Sry promoter (500 ng) along with an empty vector or expression vectors for the different GATA4 constructs in the absence (-) or presence (+) of a WT1(+KTS) expression vector (500 ng). All promoter activities are reported as fold activation over control ± S.E.M. The dotted line indicates the activation induced by WT1(+KTS) alone. *, Significantly different from the activation elicited by WT1 alone (P

Techniques Used: Transfection, Plasmid Preparation, Expressing, Construct, Activation Assay

Transcriptional properties of GATA4 on the mouse, pig, and human SRY promoters . A. Multiple consensus (but not species conserved) GATA binding motifs are present in the first 2 kilobases of the mouse, human, and pig SRY promoters; the GATA sites are indicated by the lozenges. B. Ability of GATA4 to transactivate the SRY promoter. HeLa cells were co-transfected with either a -1090 bp mouse, -1400 bp pig, or -1250 bp human SRY -luciferase promoter construct (500 ng) along with increasing amounts of a GATA4 expression vector (25, 50, or 100 ng). All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05). C. Western blot analysis of nuclear extracts (10 μg) from HeLa cells overexpressing GATA4.
Figure Legend Snippet: Transcriptional properties of GATA4 on the mouse, pig, and human SRY promoters . A. Multiple consensus (but not species conserved) GATA binding motifs are present in the first 2 kilobases of the mouse, human, and pig SRY promoters; the GATA sites are indicated by the lozenges. B. Ability of GATA4 to transactivate the SRY promoter. HeLa cells were co-transfected with either a -1090 bp mouse, -1400 bp pig, or -1250 bp human SRY -luciferase promoter construct (500 ng) along with increasing amounts of a GATA4 expression vector (25, 50, or 100 ng). All promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05). C. Western blot analysis of nuclear extracts (10 μg) from HeLa cells overexpressing GATA4.

Techniques Used: Binding Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation, Activation Assay, Western Blot

6) Product Images from "Modulation of the Pentose Phosphate Pathway Induces Endodermal Differentiation in Embryonic Stem Cells"

Article Title: Modulation of the Pentose Phosphate Pathway Induces Endodermal Differentiation in Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029321

Mechanism inducing endodermal cell differentiation. (A) RT-PCR of different lineage-specific marker genes in wt and G6pdΔ ES cells in presence of a lower oxygen concentration (5%) and in normal culture conditions (20%). (B) RT-PCR of different lineage-specific markers in differentiated wt E14 and Pgd +/− ES cells at 8, 10 and 13 days of neural differentiation. (C) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in Pgd +/− ES cells. Scale bars, 75 µm. (D) qRT-PCR for Sox17 and GATA4 in wt and G6pdΔ ES cells at day 10 after treatment with D-(-)-ribose during neural differentiation. Values are means ± SD (n = 3). * P
Figure Legend Snippet: Mechanism inducing endodermal cell differentiation. (A) RT-PCR of different lineage-specific marker genes in wt and G6pdΔ ES cells in presence of a lower oxygen concentration (5%) and in normal culture conditions (20%). (B) RT-PCR of different lineage-specific markers in differentiated wt E14 and Pgd +/− ES cells at 8, 10 and 13 days of neural differentiation. (C) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in Pgd +/− ES cells. Scale bars, 75 µm. (D) qRT-PCR for Sox17 and GATA4 in wt and G6pdΔ ES cells at day 10 after treatment with D-(-)-ribose during neural differentiation. Values are means ± SD (n = 3). * P

Techniques Used: Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Marker, Concentration Assay, Double Immunostaining, Quantitative RT-PCR

Inhibitors of the PPP induce endodermal differentiation. (A) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 in wt ES cells differentiated in presence of DHEA or 6AN. Scale bars, 50 µm. (B) qRT-PCR for Sox17 and GATA4 in wt ES cells at day 10 after treatment with DHEA or 6AN during neural differentiation. Values are means ± SD (n = 3). * P
Figure Legend Snippet: Inhibitors of the PPP induce endodermal differentiation. (A) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 in wt ES cells differentiated in presence of DHEA or 6AN. Scale bars, 50 µm. (B) qRT-PCR for Sox17 and GATA4 in wt ES cells at day 10 after treatment with DHEA or 6AN during neural differentiation. Values are means ± SD (n = 3). * P

Techniques Used: Double Immunostaining, Quantitative RT-PCR

Endodermal induction in G6pdΔ ES cells. (A) Analysis of different markers in wt and G6pdΔ ES cells during neural differentiation. Expression profiles of undifferentiated ES cells (Oct4 and Nanog), neural precursors (Nestin), neurons (NF-L), astrocytes (GFAP), mesendodermal precursors (GATA4), endodermal precursors (Sox17), and cardiac precursors (Nkx2.5) markers were analyzed by RT-PCR. RNA was isolated from cells at different days of differentiation. Lane C, positive control, RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (B) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in G6pdΔ ES cells. Scale bars, 50 µm. (C) RT-PCR analysis of GATA4, Sox17, NF-L (neural marker), TH (dopaminergic neuron marker) and GAD65 (gabaergic neuron marker) on wt, two different G6pdΔ ES cell lines, and G6pdΔ pG6pd during differentiation. Lane C, positive control, on RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (D) Western blot analysis with anti-Cripto and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated. (E) Western blot analysis with anti-phospho-Smad2 and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated.
Figure Legend Snippet: Endodermal induction in G6pdΔ ES cells. (A) Analysis of different markers in wt and G6pdΔ ES cells during neural differentiation. Expression profiles of undifferentiated ES cells (Oct4 and Nanog), neural precursors (Nestin), neurons (NF-L), astrocytes (GFAP), mesendodermal precursors (GATA4), endodermal precursors (Sox17), and cardiac precursors (Nkx2.5) markers were analyzed by RT-PCR. RNA was isolated from cells at different days of differentiation. Lane C, positive control, RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (B) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in G6pdΔ ES cells. Scale bars, 50 µm. (C) RT-PCR analysis of GATA4, Sox17, NF-L (neural marker), TH (dopaminergic neuron marker) and GAD65 (gabaergic neuron marker) on wt, two different G6pdΔ ES cell lines, and G6pdΔ pG6pd during differentiation. Lane C, positive control, on RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (D) Western blot analysis with anti-Cripto and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated. (E) Western blot analysis with anti-phospho-Smad2 and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Positive Control, Amplification, Double Immunostaining, Marker, Western Blot

Definitive and extraembryonic endoderm differentiated from G6pdΔ ES cells. (A) Immunofluorescent staining of the differentiated mouse G6pdΔ ES cells for Sox17 (red) and nuclei (blue) at 8 days of neural differentiation. PH, phase contrast images. Scale bars, 25 µm. (B) RT-PCR analysis of the endodermal markers GATA4 and extraembyonic endodermal markers Sox7 during differentiation. Lane C, positive control, RNA isolated from embryos and yolk sacs at 9,5 dpc. Amplified HPRT is shown as a positive control. (C) qRT-PCR for Pdx1 in wt and G6pdΔ ES cells at 13 days after treatment with Indolactam V from day 8 during differentiation. Values are means ± SD (n = 2). * P
Figure Legend Snippet: Definitive and extraembryonic endoderm differentiated from G6pdΔ ES cells. (A) Immunofluorescent staining of the differentiated mouse G6pdΔ ES cells for Sox17 (red) and nuclei (blue) at 8 days of neural differentiation. PH, phase contrast images. Scale bars, 25 µm. (B) RT-PCR analysis of the endodermal markers GATA4 and extraembyonic endodermal markers Sox7 during differentiation. Lane C, positive control, RNA isolated from embryos and yolk sacs at 9,5 dpc. Amplified HPRT is shown as a positive control. (C) qRT-PCR for Pdx1 in wt and G6pdΔ ES cells at 13 days after treatment with Indolactam V from day 8 during differentiation. Values are means ± SD (n = 2). * P

Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Isolation, Amplification, Quantitative RT-PCR

7) Product Images from "NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients"

Article Title: NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients

Journal: Asian Journal of Andrology

doi: 10.4103/1008-682X.159722

Expression profiles of NODAL's receptors in OA and SCO testis. ( a ): Immunohistochemical staining showed that ALK4, ALK7, and ACTR-IIB were detected in GATA4-positive Sertoli cells in OA testis. (a’): Negative control of immunocytochemistry of the OA testes, displaying nonspecific staining. ( b ): Immunohistochemical staining displayed that ALK4, ALK7, and ACTR-IIB were expressed in GATA4-positive Sertoli cells in SCO testis. No specific staining was seen in other cells. (b’): Negative control of immunohistochemistry of the OA testes without primary antibody. Scale bars = 50 μm. OA: obstructive azoospermia; SCO: Sertoli cell-only syndrome.
Figure Legend Snippet: Expression profiles of NODAL's receptors in OA and SCO testis. ( a ): Immunohistochemical staining showed that ALK4, ALK7, and ACTR-IIB were detected in GATA4-positive Sertoli cells in OA testis. (a’): Negative control of immunocytochemistry of the OA testes, displaying nonspecific staining. ( b ): Immunohistochemical staining displayed that ALK4, ALK7, and ACTR-IIB were expressed in GATA4-positive Sertoli cells in SCO testis. No specific staining was seen in other cells. (b’): Negative control of immunohistochemistry of the OA testes without primary antibody. Scale bars = 50 μm. OA: obstructive azoospermia; SCO: Sertoli cell-only syndrome.

Techniques Used: Expressing, Immunohistochemistry, Staining, Negative Control, Immunocytochemistry

Morphology of testis from OA and SCO patients and identification of the isolated human Sertoli cells, spermatogonia, spermatocytes, and spermatids. ( a ): H E staining illustrated the morphology of testicular tissues from OA (left panel) and SCO patients (right panel). Histological examination showed that seminiferous tubule from SCO testis had a reduced diameter, with only Sertoli cells along the basement membrane, compared with that of OA. Scale bars = 10 μm. ( b ): The human Sertoli cells isolated from OA and SCO patient testes expressed transcripts of GATA4 , ABP , WT1 , and FSHR but no transcripts of VASA , HSD3B , CYP17A1 , SMA , MYH11 , CD34 , and CD105 . 1 and 2 represents OA Sertoli cells and SCO Sertoli cells, respectively. ( c ): RT-PCR showed that the expresion of specific makers in isolated human spermatogonia ( GFRA1 , RET , and UCHL1 ), spermatocytes ( SCP1 and SCP3 ), and spermatids ( PRM1 , PRM2 , TP1 , and TP2 ). ( d ): Immunocytochemistry revealed that both GATA4 (red fluorescence) and WT1 (green fluorescence) were expressed in human Sertoli cells isolated from OA and SCO patients testes. The enlarged images were shown in the up-left of the pictures. Scale bar = 10 μm. ( e ): Immunocytochemistry displayed the co-expressions of GATA4 (red fluorescence) and VIMENTIN (green fluorescence) in human Sertoli cells isolated from OA and SCO patient testes. Scale bars = 10 μm. ( f ): Negtive control staining of immunocytochemistry of human Sertoli cells without primary antibody. Scale bars = 10 μm. ( g ): Immunocytochemistry displayed the co-expressions of GFRA1 (green fluorescence), PIWIL2 (green fluorescence), and ACROSIN (green fluorescence) with VASA (Red fluorescence) in isolated germ cells. ( h ): Negtive control staining of immunocytochemistry of the human germ cells without primary antibody. Scale bars = 10 μm. Notes – OA: obstructive azoospermia, SCO: Sertoli cell-only syndrome, SC: Sertoli cells; Spg: spermatogonia; Spc: spermatocytes; Spt: spermatids. Passage 2 Sertoli cells were used in this experiment.
Figure Legend Snippet: Morphology of testis from OA and SCO patients and identification of the isolated human Sertoli cells, spermatogonia, spermatocytes, and spermatids. ( a ): H E staining illustrated the morphology of testicular tissues from OA (left panel) and SCO patients (right panel). Histological examination showed that seminiferous tubule from SCO testis had a reduced diameter, with only Sertoli cells along the basement membrane, compared with that of OA. Scale bars = 10 μm. ( b ): The human Sertoli cells isolated from OA and SCO patient testes expressed transcripts of GATA4 , ABP , WT1 , and FSHR but no transcripts of VASA , HSD3B , CYP17A1 , SMA , MYH11 , CD34 , and CD105 . 1 and 2 represents OA Sertoli cells and SCO Sertoli cells, respectively. ( c ): RT-PCR showed that the expresion of specific makers in isolated human spermatogonia ( GFRA1 , RET , and UCHL1 ), spermatocytes ( SCP1 and SCP3 ), and spermatids ( PRM1 , PRM2 , TP1 , and TP2 ). ( d ): Immunocytochemistry revealed that both GATA4 (red fluorescence) and WT1 (green fluorescence) were expressed in human Sertoli cells isolated from OA and SCO patients testes. The enlarged images were shown in the up-left of the pictures. Scale bar = 10 μm. ( e ): Immunocytochemistry displayed the co-expressions of GATA4 (red fluorescence) and VIMENTIN (green fluorescence) in human Sertoli cells isolated from OA and SCO patient testes. Scale bars = 10 μm. ( f ): Negtive control staining of immunocytochemistry of human Sertoli cells without primary antibody. Scale bars = 10 μm. ( g ): Immunocytochemistry displayed the co-expressions of GFRA1 (green fluorescence), PIWIL2 (green fluorescence), and ACROSIN (green fluorescence) with VASA (Red fluorescence) in isolated germ cells. ( h ): Negtive control staining of immunocytochemistry of the human germ cells without primary antibody. Scale bars = 10 μm. Notes – OA: obstructive azoospermia, SCO: Sertoli cell-only syndrome, SC: Sertoli cells; Spg: spermatogonia; Spc: spermatocytes; Spt: spermatids. Passage 2 Sertoli cells were used in this experiment.

Techniques Used: Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry, Fluorescence, Single-particle Tracking

8) Product Images from "Distinct iPS Cells Show Different Cardiac Differentiation Efficiency"

Article Title: Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

Journal: Stem Cells International

doi: 10.1155/2013/659739

Cardiomyocyte (CM) specific gene expression profiles, as determined by RT-PCR and immunostaining, of CM derived from mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. (a) CM-associated structural protein gene expression profiles. The expression of Actn1 , Myh6 , Myh7 , Myl7 , Myl2 , and Nppb was analyzed by semiquantitative RT-PCR analysis. GAPDH was used as an internal control. (b) Immunofluorescent staining of typical CM-specific proteins on day 15 of differentiation in CM derived from ES, Nanog-iPS, and Fbx15-iPS cells. Cells were stained with Nkx2.5 (green), GATA4 (green), atrial natriuretic peptide ( ANP ; green), myosin heavy chain ( MHC ; red), and α - actinin (red). Scale bar = 50 μ m.
Figure Legend Snippet: Cardiomyocyte (CM) specific gene expression profiles, as determined by RT-PCR and immunostaining, of CM derived from mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. (a) CM-associated structural protein gene expression profiles. The expression of Actn1 , Myh6 , Myh7 , Myl7 , Myl2 , and Nppb was analyzed by semiquantitative RT-PCR analysis. GAPDH was used as an internal control. (b) Immunofluorescent staining of typical CM-specific proteins on day 15 of differentiation in CM derived from ES, Nanog-iPS, and Fbx15-iPS cells. Cells were stained with Nkx2.5 (green), GATA4 (green), atrial natriuretic peptide ( ANP ; green), myosin heavy chain ( MHC ; red), and α - actinin (red). Scale bar = 50 μ m.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Derivative Assay, Staining, Aqueous Normal-phase Chromatography

Cardiomyocyte differentiation efficiency of pluripotent stem cells and temporal gene expression patterns during cardiomyocyte differentiation. (a) Percentage of beating colonies on days 6–15 in mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. Data are the mean ± SEM ( n = 5 in all groups). ((b)–(g)) Quantitative RT-PCR analyses showing temporal gene expression patterns of the mesodermal marker Brachyury T (b), the early cardiac mesodermal marker Mesp1 (c), the cardiac-specific transcription factors Nkx2.5 (d) and Gata4 (e), and the cardiac-specific proteins Nppa (f) and Myl2 (g) in EB3 ES cells (closed squares), 20D17 Nanog-iPS cells (open circles), and WT-1 Fbx15-iPS cells (open triangles). Data are the mean ± SEM ( n = 5 in all groups).
Figure Legend Snippet: Cardiomyocyte differentiation efficiency of pluripotent stem cells and temporal gene expression patterns during cardiomyocyte differentiation. (a) Percentage of beating colonies on days 6–15 in mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. Data are the mean ± SEM ( n = 5 in all groups). ((b)–(g)) Quantitative RT-PCR analyses showing temporal gene expression patterns of the mesodermal marker Brachyury T (b), the early cardiac mesodermal marker Mesp1 (c), the cardiac-specific transcription factors Nkx2.5 (d) and Gata4 (e), and the cardiac-specific proteins Nppa (f) and Myl2 (g) in EB3 ES cells (closed squares), 20D17 Nanog-iPS cells (open circles), and WT-1 Fbx15-iPS cells (open triangles). Data are the mean ± SEM ( n = 5 in all groups).

Techniques Used: Expressing, Quantitative RT-PCR, Marker

9) Product Images from "Inhibitory effect of oxidative damage on cardiomyocyte differentiation from Wharton's jelly-derived mesenchymal stem cells"

Article Title: Inhibitory effect of oxidative damage on cardiomyocyte differentiation from Wharton's jelly-derived mesenchymal stem cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2017.5249

Immunofluorescence micrographs of the cardiac-specific proteins GATA4 and cTnT in 5-azacytidine-treated (A) WJ-MSCs and (B) BM-MSCs with or without H 2 O 2 treatment (scale bars, 100 µM). Lower fluorescence intensities of the two markers were observed in H 2 O 2 -treated cells compared with untreated cells. BM-MSCs, bone marrow-derived mesenchymal stem cells; WJ-MSCs, Wharton's jelly-derived MSCs; cTnT, cardiac troponin T.
Figure Legend Snippet: Immunofluorescence micrographs of the cardiac-specific proteins GATA4 and cTnT in 5-azacytidine-treated (A) WJ-MSCs and (B) BM-MSCs with or without H 2 O 2 treatment (scale bars, 100 µM). Lower fluorescence intensities of the two markers were observed in H 2 O 2 -treated cells compared with untreated cells. BM-MSCs, bone marrow-derived mesenchymal stem cells; WJ-MSCs, Wharton's jelly-derived MSCs; cTnT, cardiac troponin T.

Techniques Used: Immunofluorescence, Fluorescence, Derivative Assay

Fluorescence intensities of (A) GATA4 and (B) cTnT on day 3 as well as (C) GATA4 and (D) cTnT on day 7 were decreased after exposure to hydrogen peroxide (200, 500 or 1,000 µM) when compared with that in untreated cells at the same time-points. Values are expressed as the mean ± standard error of the mean of six separate experiments performed in WJ-MSCs and three separate experiments performed in BM-MSCs. # ,*P
Figure Legend Snippet: Fluorescence intensities of (A) GATA4 and (B) cTnT on day 3 as well as (C) GATA4 and (D) cTnT on day 7 were decreased after exposure to hydrogen peroxide (200, 500 or 1,000 µM) when compared with that in untreated cells at the same time-points. Values are expressed as the mean ± standard error of the mean of six separate experiments performed in WJ-MSCs and three separate experiments performed in BM-MSCs. # ,*P

Techniques Used: Fluorescence

10) Product Images from "The Transcription Factor GATA4 Is Activated by Extracellular Signal-Regulated Kinase 1- and 2-Mediated Phosphorylation of Serine 105 in Cardiomyocytes"

Article Title: The Transcription Factor GATA4 Is Activated by Extracellular Signal-Regulated Kinase 1- and 2-Mediated Phosphorylation of Serine 105 in Cardiomyocytes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.21.7460-7469.2001

GATA4-engrailed blocks MEK1-ERK1/2-induced sarcomeric organization and hypertrophy. Representative images of immunostained cardiomyocyte cultures infected with each of the indicated adenoviral constructs are shown. The left panels show anti-α-actinin antibody (orange) reactivity to demonstrate cardiomyocyte sarcomeres and gross morphology. The right panels show the same cells double immunostained for ANF (green), which appears in a perinuclear pattern.
Figure Legend Snippet: GATA4-engrailed blocks MEK1-ERK1/2-induced sarcomeric organization and hypertrophy. Representative images of immunostained cardiomyocyte cultures infected with each of the indicated adenoviral constructs are shown. The left panels show anti-α-actinin antibody (orange) reactivity to demonstrate cardiomyocyte sarcomeres and gross morphology. The right panels show the same cells double immunostained for ANF (green), which appears in a perinuclear pattern.

Techniques Used: Infection, Construct

ERK2, but not p38 and JNK, mediates phosphorylation of serine 105. (A) In vitro kinase reaction with bacterially purified wild-type (Wt) full-length GATA4 or the S105A mutant GATA4 incubated with purified ERK2 in the presence of [ 32 P]ATP and subjected to SDS-polyacrylamide gel electrophoresis. Both proteins were also incubated with purified p38α (B) and JNK1 (C) protein to assess phosphorylation. MBP and recombinant c-Jun were used as positive controls in the kinase assays. The arrow shows the migration of the full-length GST-GATA4 fusion protein, although degradation products of faster migration were also visible.
Figure Legend Snippet: ERK2, but not p38 and JNK, mediates phosphorylation of serine 105. (A) In vitro kinase reaction with bacterially purified wild-type (Wt) full-length GATA4 or the S105A mutant GATA4 incubated with purified ERK2 in the presence of [ 32 P]ATP and subjected to SDS-polyacrylamide gel electrophoresis. Both proteins were also incubated with purified p38α (B) and JNK1 (C) protein to assess phosphorylation. MBP and recombinant c-Jun were used as positive controls in the kinase assays. The arrow shows the migration of the full-length GST-GATA4 fusion protein, although degradation products of faster migration were also visible.

Techniques Used: In Vitro, Purification, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Recombinant, Migration

GATA4 contains a conserved MAPK phosphorylation site at serine 105. (A) Diagram of GATA4 showing the position of the zinc finger domains (Zn), the nuclear localization sequence and basic domain (nls), and the transcriptional activation domain (TAD). A MAPK recognition site containing serine 105 is conserved between mouse, human, and chicken GATA4. (B) Phospho-105-GATA4-specific antiserum was generated and used to examine GATA4 phosphorylation in response to α-adrenergic stimulation with PE in cultured cardiomyocytes. Phosphorylation of endogenous GATA4 was upregulated by PE in control Adβgal-infected cultures, while a similar pattern of up-regulation was observed when GATA4 was overexpressed by AdGATA4 infection. (C) Endogenous GATA4 was also phosphorylated at serine 105 in the hearts of PE-injected mice (3 h), compared to saline-treated control mice ( n = 3 in each group). GATA4 protein levels did not vary.
Figure Legend Snippet: GATA4 contains a conserved MAPK phosphorylation site at serine 105. (A) Diagram of GATA4 showing the position of the zinc finger domains (Zn), the nuclear localization sequence and basic domain (nls), and the transcriptional activation domain (TAD). A MAPK recognition site containing serine 105 is conserved between mouse, human, and chicken GATA4. (B) Phospho-105-GATA4-specific antiserum was generated and used to examine GATA4 phosphorylation in response to α-adrenergic stimulation with PE in cultured cardiomyocytes. Phosphorylation of endogenous GATA4 was upregulated by PE in control Adβgal-infected cultures, while a similar pattern of up-regulation was observed when GATA4 was overexpressed by AdGATA4 infection. (C) Endogenous GATA4 was also phosphorylated at serine 105 in the hearts of PE-injected mice (3 h), compared to saline-treated control mice ( n = 3 in each group). GATA4 protein levels did not vary.

Techniques Used: Sequencing, Activation Assay, Generated, Cell Culture, Infection, Injection, Mouse Assay

MEK1-ERK1/2 signaling promotes phosphorylation of serine 105 in GATA4. (A) Cardiomyocyte cultures were coinfected with AdGATA4 and either Adβgal, AdMEK1, AdMKK7, or AdMKK6. Twenty-four hours later, cultures were harvested and Western blotted with phospho-specific GATA4 antibody and then reprobed with GATA4 antibody. (B) Cardiomyocyte cultures were coinfected with AdGATA4 and either Adβgal, Ad-dnMEK1, Ad-dnMKK4, or Ad-dnMKK3 and stimulated with PE for 3 h. Western blotting demonstrated reduction in PE-induced serine 105 phosphorylation with dominant-negative MEK1 (asterisk) but not with other dominant-negative MAPK kinase factors. (C) Quantitation of three independent experiments demonstrates a significant inhibition of PE-induced serine 105 phosphorylation only with Ad-dnMEK1 infection. *, P
Figure Legend Snippet: MEK1-ERK1/2 signaling promotes phosphorylation of serine 105 in GATA4. (A) Cardiomyocyte cultures were coinfected with AdGATA4 and either Adβgal, AdMEK1, AdMKK7, or AdMKK6. Twenty-four hours later, cultures were harvested and Western blotted with phospho-specific GATA4 antibody and then reprobed with GATA4 antibody. (B) Cardiomyocyte cultures were coinfected with AdGATA4 and either Adβgal, Ad-dnMEK1, Ad-dnMKK4, or Ad-dnMKK3 and stimulated with PE for 3 h. Western blotting demonstrated reduction in PE-induced serine 105 phosphorylation with dominant-negative MEK1 (asterisk) but not with other dominant-negative MAPK kinase factors. (C) Quantitation of three independent experiments demonstrates a significant inhibition of PE-induced serine 105 phosphorylation only with Ad-dnMEK1 infection. *, P

Techniques Used: Western Blot, Dominant Negative Mutation, Quantitation Assay, Inhibition, Infection

ERK2 directly phosphorylates GATA4 in vitro. (A) Schematic representation of full-length GATA4 and the two different fusion proteins that were generated and purified from bacteria. (B) Polyacrylamide gel of [ 32 P]ATP-labeled proteins. Three hundred nanograms of GST-GATA4 fusion protein, GST alone, or MBP was incubated with 25 ng of activated ERK2 protein for the labeling reaction. Only MBP and the GST-G4 fusion protein containing GATA4 amino acids 80 to 250 were efficiently labeled. (C) Western blot with GATA4 phospho-specific antiserum and ERK2-mediated in vitro-labeled GST and GST-GATA4 fusion proteins (300 ng). The asterisk shows the specific increase in ERK2-mediated phosphorylation of GST-GATA4 80-250.
Figure Legend Snippet: ERK2 directly phosphorylates GATA4 in vitro. (A) Schematic representation of full-length GATA4 and the two different fusion proteins that were generated and purified from bacteria. (B) Polyacrylamide gel of [ 32 P]ATP-labeled proteins. Three hundred nanograms of GST-GATA4 fusion protein, GST alone, or MBP was incubated with 25 ng of activated ERK2 protein for the labeling reaction. Only MBP and the GST-G4 fusion protein containing GATA4 amino acids 80 to 250 were efficiently labeled. (C) Western blot with GATA4 phospho-specific antiserum and ERK2-mediated in vitro-labeled GST and GST-GATA4 fusion proteins (300 ng). The asterisk shows the specific increase in ERK2-mediated phosphorylation of GST-GATA4 80-250.

Techniques Used: In Vitro, Generated, Purification, Labeling, Incubation, Western Blot

Serine 105 in GATA4 mediates PE transcriptional responsiveness. (A) A plasmid encoding the GAL4 DNA binding domain fused to the wild-type (Wt) or S105A mutant GATA4 transcriptional activation domain (amino acids 33 to 227) was cotransfected into cardiomyocytes with the GAL4-luciferase reporter, G5E1b-luciferase. Cultures were either PE stimulated, left unstimulated, or treated with U0126 or SB202190 (SB) for 24 h. *, P
Figure Legend Snippet: Serine 105 in GATA4 mediates PE transcriptional responsiveness. (A) A plasmid encoding the GAL4 DNA binding domain fused to the wild-type (Wt) or S105A mutant GATA4 transcriptional activation domain (amino acids 33 to 227) was cotransfected into cardiomyocytes with the GAL4-luciferase reporter, G5E1b-luciferase. Cultures were either PE stimulated, left unstimulated, or treated with U0126 or SB202190 (SB) for 24 h. *, P

Techniques Used: Plasmid Preparation, Binding Assay, Mutagenesis, Activation Assay, Luciferase

GATA4-engrailed blocks MEK1-ERK1/2-induced cardiomyocyte hypertrophy. (A) Western blotting with Flag monoclonal antibody demonstrates a protein of the predicted size from AdG4-Engr-infected cardiomyocytes. (B) Cell surface areas were quantified from each of the indicated adenoviral infected cardiomyocyte cultures. *, P
Figure Legend Snippet: GATA4-engrailed blocks MEK1-ERK1/2-induced cardiomyocyte hypertrophy. (A) Western blotting with Flag monoclonal antibody demonstrates a protein of the predicted size from AdG4-Engr-infected cardiomyocytes. (B) Cell surface areas were quantified from each of the indicated adenoviral infected cardiomyocyte cultures. *, P

Techniques Used: Western Blot, Infection

ERK2-mediated phosphorylation of GATA4 enhances its DNA binding activity. (A) EMSA demonstrates that ERK2 enhances the DNA binding activity of a bacterially purified GST-GATA4 (full-length) wild-type (Wt) fusion protein but not GST-S105A mutant GATA4 (full-length). (B) Western blot analysis with phospho-specific GATA4 (serine 105) antiserum using the same protein reactions used for panel A demonstrates efficient ERK2-mediated phosphorylation of full-length GATA4, but not the S105A mutant GATA4 or GST. (C) Western blot analysis with GATA4 antibody using the same extracts from panel A demonstrates equal protein levels.
Figure Legend Snippet: ERK2-mediated phosphorylation of GATA4 enhances its DNA binding activity. (A) EMSA demonstrates that ERK2 enhances the DNA binding activity of a bacterially purified GST-GATA4 (full-length) wild-type (Wt) fusion protein but not GST-S105A mutant GATA4 (full-length). (B) Western blot analysis with phospho-specific GATA4 (serine 105) antiserum using the same protein reactions used for panel A demonstrates efficient ERK2-mediated phosphorylation of full-length GATA4, but not the S105A mutant GATA4 or GST. (C) Western blot analysis with GATA4 antibody using the same extracts from panel A demonstrates equal protein levels.

Techniques Used: Binding Assay, Activity Assay, Purification, Mutagenesis, Western Blot

11) Product Images from "Zfp322a Regulates Mouse ES Cell Pluripotency and Enhances Reprogramming Efficiency"

Article Title: Zfp322a Regulates Mouse ES Cell Pluripotency and Enhances Reprogramming Efficiency

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004038

Zfp322a can enhance OSKM reprogramming and replace Sox2. ( A ) Zfp322a enhanced reprogramming efficiency and accelerated the onset of reprogramming process. OSKM serves as control experiment. ( B ) The iPSCs generated from OSKM plus Zfp322a presented alkaline phosphatase activity. There were more AP stained colonies generated from OKSM+Zfp322a compare to OKSM. ( C ) The iPSCs expressed endogenous Oct4, Nanog, Sox2, Rex1 and SSEA-1, indicating that they were ES-cell like. Immunostaining using anti-Oct4, anti-Nanog anti-Sox2, anti-Rex1 and anti-SSEA-1 antibodies were performed with GFP + iPSCs generated from OKSM+Zfp322a. ( D ) GFP + iPSCs generated by OKSM+Zfp322a were able to express ectoderm, mesoderm and endoderm lineage markers in the EB formation assay. iPSCs were stained with anti-Nestin, anti-Gata4 and anti-alpha smooth muscle actin (SMA) antibodies and pictures were taken at 60× magnification. DAPI (blue) served as nucleus marker. ( E ) Zfp322a was able to replace Sox2, but not Oct4 or Klf4 in OSKM reprogramming process. Results from three independent experiments were presented. ( F ) iPSCs generated from OKM plus Zfp322a were positive with AP staining and more AP positive colonies were observed in OKM+Zfp322a as compared to OKSM. ( G ) iPSCs generated by OKM plus Zfp322a expressed pluripotency markers Oct4, Nanog, Sox2, Rex1 and SSEA-1. ( H ) iPSCs derived from OKM+Zfp322a could differentiate into ectoderm, mesoderm and endoderm lineages, which were showed by anti-Nestin, anti-Gata4, anti-SMA staining respectively.
Figure Legend Snippet: Zfp322a can enhance OSKM reprogramming and replace Sox2. ( A ) Zfp322a enhanced reprogramming efficiency and accelerated the onset of reprogramming process. OSKM serves as control experiment. ( B ) The iPSCs generated from OSKM plus Zfp322a presented alkaline phosphatase activity. There were more AP stained colonies generated from OKSM+Zfp322a compare to OKSM. ( C ) The iPSCs expressed endogenous Oct4, Nanog, Sox2, Rex1 and SSEA-1, indicating that they were ES-cell like. Immunostaining using anti-Oct4, anti-Nanog anti-Sox2, anti-Rex1 and anti-SSEA-1 antibodies were performed with GFP + iPSCs generated from OKSM+Zfp322a. ( D ) GFP + iPSCs generated by OKSM+Zfp322a were able to express ectoderm, mesoderm and endoderm lineage markers in the EB formation assay. iPSCs were stained with anti-Nestin, anti-Gata4 and anti-alpha smooth muscle actin (SMA) antibodies and pictures were taken at 60× magnification. DAPI (blue) served as nucleus marker. ( E ) Zfp322a was able to replace Sox2, but not Oct4 or Klf4 in OSKM reprogramming process. Results from three independent experiments were presented. ( F ) iPSCs generated from OKM plus Zfp322a were positive with AP staining and more AP positive colonies were observed in OKM+Zfp322a as compared to OKSM. ( G ) iPSCs generated by OKM plus Zfp322a expressed pluripotency markers Oct4, Nanog, Sox2, Rex1 and SSEA-1. ( H ) iPSCs derived from OKM+Zfp322a could differentiate into ectoderm, mesoderm and endoderm lineages, which were showed by anti-Nestin, anti-Gata4, anti-SMA staining respectively.

Techniques Used: Generated, Activity Assay, Staining, Immunostaining, Tube Formation Assay, Marker, Derivative Assay

12) Product Images from "All-Trans Retinoic Acid Directs Urothelial Specification of Murine Embryonic Stem Cells via GATA4/6 Signaling Mechanisms"

Article Title: All-Trans Retinoic Acid Directs Urothelial Specification of Murine Embryonic Stem Cells via GATA4/6 Signaling Mechanisms

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011513

Murine UP1B and UP2 promoters contain GATA-DNA binding sites which recruit complexes containing GATA4/6 in response to RA stimulation. Nuclear extracts from RA-treated ESCs following 9 d show distinct GATA-DNA complexes with 32 P-labeled consensus oligos specific for GATA binding to 2 kb UP1B [ A, C ] or UP2 [ B, D ] promoter fragments. Complexes were absent in spontaneously differentiating controls (C). Both complexes were inhibited by addition of respective excess unlabeled wild type (wt) but not mutant GATA oligos (m1, m2). [ C, D ] The presence of GATA4 (G4) and GATA6 (G6) in both the GATA-UP complexes was determined by supershift or immunodepletion (ID) following antibody-specific incubation (G4, G6), but not species-matched isotype control antibodies (IgG). NS represents non specific band. FP denotes excess free probe.
Figure Legend Snippet: Murine UP1B and UP2 promoters contain GATA-DNA binding sites which recruit complexes containing GATA4/6 in response to RA stimulation. Nuclear extracts from RA-treated ESCs following 9 d show distinct GATA-DNA complexes with 32 P-labeled consensus oligos specific for GATA binding to 2 kb UP1B [ A, C ] or UP2 [ B, D ] promoter fragments. Complexes were absent in spontaneously differentiating controls (C). Both complexes were inhibited by addition of respective excess unlabeled wild type (wt) but not mutant GATA oligos (m1, m2). [ C, D ] The presence of GATA4 (G4) and GATA6 (G6) in both the GATA-UP complexes was determined by supershift or immunodepletion (ID) following antibody-specific incubation (G4, G6), but not species-matched isotype control antibodies (IgG). NS represents non specific band. FP denotes excess free probe.

Techniques Used: Binding Assay, Labeling, Mutagenesis, Incubation

Nuclear GATA4 and GATA6 expression is associated with UP expression both in vitro and in vivo. [ A ] Immunoblot analysis of nuclear (NUC) and cytoplasmic (CYT) protein fractions demonstrating enrichment of GATA4 and GATA6 in nuclear extracts of wild type (WT) RA-treated ESCs following 9 d of cultivation. The degree of Ponceau S staining of membranes after transfer was used to indicate uniform sample loading. [ B ] Photomicrographs of RA-treated UP2-GFP+ cells co-stained for GFP (green, FITC) and nuclear GATA4/6 (red, Cy3) following 14 d of culture. Observed populations included: GFP/GATA4/6+ cells (denoted by white arrows), cells positive for GATA4/6 expression alone (denoted by orange arrows), and GFP+, but GATA4/6 negative cell types (denoted by yellow arrows). Scale bar = 500 µm. [ C ] Photomicrographs of adult murine (C57BL/6) bladder urothelium showing nuclear localization of GATA4/6 in superficial cells (denoted by white arrows), while exclusion in the basal and intermediate cell layers (denoted by yellow arrows). Scale bar = 40 µm. For both [ A, B ], images were merged with DAPI nuclear counterstain (blue).
Figure Legend Snippet: Nuclear GATA4 and GATA6 expression is associated with UP expression both in vitro and in vivo. [ A ] Immunoblot analysis of nuclear (NUC) and cytoplasmic (CYT) protein fractions demonstrating enrichment of GATA4 and GATA6 in nuclear extracts of wild type (WT) RA-treated ESCs following 9 d of cultivation. The degree of Ponceau S staining of membranes after transfer was used to indicate uniform sample loading. [ B ] Photomicrographs of RA-treated UP2-GFP+ cells co-stained for GFP (green, FITC) and nuclear GATA4/6 (red, Cy3) following 14 d of culture. Observed populations included: GFP/GATA4/6+ cells (denoted by white arrows), cells positive for GATA4/6 expression alone (denoted by orange arrows), and GFP+, but GATA4/6 negative cell types (denoted by yellow arrows). Scale bar = 500 µm. [ C ] Photomicrographs of adult murine (C57BL/6) bladder urothelium showing nuclear localization of GATA4/6 in superficial cells (denoted by white arrows), while exclusion in the basal and intermediate cell layers (denoted by yellow arrows). Scale bar = 40 µm. For both [ A, B ], images were merged with DAPI nuclear counterstain (blue).

Techniques Used: Expressing, In Vitro, In Vivo, Staining

GATA4 and GATA6 are crucial signaling molecules in RA-mediated upregulation of UP expression in ESCs. Real time RT-PCR analysis of uroplakin expression in WT, GATA4−/− [ A ] or GATA6−/− [ B ] ESCs cultured in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. ESC(0) = undifferentiated ESCs. Levels normalized to GAPDH expression. Mean ± SD per data point. N = 3–4 per data point. (*) = p
Figure Legend Snippet: GATA4 and GATA6 are crucial signaling molecules in RA-mediated upregulation of UP expression in ESCs. Real time RT-PCR analysis of uroplakin expression in WT, GATA4−/− [ A ] or GATA6−/− [ B ] ESCs cultured in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. ESC(0) = undifferentiated ESCs. Levels normalized to GAPDH expression. Mean ± SD per data point. N = 3–4 per data point. (*) = p

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

13) Product Images from "Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells"

Article Title: Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113262

HDAC can repress the transcriptional activity of T/Bry via physical interaction. ( A ) HDAC1 and HDAC3 interact with the T-box transcription factor T/Bry. Co-immunoprecipitation (Co-IP) was performed using control IgG or T/Bry antibody, followed by western blot analysis for HDAC1 and HDAC3. 5% Input (v/v) indicated that the ratio between the loading sample and precipitation is one to twenty. ( B ) Co-IP was performed using control IgG or HDAC1 antibody, followed by western blot analysis for T/Bry. ( C ) Co-IP was performed using control IgG or HDAC3 antibody, followed by western blot analysis for T/Bry. ( D ) HDAC3 does not interact with Gata4. Co-IP was performed using control IgG or HDAC3 antibody, followed by western blot analysis for Gata4. ( E ) A summary model shows the mechanism of HDACs in regulating the expression of mesodermal genes.
Figure Legend Snippet: HDAC can repress the transcriptional activity of T/Bry via physical interaction. ( A ) HDAC1 and HDAC3 interact with the T-box transcription factor T/Bry. Co-immunoprecipitation (Co-IP) was performed using control IgG or T/Bry antibody, followed by western blot analysis for HDAC1 and HDAC3. 5% Input (v/v) indicated that the ratio between the loading sample and precipitation is one to twenty. ( B ) Co-IP was performed using control IgG or HDAC1 antibody, followed by western blot analysis for T/Bry. ( C ) Co-IP was performed using control IgG or HDAC3 antibody, followed by western blot analysis for T/Bry. ( D ) HDAC3 does not interact with Gata4. Co-IP was performed using control IgG or HDAC3 antibody, followed by western blot analysis for Gata4. ( E ) A summary model shows the mechanism of HDACs in regulating the expression of mesodermal genes.

Techniques Used: Activity Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Expressing

Loss of HDAC1 or 3 enhances mesodermal lineage differentiation. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. ( D ) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
Figure Legend Snippet: Loss of HDAC1 or 3 enhances mesodermal lineage differentiation. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. ( D ) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

Techniques Used: Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Expressing, Two Tailed Test

Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. ( D ) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
Figure Legend Snippet: Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. ( D ) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

Techniques Used: Expressing, Staining, Over Expression, Western Blot, Quantitative RT-PCR, Immunofluorescence, Two Tailed Test

14) Product Images from "Dynamic GATA6 Expression in Primitive Endoderm Formation and Maturation in Early Mouse Embryogenesis"

Article Title: Dynamic GATA6 Expression in Primitive Endoderm Formation and Maturation in Early Mouse Embryogenesis

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

doi: 10.1002/dvdy.21703

Detection of variable GATA6 expression in visceral endoderm cells of embryonic day (E) 5.0 embryos. E5.0 embryos from timed matings of wild-type mice were analyzed by histology and immunostaining. Sequential sections stained for GATA4, GATA6, laminin,
Figure Legend Snippet: Detection of variable GATA6 expression in visceral endoderm cells of embryonic day (E) 5.0 embryos. E5.0 embryos from timed matings of wild-type mice were analyzed by histology and immunostaining. Sequential sections stained for GATA4, GATA6, laminin,

Techniques Used: Expressing, Mouse Assay, Immunostaining, Staining

A model for lineage derivation in postimplanted blastocysts. Schematic illustration shows a model for the lineage derivation in postimplantation blastocysts. It is hypothesized that induction of GATA4 and GATA6 expression leads to the origination of primitive
Figure Legend Snippet: A model for lineage derivation in postimplanted blastocysts. Schematic illustration shows a model for the lineage derivation in postimplantation blastocysts. It is hypothesized that induction of GATA4 and GATA6 expression leads to the origination of primitive

Techniques Used: Expressing

Failure of extraembryonic endoderm formation in GATA6-deficient embryos. embryonic day (E) 5.5 and E6.5 embryos from timed matings of GATA6 (+/−) mice were analyzed by histology and immunostaining. Adjacent sections were stained for GATA4, GATA6,
Figure Legend Snippet: Failure of extraembryonic endoderm formation in GATA6-deficient embryos. embryonic day (E) 5.5 and E6.5 embryos from timed matings of GATA6 (+/−) mice were analyzed by histology and immunostaining. Adjacent sections were stained for GATA4, GATA6,

Techniques Used: Mouse Assay, Immunostaining, Staining

Expression of GATA4 and GATA6 in immediately implanted blastocysts and early embryos. A – C : Fixed and paraffin-embedded uterine horns containing embryonic day (E) 4.5 (A), E4.75 (B), and E5.0 (C) embryos from timed matings of wild-type mice were
Figure Legend Snippet: Expression of GATA4 and GATA6 in immediately implanted blastocysts and early embryos. A – C : Fixed and paraffin-embedded uterine horns containing embryonic day (E) 4.5 (A), E4.75 (B), and E5.0 (C) embryos from timed matings of wild-type mice were

Techniques Used: Expressing, Mouse Assay

Derivation of GATA6- and/or GATA4-positive cells following the differentiation of embryonic stem Cells in monolayer cultures. Wild-type embryonic stem (ES) cells were cultured in the presence of 1 μM retinoic acid for 4 days on slide chambers.
Figure Legend Snippet: Derivation of GATA6- and/or GATA4-positive cells following the differentiation of embryonic stem Cells in monolayer cultures. Wild-type embryonic stem (ES) cells were cultured in the presence of 1 μM retinoic acid for 4 days on slide chambers.

Techniques Used: Cell Culture

Derivation of GATA6- and/or GATA4-positive Cells following the differentiation of embryonic stem (ES) cells in embryoid bodies. Embryoid bodies were formed following aggregation of wild-type ES cells in suspension culture for 7 days. The embryoid bodies
Figure Legend Snippet: Derivation of GATA6- and/or GATA4-positive Cells following the differentiation of embryonic stem (ES) cells in embryoid bodies. Embryoid bodies were formed following aggregation of wild-type ES cells in suspension culture for 7 days. The embryoid bodies

Techniques Used:

Absence of primitive endoderm in embryonic day (E) 4.5 GATA6-deficient embryos. E4.5 embryos from timed-matings of GATA6 (+/−) mice were analyzed by histology and immunostaining. Adjacent sections were stained for GATA4, GATA6, and Oct-3/4. A
Figure Legend Snippet: Absence of primitive endoderm in embryonic day (E) 4.5 GATA6-deficient embryos. E4.5 embryos from timed-matings of GATA6 (+/−) mice were analyzed by histology and immunostaining. Adjacent sections were stained for GATA4, GATA6, and Oct-3/4. A

Techniques Used: Mouse Assay, Immunostaining, Staining

Endoderm differentiation of GATA6-dificient embryonic stem (ES) cells. Wild-type and GATA6 (−/−) ES cells transfected with GATA4 expression plasmids (+ GATA4) or control vectors, were cultured in monolayer or suspension (Sph) as spheroids/embryoid
Figure Legend Snippet: Endoderm differentiation of GATA6-dificient embryonic stem (ES) cells. Wild-type and GATA6 (−/−) ES cells transfected with GATA4 expression plasmids (+ GATA4) or control vectors, were cultured in monolayer or suspension (Sph) as spheroids/embryoid

Techniques Used: Transfection, Expressing, Cell Culture

15) Product Images from "Cell adhesion and sorting in embryoid bodies derived from N- or E-cadherin deficient murine embryonic stem cells"

Article Title: Cell adhesion and sorting in embryoid bodies derived from N- or E-cadherin deficient murine embryonic stem cells

Journal: Biology Open

doi: 10.1242/bio.20146254

Cell adhesion molecule expression and the aggregation of wildtype and mutant ES cells. (A) Wildtype (WT), E-cadherin null (9J), and N-cadherin null (Ncad95) ES cells, with or without differentiation by retinoic acid, were analyzed by Western blot for the proteins levels of E-cadherin and N-cadherin. Oct3/4 and Dab2 levels are indicators of ES cell pluripotency or endoderm differentiation, respectively. The intensities of the signal in the Western blots were quantified using Image J program. (B,D) ES cells in monolayer cultures were analyzed by immunofluorescence microscopy by staining for Oct3/4, GATA4, and DAPI prior to (B), or following differentiation with 1 µM retinoic acid for 5 days (C). The representative individual images acquired were overlaid to produce the composed figures shown. (D) Rate of aggregation of the ES cells was determined as a measure of cell adhesive affinity. Cells were first mono-dispersed, washed with cold PBS, and then allowed to aggregate at 37°C. The aggregation of undifferentiated cells was measured using a Coulter Counter and the reduction of particle number is presented. (E) Wildtype, E-cadherin null (9J), and N-cadherin null (Ncad95) ES cells were first differentiated by treatment with retinoic acid for 4 days. The aggregation of the differentiated cells was measured using a Coulter Counter and the reduction of particle number is presented. Coulter Counter reading of particle numbers were performed using triplicate samples and the average and standard error are reported. Scale bars: 5 µm.
Figure Legend Snippet: Cell adhesion molecule expression and the aggregation of wildtype and mutant ES cells. (A) Wildtype (WT), E-cadherin null (9J), and N-cadherin null (Ncad95) ES cells, with or without differentiation by retinoic acid, were analyzed by Western blot for the proteins levels of E-cadherin and N-cadherin. Oct3/4 and Dab2 levels are indicators of ES cell pluripotency or endoderm differentiation, respectively. The intensities of the signal in the Western blots were quantified using Image J program. (B,D) ES cells in monolayer cultures were analyzed by immunofluorescence microscopy by staining for Oct3/4, GATA4, and DAPI prior to (B), or following differentiation with 1 µM retinoic acid for 5 days (C). The representative individual images acquired were overlaid to produce the composed figures shown. (D) Rate of aggregation of the ES cells was determined as a measure of cell adhesive affinity. Cells were first mono-dispersed, washed with cold PBS, and then allowed to aggregate at 37°C. The aggregation of undifferentiated cells was measured using a Coulter Counter and the reduction of particle number is presented. (E) Wildtype, E-cadherin null (9J), and N-cadherin null (Ncad95) ES cells were first differentiated by treatment with retinoic acid for 4 days. The aggregation of the differentiated cells was measured using a Coulter Counter and the reduction of particle number is presented. Coulter Counter reading of particle numbers were performed using triplicate samples and the average and standard error are reported. Scale bars: 5 µm.

Techniques Used: Expressing, Mutagenesis, Western Blot, Immunofluorescence, Microscopy, Staining

Apical polarity of primitive endoderm cells on surface of embryoid bodies. Embryoid bodies were analyzed by immunofluorescence microscopy for GATA4 (primitive endoderm marker), Dab2 (primitive endoderm marker), and megalin (primitive endoderm apical polarity marker). (A) A representative embryoid body derived from wildtype ES cells. Dab2 (cytoplasmic) and GATA4 (nuclear) mark the surface layer of primitive endoderm cells. Megalin is restricted to the apical membrane; (B) A 4-day-old embryoid body derived from E-cadherin null ES cells is shown for example. Dab2 staining marks both internal and surface primitive endoderm cells. Megalin staining indicates that primitive endoderm cells located inside the spheroid shows no polarity, and the cells located on surface display various degree of apical polarization (degree of megalin apical restriction), from less (indicated by *) to more (indicated by **) localization to apical surface. (C) A more mature (7-day-old) embryoid body derived from E-cadherin null ES cells shows strong apical polarity of megalin of the primitive endoderm cells on surface. Scale bars: 10 µm (A,B), 5 µm (C).
Figure Legend Snippet: Apical polarity of primitive endoderm cells on surface of embryoid bodies. Embryoid bodies were analyzed by immunofluorescence microscopy for GATA4 (primitive endoderm marker), Dab2 (primitive endoderm marker), and megalin (primitive endoderm apical polarity marker). (A) A representative embryoid body derived from wildtype ES cells. Dab2 (cytoplasmic) and GATA4 (nuclear) mark the surface layer of primitive endoderm cells. Megalin is restricted to the apical membrane; (B) A 4-day-old embryoid body derived from E-cadherin null ES cells is shown for example. Dab2 staining marks both internal and surface primitive endoderm cells. Megalin staining indicates that primitive endoderm cells located inside the spheroid shows no polarity, and the cells located on surface display various degree of apical polarization (degree of megalin apical restriction), from less (indicated by *) to more (indicated by **) localization to apical surface. (C) A more mature (7-day-old) embryoid body derived from E-cadherin null ES cells shows strong apical polarity of megalin of the primitive endoderm cells on surface. Scale bars: 10 µm (A,B), 5 µm (C).

Techniques Used: Immunofluorescence, Microscopy, Marker, Derivative Assay, Staining

16) Product Images from "The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4"

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

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

doi: 10.1126/science.aaa5612

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

Techniques Used:

GATA4 regulates cellular senescence
Figure Legend Snippet: GATA4 regulates cellular senescence

Techniques Used:

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

Techniques Used:

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

Techniques Used: Expressing

GATA4 regulates NF-κB
Figure Legend Snippet: GATA4 regulates NF-κB

Techniques Used:

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

Techniques Used:

17) Product Images from "Functional and molecular characterization of mouse Gata2-independent hematopoietic progenitors"

Article Title: Functional and molecular characterization of mouse Gata2-independent hematopoietic progenitors

Journal: Blood

doi: 10.1182/blood-2015-10-673749

Gata family gene expression in AGM Gata2-dependent and -independent HPCs. (A) qRT-PCR for expression of Gata1, 2, 3, 4, 5, and 6 transcription factors (normalization with Gapdh ) in E11 AGM CD31 + cKit + Venus + and CD31 + cKit + Venus − cells. n = 3. SEM shown with * P = .05 and *** P = .001. (B) Transverse section of WT E10.5 AGM immunostained for CD34 (magenta) and Gata3 (green) showing expression of Gata3 in the aortic endothelial cells and some emerging hematopoietic cells and ventral mesenchymal cells directly under the aorta. (C) Transverse section of G2V E10.5 AGM immunostained for CD34 (magenta), Gata2 (green), and Gata3 (red) showing some overlapping expression of Gata2 and Gata3 in aortic endothelial cells (arrowheads). (D) Transverse consecutive sections of E11 G2V AGM immunostained for CD34 (magenta) and Venus (green) in the top panels and for CD34 (magenta) and Gata4 (red) in the bottom panels. Gata4 expression is observed in some ventral aortic endothelial cells and emerging hematopoietic cells (arrow).
Figure Legend Snippet: Gata family gene expression in AGM Gata2-dependent and -independent HPCs. (A) qRT-PCR for expression of Gata1, 2, 3, 4, 5, and 6 transcription factors (normalization with Gapdh ) in E11 AGM CD31 + cKit + Venus + and CD31 + cKit + Venus − cells. n = 3. SEM shown with * P = .05 and *** P = .001. (B) Transverse section of WT E10.5 AGM immunostained for CD34 (magenta) and Gata3 (green) showing expression of Gata3 in the aortic endothelial cells and some emerging hematopoietic cells and ventral mesenchymal cells directly under the aorta. (C) Transverse section of G2V E10.5 AGM immunostained for CD34 (magenta), Gata2 (green), and Gata3 (red) showing some overlapping expression of Gata2 and Gata3 in aortic endothelial cells (arrowheads). (D) Transverse consecutive sections of E11 G2V AGM immunostained for CD34 (magenta) and Venus (green) in the top panels and for CD34 (magenta) and Gata4 (red) in the bottom panels. Gata4 expression is observed in some ventral aortic endothelial cells and emerging hematopoietic cells (arrow).

Techniques Used: Expressing, Quantitative RT-PCR

18) Product Images from "A Simple and Robust Method for Establishing Homogeneous Mouse Epiblast Stem Cell Lines by Wnt Inhibition"

Article Title: A Simple and Robust Method for Establishing Homogeneous Mouse Epiblast Stem Cell Lines by Wnt Inhibition

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2015.02.014

Differentiation Potential of EpiSCs Isolated by the IWP-2 Method (A and B) Expression levels of Brachyury (T) (A) and GATA4 (B) were detected in B129a4 EpiSCs isolated by the IWP-2 method when they were cultured in medium without IWP-2 for 1 week. (C) Bright-field image of EBs formed from 129Ba2 EpiSCs isolated with IWP-2 treatment. (D) Immunofluorescence images for TUBB3 (red) and NESTIN (green), detecting neural differentiation in 129 Ba2 EBs. (E–J) Hematoxylin and eosin-stained sections of teratomas from 129Ba1 EpiSCs: (E) cartilage, (F) skeletal muscle, (G) adipocytes, (H) gastrointestinal epithelium, (I) melanocytes, and (J) neural tissue. Scale bars, 50 μm (A, B, and D–J) and 0.5 mm (C). See also Figures S2 and S3 .
Figure Legend Snippet: Differentiation Potential of EpiSCs Isolated by the IWP-2 Method (A and B) Expression levels of Brachyury (T) (A) and GATA4 (B) were detected in B129a4 EpiSCs isolated by the IWP-2 method when they were cultured in medium without IWP-2 for 1 week. (C) Bright-field image of EBs formed from 129Ba2 EpiSCs isolated with IWP-2 treatment. (D) Immunofluorescence images for TUBB3 (red) and NESTIN (green), detecting neural differentiation in 129 Ba2 EBs. (E–J) Hematoxylin and eosin-stained sections of teratomas from 129Ba1 EpiSCs: (E) cartilage, (F) skeletal muscle, (G) adipocytes, (H) gastrointestinal epithelium, (I) melanocytes, and (J) neural tissue. Scale bars, 50 μm (A, B, and D–J) and 0.5 mm (C). See also Figures S2 and S3 .

Techniques Used: Isolation, Expressing, Cell Culture, Immunofluorescence, Staining

Effects of IWP-2 on Gene Expression in the 129C1 EpiSC Line Immunofluorescence images for GATA4 (red) and OCT4 (green) (A and B), SOX17 (C and D), T (E and F), and CER1 (G and H) in EpiSCs cultured without IWP-2 (A, C, E, and G) or with IWP-2 (B, D, F, and H). Nuclei were stained with TO-PRO3 (blue). Scale bar, 100 μm. See also Figures S5 and S6 .
Figure Legend Snippet: Effects of IWP-2 on Gene Expression in the 129C1 EpiSC Line Immunofluorescence images for GATA4 (red) and OCT4 (green) (A and B), SOX17 (C and D), T (E and F), and CER1 (G and H) in EpiSCs cultured without IWP-2 (A, C, E, and G) or with IWP-2 (B, D, F, and H). Nuclei were stained with TO-PRO3 (blue). Scale bar, 100 μm. See also Figures S5 and S6 .

Techniques Used: Expressing, Immunofluorescence, Cell Culture, Staining

19) Product Images from "RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects"

Article Title: RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.031534

RERE regulates the expression of GATA4 in the AV canal. (A,B) GATA4 was visualized with anti-GATA4 antibodies on sections through the AV canals of wild-type embryos and Rere om/eyes3 embryos. The immunoreactivity of GATA4 in the wild-type AV endocardial cushions was stronger than that observed in Rere om/eyes3 AV endocardial cushions. (C,D) In contrast, the levels of GATA4 expression were comparable in the AV canals of Rere flox/flox and Rere flox/flox ;Tie2-Cre embryos. These results suggest that decreased expression of RERE in cells other than the endocardium play a role in regulating the expression of GATA4 in the AV canal. Arrows and dashed lines indicate the AV endocardial cushions. Representative images are shown from the analysis of sections from three embryos of each genotype. Scale bars: 100 µm.
Figure Legend Snippet: RERE regulates the expression of GATA4 in the AV canal. (A,B) GATA4 was visualized with anti-GATA4 antibodies on sections through the AV canals of wild-type embryos and Rere om/eyes3 embryos. The immunoreactivity of GATA4 in the wild-type AV endocardial cushions was stronger than that observed in Rere om/eyes3 AV endocardial cushions. (C,D) In contrast, the levels of GATA4 expression were comparable in the AV canals of Rere flox/flox and Rere flox/flox ;Tie2-Cre embryos. These results suggest that decreased expression of RERE in cells other than the endocardium play a role in regulating the expression of GATA4 in the AV canal. Arrows and dashed lines indicate the AV endocardial cushions. Representative images are shown from the analysis of sections from three embryos of each genotype. Scale bars: 100 µm.

Techniques Used: Expressing

RERE and GATA are co-expressed in the AV canal, and Rere and Gata4 interact genetically in the development of CHD. (A-C) Sections of wild-type embryos were prepared at E10.5 and stained with anti-RERE and anti-GATA4 antibodies. RERE (red) and GATA4 (green) were colocalized in the endocardium and mesenchymal cells of the AV endocardial cushions. Dashed lines indicate the boundary between the AV endocardial cushions and the myocardium; white arrows indicate the endocardium of the AV cushion. Representative images are presented from sections obtained from four wild-type embryos. Atr, atrium; Vt, ventricle. Scale bars: 100 µm. (D,E) Gata4 +/− and Rere −/eyes3 embryos on a mixed B6/129S6 background have normal ventricular septums at E15.5. (F) E15.5 Rere −/eyes3 ; Gata4 +/− embryos on a mixed B6/129S6 background have several types of CHD including perimembranous VSDs (arrow). Scale bars: 200 µm.
Figure Legend Snippet: RERE and GATA are co-expressed in the AV canal, and Rere and Gata4 interact genetically in the development of CHD. (A-C) Sections of wild-type embryos were prepared at E10.5 and stained with anti-RERE and anti-GATA4 antibodies. RERE (red) and GATA4 (green) were colocalized in the endocardium and mesenchymal cells of the AV endocardial cushions. Dashed lines indicate the boundary between the AV endocardial cushions and the myocardium; white arrows indicate the endocardium of the AV cushion. Representative images are presented from sections obtained from four wild-type embryos. Atr, atrium; Vt, ventricle. Scale bars: 100 µm. (D,E) Gata4 +/− and Rere −/eyes3 embryos on a mixed B6/129S6 background have normal ventricular septums at E15.5. (F) E15.5 Rere −/eyes3 ; Gata4 +/− embryos on a mixed B6/129S6 background have several types of CHD including perimembranous VSDs (arrow). Scale bars: 200 µm.

Techniques Used: Staining

RERE regulates the transcription of Gata4 . (A) RT-qPCR analyses were performed using mRNA extracted from the hearts of wild-type and Rere om/eyes3 embryos at E10.5. The relative amounts of Gata4 and Erbb3 transcripts were significantly reduced in the hearts of Rere om/eyes3 embryos compared with those of wild-type embryos. Three independent RT-qPCR analyses were performed using mRNA prepared from the hearts of three different littermates. (B) A firefly luciferase reporter gene fused with a previously described 5 kb promoter of Gata4 (5kb-Gata4-Luc) ( Mazaud Guittot et al., 2007 ) was transfected into HEK293 T cells with/without an RERE -expressing vector. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. Luciferase activity driven by the 5 kb Gata4 promoter was increased by overexpression of RERE. Experiments were performed in triplicate. (C) A 5kb-Gata4-Luc plasmid was transfected into NIH3T3 cells with a nontargeting siRNA pool or an Rere siRNA pool. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. NIH3T3 cells transfected with the Rere siRNA pool showed decreased luciferase activity when compared with those transfected with a nontargeting siRNA pool. Luciferase experiments were performed in triplicate. In all graphs, data are mean±s.d. Unpaired two-tailed Student's t -test was used to determine P -values (* P
Figure Legend Snippet: RERE regulates the transcription of Gata4 . (A) RT-qPCR analyses were performed using mRNA extracted from the hearts of wild-type and Rere om/eyes3 embryos at E10.5. The relative amounts of Gata4 and Erbb3 transcripts were significantly reduced in the hearts of Rere om/eyes3 embryos compared with those of wild-type embryos. Three independent RT-qPCR analyses were performed using mRNA prepared from the hearts of three different littermates. (B) A firefly luciferase reporter gene fused with a previously described 5 kb promoter of Gata4 (5kb-Gata4-Luc) ( Mazaud Guittot et al., 2007 ) was transfected into HEK293 T cells with/without an RERE -expressing vector. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. Luciferase activity driven by the 5 kb Gata4 promoter was increased by overexpression of RERE. Experiments were performed in triplicate. (C) A 5kb-Gata4-Luc plasmid was transfected into NIH3T3 cells with a nontargeting siRNA pool or an Rere siRNA pool. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. NIH3T3 cells transfected with the Rere siRNA pool showed decreased luciferase activity when compared with those transfected with a nontargeting siRNA pool. Luciferase experiments were performed in triplicate. In all graphs, data are mean±s.d. Unpaired two-tailed Student's t -test was used to determine P -values (* P

Techniques Used: Quantitative RT-PCR, Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Cotransfection, Over Expression, Two Tailed Test

20) Product Images from "Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription"

Article Title: Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00319.2008

Western blot analysis of total and phosphorylated Akt, phosphatase and tensin homologue (PTEN), and GATA4 in H9C2 myoblast cells treated with 0.8 mM palmitic acid for 24 h. A cohort of cell had been transfected with dominant-negative (DN) Foxo3a virus
Figure Legend Snippet: Western blot analysis of total and phosphorylated Akt, phosphatase and tensin homologue (PTEN), and GATA4 in H9C2 myoblast cells treated with 0.8 mM palmitic acid for 24 h. A cohort of cell had been transfected with dominant-negative (DN) Foxo3a virus

Techniques Used: Western Blot, Transfection, Dominant Negative Mutation

21) Product Images from "Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate"

Article Title: Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.524

GATA4 is a direct substrate for caspase-1. ( a and b ) Schematic representation of rat GATA4 (accession number P46152). Alignment of GATA4 from different species shows that the putative cleavage sites YMAD168, DMFD208 and WRRD230 in boxes are conserved in human, mouse and rat. ( b ) Predicted size of GATA4 fragments cleaved by caspase-1. The depicted red fragments can be detected by the GATA4 antibody (epitope). ( c ) In vitro caspase cleavage assays. In vitro translated radiolabelled GATA4 was exposed to purified caspase-1 (C-1) and caspase-3 (C-3). Arrows indicate cleavage products by caspase-1 but not by caspase-3. ( d ) Caspase-1 cleavage of GATA4 mutants identifies D168 and D230 as cleavage sites. In vitro cleavage assays using purified caspase-1 and in vitro translated GATA4 WT and GATA4 mutants (single or double mutations as indicated in d ). Note how double mutation of D168 and D230 prevents the cleavage by caspase-1
Figure Legend Snippet: GATA4 is a direct substrate for caspase-1. ( a and b ) Schematic representation of rat GATA4 (accession number P46152). Alignment of GATA4 from different species shows that the putative cleavage sites YMAD168, DMFD208 and WRRD230 in boxes are conserved in human, mouse and rat. ( b ) Predicted size of GATA4 fragments cleaved by caspase-1. The depicted red fragments can be detected by the GATA4 antibody (epitope). ( c ) In vitro caspase cleavage assays. In vitro translated radiolabelled GATA4 was exposed to purified caspase-1 (C-1) and caspase-3 (C-3). Arrows indicate cleavage products by caspase-1 but not by caspase-3. ( d ) Caspase-1 cleavage of GATA4 mutants identifies D168 and D230 as cleavage sites. In vitro cleavage assays using purified caspase-1 and in vitro translated GATA4 WT and GATA4 mutants (single or double mutations as indicated in d ). Note how double mutation of D168 and D230 prevents the cleavage by caspase-1

Techniques Used: In Vitro, Purification, Mutagenesis

Caspase-1 is a negative regulator of GATA4 transcriptional activity. ( a ) Left panel. N-terminal truncated GATA4 proteins bind DNA. Nuclear extracts from AD293 cells transfected with the indicated GATA4 constructs were tested for their ability to bind GATA elements using EMSAs. NE: nuclear extracts, self comp: self competition with cold probe. The right panel is a western blot showing equivalent protein expression levels for all constructs. ( b ) Dose response for wild-type and truncated GATA4 protein co-transfected with the (GATA)3x-Luc promoter. ( c ) Caspase-1 cleaved GATA4 acts as dominant negative. GATA4 WT and GATA4 mut (aa 174–440) were co-transfected with the BclxL promoter. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the GATA4 WT treatment. ** P ≤0.01, *** P ≤0.001. ( d ) Effect of caspase-1 on GATA4-dependent transcription. Left panel: (GATA)3x–luc reporter (1 μ g) was co-transfected with 100 ng of native or mutant GATA4 expression vector with or without 50 and 500 ng of caspase-1 expression vector. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*) or GATA4 WT treatment ( # ). ** P ≤0.01, # P ≤0.001. Right panel: Effect of caspase-1 on the ANF promoter in response to GATA4. The amount of plasmid DNA used is the same as in left panel. The data are the mean±S.E.M. of two experiments carried out in duplicate. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*), GATA4 WT treatment ( # ) or mutant GATA4 treatment. *** P ≤0.001, # P ≤0.001, ns=not significant. Note how caspase-1 completely abrogates GATA4 activation and how mutation of the two major caspase-1 cleavage sites renders GATA4 resistant to this effect
Figure Legend Snippet: Caspase-1 is a negative regulator of GATA4 transcriptional activity. ( a ) Left panel. N-terminal truncated GATA4 proteins bind DNA. Nuclear extracts from AD293 cells transfected with the indicated GATA4 constructs were tested for their ability to bind GATA elements using EMSAs. NE: nuclear extracts, self comp: self competition with cold probe. The right panel is a western blot showing equivalent protein expression levels for all constructs. ( b ) Dose response for wild-type and truncated GATA4 protein co-transfected with the (GATA)3x-Luc promoter. ( c ) Caspase-1 cleaved GATA4 acts as dominant negative. GATA4 WT and GATA4 mut (aa 174–440) were co-transfected with the BclxL promoter. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the GATA4 WT treatment. ** P ≤0.01, *** P ≤0.001. ( d ) Effect of caspase-1 on GATA4-dependent transcription. Left panel: (GATA)3x–luc reporter (1 μ g) was co-transfected with 100 ng of native or mutant GATA4 expression vector with or without 50 and 500 ng of caspase-1 expression vector. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*) or GATA4 WT treatment ( # ). ** P ≤0.01, # P ≤0.001. Right panel: Effect of caspase-1 on the ANF promoter in response to GATA4. The amount of plasmid DNA used is the same as in left panel. The data are the mean±S.E.M. of two experiments carried out in duplicate. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*), GATA4 WT treatment ( # ) or mutant GATA4 treatment. *** P ≤0.001, # P ≤0.001, ns=not significant. Note how caspase-1 completely abrogates GATA4 activation and how mutation of the two major caspase-1 cleavage sites renders GATA4 resistant to this effect

Techniques Used: Activity Assay, Transfection, Construct, Western Blot, Expressing, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation, Activation Assay

Dox-induced GATA4 depletion is caspase-1 dependent. ( a ) Pan-caspase inhibitor restored GATA4 expression. Cardiomyocytes were treated with Dox in the presence or absence of pan-caspase inhibitor (zVAD-FMK) for 12 h and analyzed by western blot. ( b ) Caspase-1 inhibitor prevented Dox-dependent GATA4 depletion. Cardiomyocytes were treated in the presence or absence of Dox with a caspase-1 inhibitor (YVAD-CHO). Western blots were carried out to detect GATA4 and its downstream target BclxL. GAPDH was used as a loading control. Note how changes in BclxL levels parallel those of GATA4. ( c ) Effect of caspase inhibition on cardiomyocyte apoptosis. Quantification of TUNEL assays in primary cardiomyocytes treated with the indicated inhibitors. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*) or to the Dox treatment alone ( # ). *** P ≤0.001, # P ≤0.001. Note how caspase-1 inhibition is as effective as the pan-caspase inhibitor at abrogating Dox-induced apoptosis. ( d – g ) Increased activation and nuclear localization of caspase-1 in Dox-treated cardiomyocytes. ( d ) Western blots of nuclear cardiomyocyte extracts. Notice how caspase-1 is activated (lower band) after 3 and 12 h of Dox treatment. GAPDH staining was used to control for cytoplasmic contamination. ( e ) Representative images (top panel) and quantification (lower panel) of a FAM-FLICA assay measuring caspase-1 activity in control and Dox-treated cardiomyocytes. Results are shown as percent of caspase-1-positive cells. *** P ≤0.0001. In the top panel, green is active caspase-1 and blue is Hoechst staining. ( f ) and ( g ) Immunofluorescence of HL1 cells ( f ) and primary cardiomyocytes ( g ) treated with Dox for the indicated time. Caspase-1 is labeled in red, α-actinin is labeled in green and Hoechst staining is labeled in blue
Figure Legend Snippet: Dox-induced GATA4 depletion is caspase-1 dependent. ( a ) Pan-caspase inhibitor restored GATA4 expression. Cardiomyocytes were treated with Dox in the presence or absence of pan-caspase inhibitor (zVAD-FMK) for 12 h and analyzed by western blot. ( b ) Caspase-1 inhibitor prevented Dox-dependent GATA4 depletion. Cardiomyocytes were treated in the presence or absence of Dox with a caspase-1 inhibitor (YVAD-CHO). Western blots were carried out to detect GATA4 and its downstream target BclxL. GAPDH was used as a loading control. Note how changes in BclxL levels parallel those of GATA4. ( c ) Effect of caspase inhibition on cardiomyocyte apoptosis. Quantification of TUNEL assays in primary cardiomyocytes treated with the indicated inhibitors. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*) or to the Dox treatment alone ( # ). *** P ≤0.001, # P ≤0.001. Note how caspase-1 inhibition is as effective as the pan-caspase inhibitor at abrogating Dox-induced apoptosis. ( d – g ) Increased activation and nuclear localization of caspase-1 in Dox-treated cardiomyocytes. ( d ) Western blots of nuclear cardiomyocyte extracts. Notice how caspase-1 is activated (lower band) after 3 and 12 h of Dox treatment. GAPDH staining was used to control for cytoplasmic contamination. ( e ) Representative images (top panel) and quantification (lower panel) of a FAM-FLICA assay measuring caspase-1 activity in control and Dox-treated cardiomyocytes. Results are shown as percent of caspase-1-positive cells. *** P ≤0.0001. In the top panel, green is active caspase-1 and blue is Hoechst staining. ( f ) and ( g ) Immunofluorescence of HL1 cells ( f ) and primary cardiomyocytes ( g ) treated with Dox for the indicated time. Caspase-1 is labeled in red, α-actinin is labeled in green and Hoechst staining is labeled in blue

Techniques Used: Expressing, Western Blot, Inhibition, TUNEL Assay, Activation Assay, Staining, Activity Assay, Immunofluorescence, Labeling

Caspase-1 inhibition is protective against Dox cardiotoxicity in vivo . ( a ) Dox induces an increase in caspase-1 and a decrease in GATA4 staining in vivo . Immunohistochemistry of ventricular tissue sections from wild-type mice treated with Dox or vehicle. Caspase-1 staining is shown in the top panels and GATA4 staining in the bottom panels. ( b ) Caspase-1 inhibition or loss attenuates cardiomyocyte cell death in vivo . Quantification of TUNEL assays of wild-type mice treated with Dox and YVAD-CHO as well as Casp1 −/− mice treated with Dox. The results are shown as the mean±S.E.M. and analyzed by Student's T -test of wild-type control mice (*) or of wild-type Dox-treated mice ( # ). *** P ≤0.0001, ## P ≤0.001. ( c ) Effect of caspase-1 inhibition or loss on Dox induced cardiac fibrotic cardiac lesions in vivo . Trichrome staining of transverse sections of left ventricular tissue of wild-type mice treated with Dox and YVAD-CHO or Casp1 −/− mice treated with Dox. Blue staining represents fibrotic lesions
Figure Legend Snippet: Caspase-1 inhibition is protective against Dox cardiotoxicity in vivo . ( a ) Dox induces an increase in caspase-1 and a decrease in GATA4 staining in vivo . Immunohistochemistry of ventricular tissue sections from wild-type mice treated with Dox or vehicle. Caspase-1 staining is shown in the top panels and GATA4 staining in the bottom panels. ( b ) Caspase-1 inhibition or loss attenuates cardiomyocyte cell death in vivo . Quantification of TUNEL assays of wild-type mice treated with Dox and YVAD-CHO as well as Casp1 −/− mice treated with Dox. The results are shown as the mean±S.E.M. and analyzed by Student's T -test of wild-type control mice (*) or of wild-type Dox-treated mice ( # ). *** P ≤0.0001, ## P ≤0.001. ( c ) Effect of caspase-1 inhibition or loss on Dox induced cardiac fibrotic cardiac lesions in vivo . Trichrome staining of transverse sections of left ventricular tissue of wild-type mice treated with Dox and YVAD-CHO or Casp1 −/− mice treated with Dox. Blue staining represents fibrotic lesions

Techniques Used: Inhibition, In Vivo, Staining, Immunohistochemistry, Mouse Assay, TUNEL Assay

Dox-induced GATA4 depletion is independent of the ubiquitin-proteasome pathway. ( a ) Effect of time course treatment of Doxorubicin (Dox) on GATA4 (left panel), GATA6 (middle panel) and total protein (right panel) levels. Nuclear extracts were prepared from primary cardiomyocyte cultures treated for the indicated times with Dox (300 nM) and subjected to western blot analyses. ( b ) Depletion of GATA4 transcripts after 12 h of Dox treatment. Cardiomyocytes were treated for the indicated times with Dox. RNA was subjected to real-time PCR. GATA4 mRNA levels were normalized to S16 mRNA. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the 3-h Dox treatment ( n =3). ** P ≤0.01, *** P ≤0.001. ( c ) Depletion of GATA4 protein by Dox is not prevented by a proteasome inhibitor. Cardiomyocytes were treated with Dox for 12 h in the presence or absence of 10 μ M proteasome inhibitor MG132. Nuclear extracts were subjected to western blot to detect GATA4 protein. P300 was used as a control. ( d ) A putative cleavage site in the N-terminal region of GATA4. Transient transfection was carried out in HL-1 atrial cardiomyocytes using GATA4 WT and a GATA4 N-terminal deletion (201–440) mutant. Nuclear extracts were subjected to western blot analysis using anti-HA and anti-GATA4 antibodies to detect N- and C-terminal fragments, respectively
Figure Legend Snippet: Dox-induced GATA4 depletion is independent of the ubiquitin-proteasome pathway. ( a ) Effect of time course treatment of Doxorubicin (Dox) on GATA4 (left panel), GATA6 (middle panel) and total protein (right panel) levels. Nuclear extracts were prepared from primary cardiomyocyte cultures treated for the indicated times with Dox (300 nM) and subjected to western blot analyses. ( b ) Depletion of GATA4 transcripts after 12 h of Dox treatment. Cardiomyocytes were treated for the indicated times with Dox. RNA was subjected to real-time PCR. GATA4 mRNA levels were normalized to S16 mRNA. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the 3-h Dox treatment ( n =3). ** P ≤0.01, *** P ≤0.001. ( c ) Depletion of GATA4 protein by Dox is not prevented by a proteasome inhibitor. Cardiomyocytes were treated with Dox for 12 h in the presence or absence of 10 μ M proteasome inhibitor MG132. Nuclear extracts were subjected to western blot to detect GATA4 protein. P300 was used as a control. ( d ) A putative cleavage site in the N-terminal region of GATA4. Transient transfection was carried out in HL-1 atrial cardiomyocytes using GATA4 WT and a GATA4 N-terminal deletion (201–440) mutant. Nuclear extracts were subjected to western blot analysis using anti-HA and anti-GATA4 antibodies to detect N- and C-terminal fragments, respectively

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis

HSP70 physically interacts with GATA4 and rescues caspase-1 inhibition. ( a and b ) HSP70 co-immunoprecipitates with GATA4. Nuclear extracts from AD293 cells transfected with HA-GATA4 and/or Flag-HSP70-GFP were immunoprecipitated with anti-Flag antibody, separated on a 10% (vol/vol) SDS-PAGE and immunoblotted with anti-HA, anti-Flag and anti-Nucleolin antibodies. ( c and d ) HSP70 interacts directly with the N-terminal of GATA4. GST and GST bound GATA4 aa 2–207, aa 130–170 and aa 329–440 fusion proteins were incubated with in vitro translated HSP70. Bound proteins were resolved using SDS-PAGE (12% vol/vol) and revealed using autoradiography. Fusion protein inputs ( c ) were resolved using SDS-PAGE (12% vol/vol) and stained using coomassie blue. Astrices indicate fusion protein bands. ( e ) HSP70 prevents caspase-1 mediated GATA4 processing. NIH 3T3 cells were transfected with expression vectors for GATA4, caspase-1 and/or HSP70. Nuclear extracts were analyzed by western blot. ( f ) Hsp70 rescues caspase-1 mediated inhibition of GATA4 transcriptional activity. Transfection into NIH3T3 cells of the GATA-dependent Nppb promoter –Luc reporter along with the indicated expression vectors for GATA4, caspase-1 and HSP70 and activations thereof. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*) or GATA4 treatment ( # ). *** P ≤0.001, # P ≤0.001 ( n =3). Note how caspase-1 inhibits GATA4 transactivation in the absence but not in the presence of HSP70
Figure Legend Snippet: HSP70 physically interacts with GATA4 and rescues caspase-1 inhibition. ( a and b ) HSP70 co-immunoprecipitates with GATA4. Nuclear extracts from AD293 cells transfected with HA-GATA4 and/or Flag-HSP70-GFP were immunoprecipitated with anti-Flag antibody, separated on a 10% (vol/vol) SDS-PAGE and immunoblotted with anti-HA, anti-Flag and anti-Nucleolin antibodies. ( c and d ) HSP70 interacts directly with the N-terminal of GATA4. GST and GST bound GATA4 aa 2–207, aa 130–170 and aa 329–440 fusion proteins were incubated with in vitro translated HSP70. Bound proteins were resolved using SDS-PAGE (12% vol/vol) and revealed using autoradiography. Fusion protein inputs ( c ) were resolved using SDS-PAGE (12% vol/vol) and stained using coomassie blue. Astrices indicate fusion protein bands. ( e ) HSP70 prevents caspase-1 mediated GATA4 processing. NIH 3T3 cells were transfected with expression vectors for GATA4, caspase-1 and/or HSP70. Nuclear extracts were analyzed by western blot. ( f ) Hsp70 rescues caspase-1 mediated inhibition of GATA4 transcriptional activity. Transfection into NIH3T3 cells of the GATA-dependent Nppb promoter –Luc reporter along with the indicated expression vectors for GATA4, caspase-1 and HSP70 and activations thereof. The results are shown as mean±S.E.M. and analyzed by one-way ANOVA with Bonferroni post-test relative to the control (*) or GATA4 treatment ( # ). *** P ≤0.001, # P ≤0.001 ( n =3). Note how caspase-1 inhibits GATA4 transactivation in the absence but not in the presence of HSP70

Techniques Used: Inhibition, Transfection, Immunoprecipitation, SDS Page, Incubation, In Vitro, Autoradiography, Staining, Expressing, Western Blot, Activity Assay

22) Product Images from "GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development"

Article Title: GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development

Journal: Nature Communications

doi: 10.1038/ncomms4680

GATA sites are required to mediate repression of AV canal genes in chamber myocardium. Localization of transgene expression in E11.5 mouse hearts. ( a , b ) The cTnT transgene is expressed both in the AV canal myocardium and the chamber myocardium. ( c , d ) cGata6-cTnT transgenics show predominant expression in the AV canal, similar to the pattern of the Tbx2-cTnT transgenes ( e , f ). ( g , h ) Mutation of the two GATA sites in the Tbx2 regulatory region removes the repression in the chambers. Ratios denote proportion of embryos showing chamber repression. Scale bar represents 100 μm. (la) left atrium; (ra) right atrium; (avc) AV canal; (lv) left ventricle; (rv) right ventricle; (oft) outflow tract. ( i ) Browser view of Tbx2 gene locus with ChIP-seq profiles of Gata4 (ref. 18 ) (black) and p300 (ref. 24 ) (black) in heart, Smad4 (grey) in human ESC 31 and Hey2 (blue) in mouse ESC. The 380 bp Tbx2 (−2700/−2300) enhancer region is depicted as a black box.
Figure Legend Snippet: GATA sites are required to mediate repression of AV canal genes in chamber myocardium. Localization of transgene expression in E11.5 mouse hearts. ( a , b ) The cTnT transgene is expressed both in the AV canal myocardium and the chamber myocardium. ( c , d ) cGata6-cTnT transgenics show predominant expression in the AV canal, similar to the pattern of the Tbx2-cTnT transgenes ( e , f ). ( g , h ) Mutation of the two GATA sites in the Tbx2 regulatory region removes the repression in the chambers. Ratios denote proportion of embryos showing chamber repression. Scale bar represents 100 μm. (la) left atrium; (ra) right atrium; (avc) AV canal; (lv) left ventricle; (rv) right ventricle; (oft) outflow tract. ( i ) Browser view of Tbx2 gene locus with ChIP-seq profiles of Gata4 (ref. 18 ) (black) and p300 (ref. 24 ) (black) in heart, Smad4 (grey) in human ESC 31 and Hey2 (blue) in mouse ESC. The 380 bp Tbx2 (−2700/−2300) enhancer region is depicted as a black box.

Techniques Used: Expressing, Mutagenesis, Chromatin Immunoprecipitation

The Gata4-Smad-Hat complex induces H3K27 acetylation and AV canal gene activation. ( a ) Luciferase reporter assays on the 102 bp cGata6 enhancer. Plasmids encoding full-length Gata4 protein, the luciferase reporter construct with a minimal promoter (minP) or containing the cGata6 enhancer with the same minimal promoter ( cGata6_minP ) were co-transfected in C2C12 cells. The cells were incubated with or without the recombinant human BMP2. Statistical test was conducted using two- and three-way analysis of variance (ANOVA) (mean±s.e.m., n =3, # P
Figure Legend Snippet: The Gata4-Smad-Hat complex induces H3K27 acetylation and AV canal gene activation. ( a ) Luciferase reporter assays on the 102 bp cGata6 enhancer. Plasmids encoding full-length Gata4 protein, the luciferase reporter construct with a minimal promoter (minP) or containing the cGata6 enhancer with the same minimal promoter ( cGata6_minP ) were co-transfected in C2C12 cells. The cells were incubated with or without the recombinant human BMP2. Statistical test was conducted using two- and three-way analysis of variance (ANOVA) (mean±s.e.m., n =3, # P

Techniques Used: HAT Assay, Activation Assay, Luciferase, Construct, Transfection, Incubation, Recombinant

The Gata4/Hey/Hdac complex induces H3K27 deacetylation and AV canal gene repression. ( a ) Luciferase reporter assays on 102 bp cGata6 , the 380 bp Tbx2 , 660 bp Cx30.2 and the 356 bp cTnI genomic fragments. Constructs were co-transfected with Gata4, Alk3CA, Smad1 and Smad4, HDAC1 and 2 expression vectors into Cos-7 cells (mean±s.e.m., n =6, * P
Figure Legend Snippet: The Gata4/Hey/Hdac complex induces H3K27 deacetylation and AV canal gene repression. ( a ) Luciferase reporter assays on 102 bp cGata6 , the 380 bp Tbx2 , 660 bp Cx30.2 and the 356 bp cTnI genomic fragments. Constructs were co-transfected with Gata4, Alk3CA, Smad1 and Smad4, HDAC1 and 2 expression vectors into Cos-7 cells (mean±s.e.m., n =6, * P

Techniques Used: Luciferase, Construct, Transfection, Expressing

GATA sites serve as a scaffold to recruit functionally distinct complexes. ( a , b ) In vitro reporter assays with the cGata6 (102 bp) and Tbx2 (380 bp) luciferase constructs containing mutations for GATA sites ( cGata6muGATA, Tbx2muGATA ). Cos-7 cells co-transfected with Gata4, Alk3CA, Smad1 and 4, Hey1, 2, HDAC1 and 2 expressing vectors or not were treated in the absence or presence of 30 nmol l −1 TSA (mean±s.e.m., n =3, # P
Figure Legend Snippet: GATA sites serve as a scaffold to recruit functionally distinct complexes. ( a , b ) In vitro reporter assays with the cGata6 (102 bp) and Tbx2 (380 bp) luciferase constructs containing mutations for GATA sites ( cGata6muGATA, Tbx2muGATA ). Cos-7 cells co-transfected with Gata4, Alk3CA, Smad1 and 4, Hey1, 2, HDAC1 and 2 expressing vectors or not were treated in the absence or presence of 30 nmol l −1 TSA (mean±s.e.m., n =3, # P

Techniques Used: In Vitro, Luciferase, Construct, Transfection, Expressing

23) Product Images from "Senescence as a novel mechanism involved in β-adrenergic receptor mediated cardiac hypertrophy"

Article Title: Senescence as a novel mechanism involved in β-adrenergic receptor mediated cardiac hypertrophy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0182668

Expression of CDKIs and GATA4 increased in ISO-treated cells. (A) The protein level of cell cycle inhibitors and Eif5 (loading control) was examined by western blotting. (B) The protein level was quantified by densitometry. (C) The protein level of GATA4 and Eif5 was examined by western blotting and (D) quantified by densitometry. The gene expression of SASP factors (E) and (F) was examined with the use of quantitative RT-PCR. Data are means ± SEM (n = 6; *P
Figure Legend Snippet: Expression of CDKIs and GATA4 increased in ISO-treated cells. (A) The protein level of cell cycle inhibitors and Eif5 (loading control) was examined by western blotting. (B) The protein level was quantified by densitometry. (C) The protein level of GATA4 and Eif5 was examined by western blotting and (D) quantified by densitometry. The gene expression of SASP factors (E) and (F) was examined with the use of quantitative RT-PCR. Data are means ± SEM (n = 6; *P

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

24) Product Images from "The transcription factor GATA4 promotes myocardial regeneration in neonatal mice"

Article Title: The transcription factor GATA4 promotes myocardial regeneration in neonatal mice

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201606602

Reduced GATA 4 levels entail diminished cardiomyocyte proliferation in vitro Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 h. Number of cardiomyocytes (CM) in a defined low‐magnification (50×) microscopic field before the adenoviral infection and 48 h later. **** P
Figure Legend Snippet: Reduced GATA 4 levels entail diminished cardiomyocyte proliferation in vitro Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 h. Number of cardiomyocytes (CM) in a defined low‐magnification (50×) microscopic field before the adenoviral infection and 48 h later. **** P

Techniques Used: In Vitro, Western Blot, Expressing, shRNA, Infection

Cardiac GATA 4 becomes downregulated within the first postnatal week and is necessary for myocardial regeneration Cardiac GATA4 protein abundance analyzed by immunoblotting. GAPDH was the loading control. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4‐overexpressing adenovirus. Densitometric quantification of the immunoblot shown in (A); *** P = 0.0003 for P1 vs. P7 and P1 vs. P21; *** P
Figure Legend Snippet: Cardiac GATA 4 becomes downregulated within the first postnatal week and is necessary for myocardial regeneration Cardiac GATA4 protein abundance analyzed by immunoblotting. GAPDH was the loading control. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4‐overexpressing adenovirus. Densitometric quantification of the immunoblot shown in (A); *** P = 0.0003 for P1 vs. P7 and P1 vs. P21; *** P

Techniques Used: Positive Control, Infection

Additional characterization of GATA 4 overexpression and IL ‐13 in neonatal hearts Heart weight/body weight (BW) ratio of the indicated mice 7 days after sham surgery or cryoinfarction and application of adenoviruses as shown. Lung weight/body weight ratio of the indicated mice 7 days after sham surgery or cryoinfarction and application of adenoviruses as shown. Cardiomyocyte (CM) cross‐sectional area assessed 7 days after cryoinfarction in the indicated mice. Quantification of myocardial macrophage abundance (positive for F4/80) remote of the infarcted area of the indicated mice. Quantification of myocardial macrophage abundance (positive for F4/80) within the infarcted area of the indicated mice. Myocardial immunofluorescence staining for αSMA in mice 7 days after cryoinjury and treatment with the indicated viruses. Quantification of small αSMA‐positive small conductance vessel (˜20–50 μm in diameter) in the myocardium of mice as shown. Scale bars: 50μm. Scheme of the mouse Il13 promoter region (˜700 bp) before the ATG translational start sites, GATA binding regions are marked by a red arrow, and the primers used are indicated by blue or black arrows. On the left, the results of a chromatin immunoprecipitation assay from neonatal hearts 7 days after cryoinjury and after immunoprecipitation with control IgG or GATA4 are shown. * P = 0.0197 for primer 1 and * P = 0.0142 for primer 2. Representative immunoblots for cyclin A2 and GAPDH from hearts of the mice as indicated (con = control, KO = CM‐G4‐KO). Representative immunoblots for cenpa and GAPDH from hearts of the mice as indicated. All lanes were run on the same gel but were non‐contiguous where indicated by the gray lines. Data information: (A–G) The number within bars indicates the number of mice analyzed in that particular group. All data are expressed as mean ± SEM. Unpaired Student's t ‐test was used to compare groups.
Figure Legend Snippet: Additional characterization of GATA 4 overexpression and IL ‐13 in neonatal hearts Heart weight/body weight (BW) ratio of the indicated mice 7 days after sham surgery or cryoinfarction and application of adenoviruses as shown. Lung weight/body weight ratio of the indicated mice 7 days after sham surgery or cryoinfarction and application of adenoviruses as shown. Cardiomyocyte (CM) cross‐sectional area assessed 7 days after cryoinfarction in the indicated mice. Quantification of myocardial macrophage abundance (positive for F4/80) remote of the infarcted area of the indicated mice. Quantification of myocardial macrophage abundance (positive for F4/80) within the infarcted area of the indicated mice. Myocardial immunofluorescence staining for αSMA in mice 7 days after cryoinjury and treatment with the indicated viruses. Quantification of small αSMA‐positive small conductance vessel (˜20–50 μm in diameter) in the myocardium of mice as shown. Scale bars: 50μm. Scheme of the mouse Il13 promoter region (˜700 bp) before the ATG translational start sites, GATA binding regions are marked by a red arrow, and the primers used are indicated by blue or black arrows. On the left, the results of a chromatin immunoprecipitation assay from neonatal hearts 7 days after cryoinjury and after immunoprecipitation with control IgG or GATA4 are shown. * P = 0.0197 for primer 1 and * P = 0.0142 for primer 2. Representative immunoblots for cyclin A2 and GAPDH from hearts of the mice as indicated (con = control, KO = CM‐G4‐KO). Representative immunoblots for cenpa and GAPDH from hearts of the mice as indicated. All lanes were run on the same gel but were non‐contiguous where indicated by the gray lines. Data information: (A–G) The number within bars indicates the number of mice analyzed in that particular group. All data are expressed as mean ± SEM. Unpaired Student's t ‐test was used to compare groups.

Techniques Used: Over Expression, Mouse Assay, Immunofluorescence, Staining, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Western Blot

GATA 4 is necessary for cardiac regeneration after cryoinjury Cardiac GATA4 protein abundance analyzed by immunoblotting in mice treated as indicated. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4‐overexpressing adenovirus. Densitometric quantification of the immunoblot shown in (A); *** P = 0.0008 for sham vs. cryoinfarction and *** P
Figure Legend Snippet: GATA 4 is necessary for cardiac regeneration after cryoinjury Cardiac GATA4 protein abundance analyzed by immunoblotting in mice treated as indicated. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4‐overexpressing adenovirus. Densitometric quantification of the immunoblot shown in (A); *** P = 0.0008 for sham vs. cryoinfarction and *** P

Techniques Used: Mouse Assay, Positive Control, Infection

GATA 4 overexpression triggers cardiomyocyte proliferation in vitro and improves myocardial regeneration at P7 in vivo Representative immunoblot for GATA4 and GAPDH from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4‐expressing adenovirus (Ad.GATA4). Quantification of BrdU incorporation, pH3 labeling, and cytokinesis in neonatal cardiomyocytes treated as shown. * P = 0.0272 for BrdU incorporation, * P = 0.0329 for pH3 labeling, * P = 0.0217 for cytokinesis. Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental timescale above. A quantification of the immunoblot from (C) is shown; ** P = 0.0062. Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E′). Scale bars: 20 μm. **** P
Figure Legend Snippet: GATA 4 overexpression triggers cardiomyocyte proliferation in vitro and improves myocardial regeneration at P7 in vivo Representative immunoblot for GATA4 and GAPDH from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4‐expressing adenovirus (Ad.GATA4). Quantification of BrdU incorporation, pH3 labeling, and cytokinesis in neonatal cardiomyocytes treated as shown. * P = 0.0272 for BrdU incorporation, * P = 0.0329 for pH3 labeling, * P = 0.0217 for cytokinesis. Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental timescale above. A quantification of the immunoblot from (C) is shown; ** P = 0.0062. Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E′). Scale bars: 20 μm. **** P

Techniques Used: Over Expression, In Vitro, In Vivo, Expressing, BrdU Incorporation Assay, Labeling, Mouse Assay, Staining

25) Product Images from "Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression"

Article Title: Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201109067

Model of FAK function downstream of TNF-α in the regulation of VCAM-1 expression. TNF-α binding to cell surface receptors triggers intracellular signaling cascade activation of MAPKs and NF-κB. This leads to alterations in gene transcription of targets such as VCAM-1 that is regulated in part by combined effects of AP-1, GATA, and NF-κB transcription factors. Inhibition of FAK prevents TNF-α–induced MAPK activation and the inhibition of GATA4 Ser105 phosphorylation. Inhibited FAK (FAK-KD) accumulates in the nucleus, binds directly to GATA4, and promotes increased GATA4 ubiquitination and proteasomal degradation via interactions with the CHIP E3 ubiquitin ligase. Impairment in both FAK-mediated MAPK activation and GATA4 stability prevent cytokine-stimulated VCAM-1 transcription and reveal novel anti-inflammatory effects of FAK inhibition.
Figure Legend Snippet: Model of FAK function downstream of TNF-α in the regulation of VCAM-1 expression. TNF-α binding to cell surface receptors triggers intracellular signaling cascade activation of MAPKs and NF-κB. This leads to alterations in gene transcription of targets such as VCAM-1 that is regulated in part by combined effects of AP-1, GATA, and NF-κB transcription factors. Inhibition of FAK prevents TNF-α–induced MAPK activation and the inhibition of GATA4 Ser105 phosphorylation. Inhibited FAK (FAK-KD) accumulates in the nucleus, binds directly to GATA4, and promotes increased GATA4 ubiquitination and proteasomal degradation via interactions with the CHIP E3 ubiquitin ligase. Impairment in both FAK-mediated MAPK activation and GATA4 stability prevent cytokine-stimulated VCAM-1 transcription and reveal novel anti-inflammatory effects of FAK inhibition.

Techniques Used: Expressing, Binding Assay, Activation Assay, Inhibition, Chromatin Immunoprecipitation

FAK-enhanced GATA4 polyubiquitination is dependent on CHIP and FAK-FERM nuclear localization. (A) FAK-WT, Mdm2 −/− p53 −/− , and CHIP −/− MEFs were transfected with flag-tagged GATA4 and treated with MG132 (40 µM, 3 h) and FAK-I (1 µM PF271), as indicated. Flag tag immunoprecipitates (IPs) were evaluated by anti-ubiquitin (Ub) and GATA4 immunoblotting and show no ubiquitination of GATA4 in CHIP −/− MEFs. (B) CHIP −/− MEFs were transfected with HA-tagged CHIP and flag-tagged GATA4 and treated with MG132 (40 µM, 3 h) and FAK-I (1 µM PF271). Flag tag immunoprecipitates were evaluated by anti-ubiquitin and GATA4 immunoblotting and show rescue of GATA4 ubiquitination by CHIP reexpression. Lysates show HA-CHIP and actin expression. (C) 293T cells were cotransfected with GFP-tagged FAK, FAK-FERM, and FAK-CT with HA-CHIP. Coimmunoprecipitation analyses with antibodies to GFP reveal FAK-FERM and CHIP association by immunoblotting. Lysates show equal levels of GFP-FAK and HA-CHIP expression. (D) 293T cells were transfected with GFP-tagged FAK-FERM WT or FAK-FERM R177A/R178A, flag-tagged GATA4, and His-tagged ubiquitin and denatured lysates (8 M urea) purified by nickel agarose affinity binding. GFP-FERM and Flag-GATA4 (with actin and GAPDH as loading controls; left), total eluted ubiquitinated proteins (middle), and mono-, di-, and polyubiquitinated GATA4 (right), as determined by immunoblotting, are shown. (E) FAK-WT MEFs pretreated with DMSO or FAK-I (1 µM PF271, 30 min) were stimulated with 10 ng/ml TNF-α for the indicated times, and lysates were prepared for immunoblotting. Blots for activated GATA4 (pS105), total GATA4, activated FAK (pY397), and actin are shown.
Figure Legend Snippet: FAK-enhanced GATA4 polyubiquitination is dependent on CHIP and FAK-FERM nuclear localization. (A) FAK-WT, Mdm2 −/− p53 −/− , and CHIP −/− MEFs were transfected with flag-tagged GATA4 and treated with MG132 (40 µM, 3 h) and FAK-I (1 µM PF271), as indicated. Flag tag immunoprecipitates (IPs) were evaluated by anti-ubiquitin (Ub) and GATA4 immunoblotting and show no ubiquitination of GATA4 in CHIP −/− MEFs. (B) CHIP −/− MEFs were transfected with HA-tagged CHIP and flag-tagged GATA4 and treated with MG132 (40 µM, 3 h) and FAK-I (1 µM PF271). Flag tag immunoprecipitates were evaluated by anti-ubiquitin and GATA4 immunoblotting and show rescue of GATA4 ubiquitination by CHIP reexpression. Lysates show HA-CHIP and actin expression. (C) 293T cells were cotransfected with GFP-tagged FAK, FAK-FERM, and FAK-CT with HA-CHIP. Coimmunoprecipitation analyses with antibodies to GFP reveal FAK-FERM and CHIP association by immunoblotting. Lysates show equal levels of GFP-FAK and HA-CHIP expression. (D) 293T cells were transfected with GFP-tagged FAK-FERM WT or FAK-FERM R177A/R178A, flag-tagged GATA4, and His-tagged ubiquitin and denatured lysates (8 M urea) purified by nickel agarose affinity binding. GFP-FERM and Flag-GATA4 (with actin and GAPDH as loading controls; left), total eluted ubiquitinated proteins (middle), and mono-, di-, and polyubiquitinated GATA4 (right), as determined by immunoblotting, are shown. (E) FAK-WT MEFs pretreated with DMSO or FAK-I (1 µM PF271, 30 min) were stimulated with 10 ng/ml TNF-α for the indicated times, and lysates were prepared for immunoblotting. Blots for activated GATA4 (pS105), total GATA4, activated FAK (pY397), and actin are shown.

Techniques Used: Chromatin Immunoprecipitation, Transfection, FLAG-tag, Expressing, Purification, Binding Assay

FAK inhibition decreases GATA4 levels needed for TNF-α–induced VCAM-1 expression. (A) Steady-state GATA4 and GATA6 levels in FAK-WT and FAK-KD MEFs, as determined by immunoblotting with actin as a control. (B) FAK-WT MEFs treated with DMSO or FAK-I (1 µM PF271, 6 h) and lysates blotted for GATA4 or GATA6. Anti-GAPDH blotting is shown as loading controls. (C) GATA4 mRNA levels to GAPDH were determined by Q-PCR (±SD; n = 3) in experiments, as described in B. (D) Rescue of TNF-α–induced VCAM-1 expression in FAK-KD MEFs by combined FAK-WT and GATA4 expression. Combinations of GFP-FAK and GATA4 were transfected into FAK-KD MEFs. After 24 h, cells were stimulated with 10 ng/ml TNF-α, as indicated, and FAK, VCAM-1, and GATA4 immunoblotting was performed at 40 h. (E) FAK-WT MEFs were transfected with Scr or GATA4 (G4) siRNA and, after 48 h, stimulated with TNF-α (10 ng/ml, 6 h), and immunoblotting was performed for VCAM-1 and GATA4. Anti-actin and anti-GAPDH blotting are shown as loading controls. (F) Densitometry analyses of VCAM-1 protein levels relative to actin, as described in E. (±SD; n = 2; ***, P
Figure Legend Snippet: FAK inhibition decreases GATA4 levels needed for TNF-α–induced VCAM-1 expression. (A) Steady-state GATA4 and GATA6 levels in FAK-WT and FAK-KD MEFs, as determined by immunoblotting with actin as a control. (B) FAK-WT MEFs treated with DMSO or FAK-I (1 µM PF271, 6 h) and lysates blotted for GATA4 or GATA6. Anti-GAPDH blotting is shown as loading controls. (C) GATA4 mRNA levels to GAPDH were determined by Q-PCR (±SD; n = 3) in experiments, as described in B. (D) Rescue of TNF-α–induced VCAM-1 expression in FAK-KD MEFs by combined FAK-WT and GATA4 expression. Combinations of GFP-FAK and GATA4 were transfected into FAK-KD MEFs. After 24 h, cells were stimulated with 10 ng/ml TNF-α, as indicated, and FAK, VCAM-1, and GATA4 immunoblotting was performed at 40 h. (E) FAK-WT MEFs were transfected with Scr or GATA4 (G4) siRNA and, after 48 h, stimulated with TNF-α (10 ng/ml, 6 h), and immunoblotting was performed for VCAM-1 and GATA4. Anti-actin and anti-GAPDH blotting are shown as loading controls. (F) Densitometry analyses of VCAM-1 protein levels relative to actin, as described in E. (±SD; n = 2; ***, P

Techniques Used: Inhibition, Expressing, Polymerase Chain Reaction, Transfection

Inhibited FAK is nuclear localized, and FAK-FERM binds GATA4 to promote GATA4 ubiquitination in cells. (A) FAK-WT MEFs treated with DMSO, FAK-I (1 µM PF271), or FAK-I with MG132 (40 µM) for 12 h and lysates blotted for GATA4 and actin. (B) FAK-WT and FAK-KD MEF lysates were separated into cytosolic (C) or nuclear (N) fractions and immunoblotted for FAK, PARP, GATA4, and GAPDH. PARP and GAPDH are nuclear and cytosolic markers, respectively. (C) FAK inhibition promotes FAK nuclear localization within 3 h. FAK-WT MEFs were treated with FAK-I (1 µM PF271) for the indicated times, and fractionated lysates were immunoblotted for FAK, PARP, and GAPDH. (D) 293T cells were transduced with Ad-TA, the indicated HA- or Myc-tagged FAK-WT, FAK-KD, FAK-FERM, and FAK-CT constructs, and association with endogenous GATA4 was determined by coimmunoprecipitation (IP). Immunoblotting shows expression of FAK constructs or actin (top) and FAK-FERM association with GATA4 (bottom). (E) GATA4 directly binds FAK. In vitro translated GFP–tandem affinity probe (TAP), FAK-WT, FAK-KD, TAP-FAK-FERM, and TAP-FAK kinase domain (386–686) were used in a direct binding assay with GST or GST fusions of GATA4 N terminus or GATA4 C terminus. Streptavidin (Strept)-HRP analyses show the amount of FAK bound (left) or 10% of input (right). (F) FAK-FERM enhances GATA4 ubiquitination. FAK-WT MEFs were transduced with Ad-TA, the indicated FAK constructs, and flag-tagged GATA4 and treated with MG132 (40 µM, 3 h). Whole-cell lysates were analyzed for expression of FAK or actin (left), and flag tag immunoprecipitates (antibody coupled to beads) were evaluated by anti-ubiquitin (Ub) and GATA4 immunoblotting.
Figure Legend Snippet: Inhibited FAK is nuclear localized, and FAK-FERM binds GATA4 to promote GATA4 ubiquitination in cells. (A) FAK-WT MEFs treated with DMSO, FAK-I (1 µM PF271), or FAK-I with MG132 (40 µM) for 12 h and lysates blotted for GATA4 and actin. (B) FAK-WT and FAK-KD MEF lysates were separated into cytosolic (C) or nuclear (N) fractions and immunoblotted for FAK, PARP, GATA4, and GAPDH. PARP and GAPDH are nuclear and cytosolic markers, respectively. (C) FAK inhibition promotes FAK nuclear localization within 3 h. FAK-WT MEFs were treated with FAK-I (1 µM PF271) for the indicated times, and fractionated lysates were immunoblotted for FAK, PARP, and GAPDH. (D) 293T cells were transduced with Ad-TA, the indicated HA- or Myc-tagged FAK-WT, FAK-KD, FAK-FERM, and FAK-CT constructs, and association with endogenous GATA4 was determined by coimmunoprecipitation (IP). Immunoblotting shows expression of FAK constructs or actin (top) and FAK-FERM association with GATA4 (bottom). (E) GATA4 directly binds FAK. In vitro translated GFP–tandem affinity probe (TAP), FAK-WT, FAK-KD, TAP-FAK-FERM, and TAP-FAK kinase domain (386–686) were used in a direct binding assay with GST or GST fusions of GATA4 N terminus or GATA4 C terminus. Streptavidin (Strept)-HRP analyses show the amount of FAK bound (left) or 10% of input (right). (F) FAK-FERM enhances GATA4 ubiquitination. FAK-WT MEFs were transduced with Ad-TA, the indicated FAK constructs, and flag-tagged GATA4 and treated with MG132 (40 µM, 3 h). Whole-cell lysates were analyzed for expression of FAK or actin (left), and flag tag immunoprecipitates (antibody coupled to beads) were evaluated by anti-ubiquitin (Ub) and GATA4 immunoblotting.

Techniques Used: Inhibition, Transduction, Construct, Expressing, In Vitro, Binding Assay, FLAG-tag

26) Product Images from "Sin3a is essential for the genome integrity and viability of pluripotent cells"

Article Title: Sin3a is essential for the genome integrity and viability of pluripotent cells

Journal: Developmental Biology

doi: 10.1016/j.ydbio.2011.12.019

Loss of ICM cells in peri-implantation Sin3a -null embryos. A. Heterozygote and null 4.5 dpc embryos stained for Eomes (green), Oct4 (red) and Gata4 (purple). Scale bar = 50 μm. B. 5.5 dpc embryos of indicated genotypes stained for Eomes (green), Oct4 (red) and DAPI (blue). Scale bar = 100 μm. C. Sin3a +/− and Sin3a −/− embryos recovered after two days of diapause stained for Eomes (green), in the left hand panel, and Oct4 (red), Gata4 (white) and DAPI (blue) in the middle panel. Phase image of the embryos is shown in the right hand panel. Genotypes are indicated to the right. Scale bar = 50 μm.
Figure Legend Snippet: Loss of ICM cells in peri-implantation Sin3a -null embryos. A. Heterozygote and null 4.5 dpc embryos stained for Eomes (green), Oct4 (red) and Gata4 (purple). Scale bar = 50 μm. B. 5.5 dpc embryos of indicated genotypes stained for Eomes (green), Oct4 (red) and DAPI (blue). Scale bar = 100 μm. C. Sin3a +/− and Sin3a −/− embryos recovered after two days of diapause stained for Eomes (green), in the left hand panel, and Oct4 (red), Gata4 (white) and DAPI (blue) in the middle panel. Phase image of the embryos is shown in the right hand panel. Genotypes are indicated to the right. Scale bar = 50 μm.

Techniques Used: Staining

27) Product Images from "Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy"

Article Title: Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-2121-0

Activation of GATA4 mediates cardiac hypertrophy in Kindlin-2 cKO mice. a Wild-type Kindlin-2 or cKO mice were injected with saline or ISO. Total protein was extracted from heart tissue for western blot (left panel). Protein bands on the left were scanned and relative band intensities were normalized (right panel). b Immunohistochemical staining showed GATA4 level (Scale bar = 50 µm). c Lysates from heart tissue of wild-type mice were prepared for Co-IP. d Lysates were extracted from heart tissue of wild-type and Kindlin-2 cKO mice for ChIP assays. Means ± S.D. * P
Figure Legend Snippet: Activation of GATA4 mediates cardiac hypertrophy in Kindlin-2 cKO mice. a Wild-type Kindlin-2 or cKO mice were injected with saline or ISO. Total protein was extracted from heart tissue for western blot (left panel). Protein bands on the left were scanned and relative band intensities were normalized (right panel). b Immunohistochemical staining showed GATA4 level (Scale bar = 50 µm). c Lysates from heart tissue of wild-type mice were prepared for Co-IP. d Lysates were extracted from heart tissue of wild-type and Kindlin-2 cKO mice for ChIP assays. Means ± S.D. * P

Techniques Used: Activation Assay, Mouse Assay, Injection, Western Blot, Immunohistochemistry, Staining, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation

A working model for the role of Kindlin-2 in suppressing GATA4.
Figure Legend Snippet: A working model for the role of Kindlin-2 in suppressing GATA4.

Techniques Used:

Kindlin-2 suppresses expression of GATA4. a GO analysis of differentially expressed genes in RNAseq. b 11 differentially expressed genes (log2 Fold > 1) in cardiovascular disease-related genes. c Control or Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes for 48 h, followed western blot. d Protein bands were scanned and relative band intensities were normalized to each GAPDH band. The column diagrams represent average relative band intensity with standard error from three independent experiments. e Cardiomyocytes were infected by Kindlin-2 or control adenovirus, followed western blot. f Relative band intensities were analyzed. g Control or Kindlin-2 siRNA was transfected into cardiomyocytes and RT-qPCR detected Kindlin-2 and GATA4 mRNA level. h Adenovirus carrying Kindlin-2 or control was transfected into cardiomyocytes, followed RT-qPCR. i Schematic diagrams of the regions for ChIP assay. j Lysates were extracted from mouse cardiac tissues for ChIP assays using anti-Kindlin-2 antibody. Q-PCR assay was then performed to quantify ChIP-enriched DNA using the three primers.
Figure Legend Snippet: Kindlin-2 suppresses expression of GATA4. a GO analysis of differentially expressed genes in RNAseq. b 11 differentially expressed genes (log2 Fold > 1) in cardiovascular disease-related genes. c Control or Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes for 48 h, followed western blot. d Protein bands were scanned and relative band intensities were normalized to each GAPDH band. The column diagrams represent average relative band intensity with standard error from three independent experiments. e Cardiomyocytes were infected by Kindlin-2 or control adenovirus, followed western blot. f Relative band intensities were analyzed. g Control or Kindlin-2 siRNA was transfected into cardiomyocytes and RT-qPCR detected Kindlin-2 and GATA4 mRNA level. h Adenovirus carrying Kindlin-2 or control was transfected into cardiomyocytes, followed RT-qPCR. i Schematic diagrams of the regions for ChIP assay. j Lysates were extracted from mouse cardiac tissues for ChIP assays using anti-Kindlin-2 antibody. Q-PCR assay was then performed to quantify ChIP-enriched DNA using the three primers.

Techniques Used: Expressing, Transfection, Western Blot, Infection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction

SUV39H1 mediated suppression of Kindlin-2 on GATA4. a Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by Western blot. b Relative band intensities of western blot were analyzed. c Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by RT-qPCR. d HEK293A cells were treated with the SUV39H1 inhibitor Chaetomin at various doses for 48 h, and GATA4 was measured by western blot. e Relative band intensities of western blot were analyzed. f HEK293A cells were treated with Chaetomin and GATA4 was detected by RT-PCR. g Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. Means ± S.D. ** indicates p
Figure Legend Snippet: SUV39H1 mediated suppression of Kindlin-2 on GATA4. a Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by Western blot. b Relative band intensities of western blot were analyzed. c Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by RT-qPCR. d HEK293A cells were treated with the SUV39H1 inhibitor Chaetomin at various doses for 48 h, and GATA4 was measured by western blot. e Relative band intensities of western blot were analyzed. f HEK293A cells were treated with Chaetomin and GATA4 was detected by RT-PCR. g Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. Means ± S.D. ** indicates p

Techniques Used: Transfection, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation

Kindlin-2 prevents cardiomyocytes from hypertrophy through suppressing GATA4. a–c Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO treatment (5 µm, 24 h). The efficacy of Kindlin-2 overexpression was detected by Western blot a . Representative images of cardiomyocytes stained with anti-α-actinin2 (Alexa Fluor 568) b . Cell surface areas were measured c . d – f Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes plus ISO treatment with or without knockdown of GATA4 and GATA6 alone or in combination. The efficacy of Kindlin-2 siRNA was detected by Western blotting d . Representative images of cardiomyocytes stained with anti-α-actinin2 e . Cell surface areas were measured f . g Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO, followed by Western blot (left panel). Relative band intensities were analyzed (right panel). h Cardiomyocytes were transfected with control or Kindlin-2 siRNA plus ISO treatment, followed Western blot (left panel). Relative band intensities were analyzed (right panel).
Figure Legend Snippet: Kindlin-2 prevents cardiomyocytes from hypertrophy through suppressing GATA4. a–c Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO treatment (5 µm, 24 h). The efficacy of Kindlin-2 overexpression was detected by Western blot a . Representative images of cardiomyocytes stained with anti-α-actinin2 (Alexa Fluor 568) b . Cell surface areas were measured c . d – f Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes plus ISO treatment with or without knockdown of GATA4 and GATA6 alone or in combination. The efficacy of Kindlin-2 siRNA was detected by Western blotting d . Representative images of cardiomyocytes stained with anti-α-actinin2 e . Cell surface areas were measured f . g Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO, followed by Western blot (left panel). Relative band intensities were analyzed (right panel). h Cardiomyocytes were transfected with control or Kindlin-2 siRNA plus ISO treatment, followed Western blot (left panel). Relative band intensities were analyzed (right panel).

Techniques Used: Infection, Over Expression, Western Blot, Staining, Transfection

28) Product Images from "Generation of Induced Pluripotent Stem Cells in Rabbits"

Article Title: Generation of Induced Pluripotent Stem Cells in Rabbits

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.150540

Differentiation of rabbit iPS cells in vitro ( A ) and in vivo ( B ). Aa , embryoid bodies formed from iPS cells under differentiation conditions at day 5. After replating on gelatin-coated dishes, they further differentiated into a variety of cell types from the three basic germ layers expressing specific markers, as detected by immunostaining. Ab , neural cells (ectoderm) expressing βIII-tubulin ( green ) and glial fibrillary acidic protein ( red ). Ac , smooth muscle cells (mesoderm) expressing α-smooth muscle myosin ( green ). Ad , endodermal cells expressing GATA4 ( red ). The cells were counterstained with DAPI. Representative images were prepared from iPS-L1 cells at passage 13. B , teratoma formation by rabbit iPS cells (iPS-L1 at passage 8). Various tissues of the three germ layer origins are identified: epidermis ( a , ectoderm), bone ( b , mesoderm), glands ( c , endoderm), neural tissue ( d , ectoderm), muscle fibers ( e , mesoderm), and epithelium with goblet cells ( f , endoderm). Scale bar , 100 μm.
Figure Legend Snippet: Differentiation of rabbit iPS cells in vitro ( A ) and in vivo ( B ). Aa , embryoid bodies formed from iPS cells under differentiation conditions at day 5. After replating on gelatin-coated dishes, they further differentiated into a variety of cell types from the three basic germ layers expressing specific markers, as detected by immunostaining. Ab , neural cells (ectoderm) expressing βIII-tubulin ( green ) and glial fibrillary acidic protein ( red ). Ac , smooth muscle cells (mesoderm) expressing α-smooth muscle myosin ( green ). Ad , endodermal cells expressing GATA4 ( red ). The cells were counterstained with DAPI. Representative images were prepared from iPS-L1 cells at passage 13. B , teratoma formation by rabbit iPS cells (iPS-L1 at passage 8). Various tissues of the three germ layer origins are identified: epidermis ( a , ectoderm), bone ( b , mesoderm), glands ( c , endoderm), neural tissue ( d , ectoderm), muscle fibers ( e , mesoderm), and epithelium with goblet cells ( f , endoderm). Scale bar , 100 μm.

Techniques Used: In Vitro, In Vivo, Expressing, Immunostaining

29) Product Images from "FGF/MAPK signaling sets the switching threshold of a bistable circuit controlling cell fate decisions in embryonic stem cells"

Article Title: FGF/MAPK signaling sets the switching threshold of a bistable circuit controlling cell fate decisions in embryonic stem cells

Journal: Development (Cambridge, England)

doi: 10.1242/dev.127530

Expression of endogenous markers of PrE-like differentiation following transient expression of GATA6-FLAG and GATA4-FLAG. (A) Experimental approach. Doxycycline-induced transgene expression creates a GATA6/NANOG co-expression state in ESCs similar to the situation in the ICM, from which cells can embark on PrE-like differentiation, or return to the NANOG-positive state. (B) Immunostaining (upper panel) and quantification (lower panel) of untreated (left) or doxycycline-treated (right) inducible ESCs indicates co-expression of NANOG and GATA6-FLAG in individual cells after 6 h of doxycycline treatment. Co-expression is limited because of heterogeneous NANOG and GATA6-FLAG expression in the presence of serum and feeders. (C) Immunostaining (upper panels) and quantification (lower panels) of NANOG and GATA4 expression 24 h after the end of a 6 h doxycycline pulse. GATA4 expression depends on doxycycline treatment, and is mutually exclusive with NANOG expression. (D-F) Flow cytometry of cells immunostained for endogenous GATA4 and GATA6 one day after transient GATA4-FLAG (D) or GATA6-FLAG (E) expression. (F) Overlay of D and E. Scale bars: 50 µm.
Figure Legend Snippet: Expression of endogenous markers of PrE-like differentiation following transient expression of GATA6-FLAG and GATA4-FLAG. (A) Experimental approach. Doxycycline-induced transgene expression creates a GATA6/NANOG co-expression state in ESCs similar to the situation in the ICM, from which cells can embark on PrE-like differentiation, or return to the NANOG-positive state. (B) Immunostaining (upper panel) and quantification (lower panel) of untreated (left) or doxycycline-treated (right) inducible ESCs indicates co-expression of NANOG and GATA6-FLAG in individual cells after 6 h of doxycycline treatment. Co-expression is limited because of heterogeneous NANOG and GATA6-FLAG expression in the presence of serum and feeders. (C) Immunostaining (upper panels) and quantification (lower panels) of NANOG and GATA4 expression 24 h after the end of a 6 h doxycycline pulse. GATA4 expression depends on doxycycline treatment, and is mutually exclusive with NANOG expression. (D-F) Flow cytometry of cells immunostained for endogenous GATA4 and GATA6 one day after transient GATA4-FLAG (D) or GATA6-FLAG (E) expression. (F) Overlay of D and E. Scale bars: 50 µm.

Techniques Used: Expressing, Immunostaining, Flow Cytometry, Cytometry

A GATA4-mCherry threshold dose determines PrE-like differentiation. (A) Flow cytometry for Gata6:H2B-Venus fluorescence one day after transient GATA4-mCherry expression triggered by doxycycline pulses of indicated lengths. (B) Quantitative analysis of results from A. Data points show mean±s.d. from three independent experiments. (C) Cells sorted for low (middle) and high (right) GATA4-mCherry expression levels after a 6 h doxycycline pulse and immunostained 30 h after re-plating. Unsorted control is on the left. Scale bar: 50 µm. (D) Flow cytometry of cells treated as in C stained for GATA6 expression. Purification increases the proportion of differentiating cells in both sorted pools compared with the unsorted control, and a larger proportion of GATA4-mCherry high cells activate GATA6 expression compared with GATA4-mCherry low cells. (E) GATA4-mCherry fluorescence traces of cells filmed during and after a 6 h doxycycline pulse. Color-code of individual traces is informed by immunostaining for NANOG and GATA6 at the end of the time-lapse. Area shaded in light green indicates duration of doxycycline pulse, red bar indicates time point analyzed in F,G, and black curve indicates optimal threshold calculated by ROC. See also Movie 2 and Fig. S9 . (F) GATA4-mCherry fluorescence intensity of cells from the experiment shown in E at a single time-point (red vertical line in E). Optimal threshold to predict fate choice estimated by ROC analysis is indicated by a black dotted line. (G) ROC curve for the time-point shown in F. The optimal threshold maximizing the difference between the true positive and false positive prediction rate (TPR and FPR) is indicated by a red dot. (H) Area under the curve (AUC) values from ROC analysis in all time frames of the experiment shown in E. Error margin indicates s.d. determined by bootstrapping ( n =1000).
Figure Legend Snippet: A GATA4-mCherry threshold dose determines PrE-like differentiation. (A) Flow cytometry for Gata6:H2B-Venus fluorescence one day after transient GATA4-mCherry expression triggered by doxycycline pulses of indicated lengths. (B) Quantitative analysis of results from A. Data points show mean±s.d. from three independent experiments. (C) Cells sorted for low (middle) and high (right) GATA4-mCherry expression levels after a 6 h doxycycline pulse and immunostained 30 h after re-plating. Unsorted control is on the left. Scale bar: 50 µm. (D) Flow cytometry of cells treated as in C stained for GATA6 expression. Purification increases the proportion of differentiating cells in both sorted pools compared with the unsorted control, and a larger proportion of GATA4-mCherry high cells activate GATA6 expression compared with GATA4-mCherry low cells. (E) GATA4-mCherry fluorescence traces of cells filmed during and after a 6 h doxycycline pulse. Color-code of individual traces is informed by immunostaining for NANOG and GATA6 at the end of the time-lapse. Area shaded in light green indicates duration of doxycycline pulse, red bar indicates time point analyzed in F,G, and black curve indicates optimal threshold calculated by ROC. See also Movie 2 and Fig. S9 . (F) GATA4-mCherry fluorescence intensity of cells from the experiment shown in E at a single time-point (red vertical line in E). Optimal threshold to predict fate choice estimated by ROC analysis is indicated by a black dotted line. (G) ROC curve for the time-point shown in F. The optimal threshold maximizing the difference between the true positive and false positive prediction rate (TPR and FPR) is indicated by a red dot. (H) Area under the curve (AUC) values from ROC analysis in all time frames of the experiment shown in E. Error margin indicates s.d. determined by bootstrapping ( n =1000).

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Expressing, Staining, Purification, Immunostaining

Transient GATA4-mCherry expression induces stable expression of PrE marker genes. (A) Immunostaining for Venus and GATA6 protein in Gata6:H2B-Venus reporter cells 24 h after a 6 h pulse of doxycycline-induced GATA4-mCherry expression. Scale bar: 50 µm. (B) Flow cytometry of GATA6-reporter cells treated as in A. (C) Flow cytometry detecting Gata6:H2B-Venus expression at indicated time-points after a 6 h pulse of GATA4-mCherry induction. (D) Percentages of Venus high cells at different times after a 6 h doxycycline pulse. Data points represent mean±s.d. from three independent experiments. (E) Flow cytometry of Venus high cells sorted 18 h after the end of a 6 h doxycycline pulse (royal blue), cultured for 48 h and analyzed for Venus expression. (F) Venus fluorescence intensity values of individual GATA6-reporter cells tracked in time-lapse movies during and following a 6 h doxycycline pulse (light green shaded area). Color-code is informed by hierarchical clustering based on Venus expression levels. Small panels on the right show traces for each cluster separately. See also Fig. S6 and Movie 1 .
Figure Legend Snippet: Transient GATA4-mCherry expression induces stable expression of PrE marker genes. (A) Immunostaining for Venus and GATA6 protein in Gata6:H2B-Venus reporter cells 24 h after a 6 h pulse of doxycycline-induced GATA4-mCherry expression. Scale bar: 50 µm. (B) Flow cytometry of GATA6-reporter cells treated as in A. (C) Flow cytometry detecting Gata6:H2B-Venus expression at indicated time-points after a 6 h pulse of GATA4-mCherry induction. (D) Percentages of Venus high cells at different times after a 6 h doxycycline pulse. Data points represent mean±s.d. from three independent experiments. (E) Flow cytometry of Venus high cells sorted 18 h after the end of a 6 h doxycycline pulse (royal blue), cultured for 48 h and analyzed for Venus expression. (F) Venus fluorescence intensity values of individual GATA6-reporter cells tracked in time-lapse movies during and following a 6 h doxycycline pulse (light green shaded area). Color-code is informed by hierarchical clustering based on Venus expression levels. Small panels on the right show traces for each cluster separately. See also Fig. S6 and Movie 1 .

Techniques Used: Expressing, Marker, Immunostaining, Flow Cytometry, Cytometry, Cell Culture, Fluorescence

MAPK signaling controls the proportion of cells with PrE-like differentiation. (A) Immunoblot detecting phosphorylated ERK (top) and total ERK (bottom) in cells grown for 3 days in the presence of serum and 1 µM PD03 1 h after transfer into medium containing indicated concentrations of PD03. (B) Immunostaining of ESCs after a 6 h pulse of doxycycline-induced GATA4-mCherry expression and 24 h of differentiation in the indicated concentrations of PD03. Scale bars: 50 µm. (C) Flow cytometry of GATA6 expression in cells treated as in B. (D) Plot of relative Erk phosphorylation levels versus percentage of GATA6-positive cells for different concentrations of PD03. Data points show mean±s.d. from three independent experiments per condition. (E) GATA4-mCherry fluorescence traces of cells filmed during and after a 6 h doxycycline pulse in the absence of PD03 (top) or with 62.5 nM PD03 (bottom). Color-code of individual traces is informed by immunostaining for NANOG and GATA6 at the end of the time-lapse. Light green shaded area indicates presence of doxycycline, black trace indicates optimal threshold estimated by ROC, and insets show traces for cells with GATA6- and NANOG-positive progeny separately. (F) GATA4-mCherry fluorescence intensity of cells from the experiment shown in E at a single time-point 1 h after removal of doxycycline in the absence of PD03 (left) and in 62.5 nM PD03 (right). (G) AUC values determined by ROC analysis of the dataset shown in E for no PD03 (red) and 62.5 nM PD03 (green). AUC values decay after ∼20 h as a result of the very low levels of GATA4-mCherry expression at the end of this experiment. Error margin indicates s.d. determined by bootstrapping ( n =1000). (H) The optimal threshold to predict differentiation from GATA4-mCherry expression in the absence of PD03 (red) and in the presence of 62.5 nM PD03 (green) increases with decreasing MAPK signaling. Error margins indicate s.d. determined by bootstrapping ( n =1000).
Figure Legend Snippet: MAPK signaling controls the proportion of cells with PrE-like differentiation. (A) Immunoblot detecting phosphorylated ERK (top) and total ERK (bottom) in cells grown for 3 days in the presence of serum and 1 µM PD03 1 h after transfer into medium containing indicated concentrations of PD03. (B) Immunostaining of ESCs after a 6 h pulse of doxycycline-induced GATA4-mCherry expression and 24 h of differentiation in the indicated concentrations of PD03. Scale bars: 50 µm. (C) Flow cytometry of GATA6 expression in cells treated as in B. (D) Plot of relative Erk phosphorylation levels versus percentage of GATA6-positive cells for different concentrations of PD03. Data points show mean±s.d. from three independent experiments per condition. (E) GATA4-mCherry fluorescence traces of cells filmed during and after a 6 h doxycycline pulse in the absence of PD03 (top) or with 62.5 nM PD03 (bottom). Color-code of individual traces is informed by immunostaining for NANOG and GATA6 at the end of the time-lapse. Light green shaded area indicates presence of doxycycline, black trace indicates optimal threshold estimated by ROC, and insets show traces for cells with GATA6- and NANOG-positive progeny separately. (F) GATA4-mCherry fluorescence intensity of cells from the experiment shown in E at a single time-point 1 h after removal of doxycycline in the absence of PD03 (left) and in 62.5 nM PD03 (right). (G) AUC values determined by ROC analysis of the dataset shown in E for no PD03 (red) and 62.5 nM PD03 (green). AUC values decay after ∼20 h as a result of the very low levels of GATA4-mCherry expression at the end of this experiment. Error margin indicates s.d. determined by bootstrapping ( n =1000). (H) The optimal threshold to predict differentiation from GATA4-mCherry expression in the absence of PD03 (red) and in the presence of 62.5 nM PD03 (green) increases with decreasing MAPK signaling. Error margins indicate s.d. determined by bootstrapping ( n =1000).

Techniques Used: Immunostaining, Expressing, Flow Cytometry, Cytometry, Fluorescence

Culture conditions affect responsiveness to GATA4-mCherry expression. (A) Immunostaining of ESCs cultured for indicated times in 2i+LIF before a 6 h pulse of GATA4-mCherry expression followed by a 24 h chase in medium containing serum+LIF. (B) Flow cytometry of cells treated as in A and stained for GATA6. (C) Percentage of GATA6-positive cells (black) and ratio of the percentages of GATA6-positive and mCherry-positive cells (red) for different durations of pre-culture in 2i+LIF. Data averaged from three (% GATA6-positive) or two (ratios) independent experiments, errors bars state s.d. (D) Immunostaining of ESCs cultured for 3 days in the indicated media before a 6 h pulse of GATA4-mCherry expression followed by a 24 h chase in medium containing serum+LIF. Chi, CHIR99021. (E) Flow cytometry of cells treated as in D stained for GATA6. (F) Average percentage of GATA6-positive cells (black) and ratio of the percentages of GATA6-positive and mCherry-positive cells (red) for different pre-culture media. Data averaged from three (% GATA6-positive) or two (ratios) independent experiments, errors bars indicate s.d. Scale bars: 50 µm.
Figure Legend Snippet: Culture conditions affect responsiveness to GATA4-mCherry expression. (A) Immunostaining of ESCs cultured for indicated times in 2i+LIF before a 6 h pulse of GATA4-mCherry expression followed by a 24 h chase in medium containing serum+LIF. (B) Flow cytometry of cells treated as in A and stained for GATA6. (C) Percentage of GATA6-positive cells (black) and ratio of the percentages of GATA6-positive and mCherry-positive cells (red) for different durations of pre-culture in 2i+LIF. Data averaged from three (% GATA6-positive) or two (ratios) independent experiments, errors bars state s.d. (D) Immunostaining of ESCs cultured for 3 days in the indicated media before a 6 h pulse of GATA4-mCherry expression followed by a 24 h chase in medium containing serum+LIF. Chi, CHIR99021. (E) Flow cytometry of cells treated as in D stained for GATA6. (F) Average percentage of GATA6-positive cells (black) and ratio of the percentages of GATA6-positive and mCherry-positive cells (red) for different pre-culture media. Data averaged from three (% GATA6-positive) or two (ratios) independent experiments, errors bars indicate s.d. Scale bars: 50 µm.

Techniques Used: Expressing, Immunostaining, Cell Culture, Flow Cytometry, Cytometry, Staining

A simple mutual repression circuit recapitulates experimentally observed expression dynamics. (A) Connectivity of the mutual repression circuit used in dynamic simulations. (B) Phase portrait depicting the autonomous dynamics of the system in A. Intersections of the NANOG nullcline (blue) and the GATA nullcline (orange) define three steady states, two of which are stable and correspond to the NANOG high , GATA low Epi state (green dot) or the NANOG low , GATA high PrE state (purple). (C-E) Simulated time traces of endogenous GATA G (C), exogenous GATA4-mCherry G X (D), and NANOG N expression (E). Color code is informed by final GATA expression levels in C (GATA high , purple; GATA low , green), and corresponding traces have same color in C, D and E. (F) Average NANOG-Venus expression levels in cells carrying a NANOG-Venus translational reporter during and after 6 h of doxycycline-induced GATA4-mCherry expression. Cells were classified as GATA6-positive and NANOG-positive by immunostaining after the time-lapse. Presence of doxycycline is indicated by light green shading. Purple and green lines and shaded areas indicate mean NANOG-Venus fluorescence levels±s.d. Insets show histograms for distributions of NANOG-Venus fluorescence in the two classes of cells at 6 h and 16 h after the start of recording; *** P ≤0.0001 (Mann–Whitney U-test). See also Movie 4 .
Figure Legend Snippet: A simple mutual repression circuit recapitulates experimentally observed expression dynamics. (A) Connectivity of the mutual repression circuit used in dynamic simulations. (B) Phase portrait depicting the autonomous dynamics of the system in A. Intersections of the NANOG nullcline (blue) and the GATA nullcline (orange) define three steady states, two of which are stable and correspond to the NANOG high , GATA low Epi state (green dot) or the NANOG low , GATA high PrE state (purple). (C-E) Simulated time traces of endogenous GATA G (C), exogenous GATA4-mCherry G X (D), and NANOG N expression (E). Color code is informed by final GATA expression levels in C (GATA high , purple; GATA low , green), and corresponding traces have same color in C, D and E. (F) Average NANOG-Venus expression levels in cells carrying a NANOG-Venus translational reporter during and after 6 h of doxycycline-induced GATA4-mCherry expression. Cells were classified as GATA6-positive and NANOG-positive by immunostaining after the time-lapse. Presence of doxycycline is indicated by light green shading. Purple and green lines and shaded areas indicate mean NANOG-Venus fluorescence levels±s.d. Insets show histograms for distributions of NANOG-Venus fluorescence in the two classes of cells at 6 h and 16 h after the start of recording; *** P ≤0.0001 (Mann–Whitney U-test). See also Movie 4 .

Techniques Used: Expressing, Immunostaining, Fluorescence, MANN-WHITNEY

30) Product Images from "Spontaneously beating cardiomyocytes derived from white mature adipocytes"

Article Title: Spontaneously beating cardiomyocytes derived from white mature adipocytes

Journal: Cardiovascular Research

doi: 10.1093/cvr/cvp267

Immunocytochemical analysis of cardiomyocyte-specific markers. (Left panels) Cells were stained for sarcomeric actin (Sr, green), or double-stained for sarcomeric actin and GATA4 (red). (Middle panels) Cells were stained for Nkx2.5 (green), or connexin
Figure Legend Snippet: Immunocytochemical analysis of cardiomyocyte-specific markers. (Left panels) Cells were stained for sarcomeric actin (Sr, green), or double-stained for sarcomeric actin and GATA4 (red). (Middle panels) Cells were stained for Nkx2.5 (green), or connexin

Techniques Used: Staining

Immunocytochemical analysis of cardiomyocyte-specific markers. (Left panels) Cells were stained for sarcomeric actin (Sr, green) or troponin I (green). (Middle panels) Cells were double-stained for GATA4 (red) together with sarcomeric actin or troponin
Figure Legend Snippet: Immunocytochemical analysis of cardiomyocyte-specific markers. (Left panels) Cells were stained for sarcomeric actin (Sr, green) or troponin I (green). (Middle panels) Cells were double-stained for GATA4 (red) together with sarcomeric actin or troponin

Techniques Used: Staining

31) Product Images from "Influence of Gestational Overfeeding on Cardiac Morphometry and Hypertrophic Protein Markers in Fetal Sheep"

Article Title: Influence of Gestational Overfeeding on Cardiac Morphometry and Hypertrophic Protein Markers in Fetal Sheep

Journal: The Journal of nutritional biochemistry

doi: 10.1016/j.jnutbio.2009.11.006

Protein expression of GATA4, ANF and calcineurin A in ventricles from fetus of control and overfed ewes. Panel A: Representative GATA4, pGATA4, ANF and calcineurin A gel bands using specific antibodies. GAPDH was used as the loading control (1:1,000); Panel B: GATA4; Panel C: p-GATA4; Panel D: p-GATA4-to-GATA4 ratio; Panel E: ANF; and Panel F: calcineurin A from both ventricles in control and overfed groups. Mean ± SEM, * p
Figure Legend Snippet: Protein expression of GATA4, ANF and calcineurin A in ventricles from fetus of control and overfed ewes. Panel A: Representative GATA4, pGATA4, ANF and calcineurin A gel bands using specific antibodies. GAPDH was used as the loading control (1:1,000); Panel B: GATA4; Panel C: p-GATA4; Panel D: p-GATA4-to-GATA4 ratio; Panel E: ANF; and Panel F: calcineurin A from both ventricles in control and overfed groups. Mean ± SEM, * p

Techniques Used: Expressing

32) Product Images from "GATA4 inhibits expression of the tryptophan oxygenase gene by binding to the TATA box in fetal hepatocytes"

Article Title: GATA4 inhibits expression of the tryptophan oxygenase gene by binding to the TATA box in fetal hepatocytes

Journal: Cytotechnology

doi: 10.1007/s10616-007-9120-1

The binding of GATA4 and TBP to the downstream TATA sequence. A gel shift assay was performed with purified GST-fused GATA4 (+: 1 μg, ++: 2 μg) and TBP (+: 1 μg, ++: 2 μg). Arrows indicate the specific shifted bands. As a negative control, purified GST protein was used. Non-labeled oligonucleotide was added at a 100-fold molar excess as a competitor
Figure Legend Snippet: The binding of GATA4 and TBP to the downstream TATA sequence. A gel shift assay was performed with purified GST-fused GATA4 (+: 1 μg, ++: 2 μg) and TBP (+: 1 μg, ++: 2 μg). Arrows indicate the specific shifted bands. As a negative control, purified GST protein was used. Non-labeled oligonucleotide was added at a 100-fold molar excess as a competitor

Techniques Used: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Purification, Negative Control, Labeling

GATA4 is required for repression of the TO gene in fetal hepatocytes. siRNAs for rat GATA4 were transfected to E17 fetal hepatocytes at 50 nM. RT-PCR assays were carried out with RNAs extracted from hepatocytes 48 h post-transfection. As a negative control, scrambled siRNA was used
Figure Legend Snippet: GATA4 is required for repression of the TO gene in fetal hepatocytes. siRNAs for rat GATA4 were transfected to E17 fetal hepatocytes at 50 nM. RT-PCR assays were carried out with RNAs extracted from hepatocytes 48 h post-transfection. As a negative control, scrambled siRNA was used

Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Negative Control

GATA4 exists in the downstream TATA region in fetal hepatocytes. A ChIP assay was performed with fetal and adult hepatocytes. As a negative control, rabbit normal IgG was used. The ratio of precipitated/input DNA is shown below the result of ChIP
Figure Legend Snippet: GATA4 exists in the downstream TATA region in fetal hepatocytes. A ChIP assay was performed with fetal and adult hepatocytes. As a negative control, rabbit normal IgG was used. The ratio of precipitated/input DNA is shown below the result of ChIP

Techniques Used: Chromatin Immunoprecipitation, Negative Control

GATA4 represses the TO promoter. C33A cells were transfected with the pGL3-TO promoter plasmid together with the pGL3-EF-renilla, GR, and GATA expression plasmids. Luciferase activity was measured 48 h post-transfection. Values represent the average of three independent experiments (±SE)
Figure Legend Snippet: GATA4 represses the TO promoter. C33A cells were transfected with the pGL3-TO promoter plasmid together with the pGL3-EF-renilla, GR, and GATA expression plasmids. Luciferase activity was measured 48 h post-transfection. Values represent the average of three independent experiments (±SE)

Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

33) Product Images from "Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes"

Article Title: Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

Journal: Stem Cells International

doi: 10.1155/2016/6029271

Maintenance of GATA4-positive feeder cells and germ cell-derived colonies (GDCs) in subculture at passages 1 and 10. GDC cells (labeled red) in passages 1 and 10 were maintained in the colonies in passages 2 and 11 ((A) and (C)). Panels (B) and (D) are bright-field images of panels (A) and (C), respectively. GATA4 and PGP 9.5 double staining in neonatal pig testis tissue (E). Panel (F) shows nuclear staining of panel (E). Total testicular cells were stained with antibodies against GATA4 (green) and PGP 9.5 (red) (G). Nuclear staining and bright-field images are shown in panels (H) and (I). Panel (J) shows negative controls on feeder cells attached to the wells. Panels (K) and (L) show GATA4 staining of old feeder cells (OFCs) and fresh feeder cells (FFCs), respectively, in passage 2. White and yellow arrows indicate PGP 9.5- and GATA4-positive cells, respectively; green arrows indicate PGP 9.5- and GATA4-negative cells. Scale bars indicate 50 μ m in panels (A)–(F) and 10 μ m in panels (G)–(L). Panel (M) shows PCR results for GATA4 in OFCs and FFCs in passage 2.
Figure Legend Snippet: Maintenance of GATA4-positive feeder cells and germ cell-derived colonies (GDCs) in subculture at passages 1 and 10. GDC cells (labeled red) in passages 1 and 10 were maintained in the colonies in passages 2 and 11 ((A) and (C)). Panels (B) and (D) are bright-field images of panels (A) and (C), respectively. GATA4 and PGP 9.5 double staining in neonatal pig testis tissue (E). Panel (F) shows nuclear staining of panel (E). Total testicular cells were stained with antibodies against GATA4 (green) and PGP 9.5 (red) (G). Nuclear staining and bright-field images are shown in panels (H) and (I). Panel (J) shows negative controls on feeder cells attached to the wells. Panels (K) and (L) show GATA4 staining of old feeder cells (OFCs) and fresh feeder cells (FFCs), respectively, in passage 2. White and yellow arrows indicate PGP 9.5- and GATA4-positive cells, respectively; green arrows indicate PGP 9.5- and GATA4-negative cells. Scale bars indicate 50 μ m in panels (A)–(F) and 10 μ m in panels (G)–(L). Panel (M) shows PCR results for GATA4 in OFCs and FFCs in passage 2.

Techniques Used: Derivative Assay, Labeling, Double Staining, Staining, Polymerase Chain Reaction

34) Product Images from "Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst"

Article Title: Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

Journal: Development (Cambridge, England)

doi: 10.1242/dev.096875

Provision of ESCs to IOD embryos prior to Oct4 deletion. (A) Maximum projection confocal images of embryos injected with eight H2B Tomato ESCs each at the 8-cell stage, followed by culture with 4-OHT for 24 hours, then without for a further 24 (top panel) or 48 (bottom panel) hours prior to immunohistochemistry for Oct4 (white), Sox17 (green) or Gata4 (blue). The red channel shows H2B Tomato from the injected ESCs. (B) Percentage of Sox17 + cells. Each dot represents an embryo. (C) Representative digital micrographs of a chimaeric E7 egg cylinder generated from an Oct4 -deleted IOD embryo injected with ESCs. Left, brightfield; right, fluorescence. Scale bar: 100 μm.
Figure Legend Snippet: Provision of ESCs to IOD embryos prior to Oct4 deletion. (A) Maximum projection confocal images of embryos injected with eight H2B Tomato ESCs each at the 8-cell stage, followed by culture with 4-OHT for 24 hours, then without for a further 24 (top panel) or 48 (bottom panel) hours prior to immunohistochemistry for Oct4 (white), Sox17 (green) or Gata4 (blue). The red channel shows H2B Tomato from the injected ESCs. (B) Percentage of Sox17 + cells. Each dot represents an embryo. (C) Representative digital micrographs of a chimaeric E7 egg cylinder generated from an Oct4 -deleted IOD embryo injected with ESCs. Left, brightfield; right, fluorescence. Scale bar: 100 μm.

Techniques Used: Injection, Immunohistochemistry, Generated, Fluorescence

35) Product Images from "Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice"

Article Title: Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0212367

Repression of initial follicular growth in ovarian grafts under androgen-excess host conditions. (A, B) Anti-GATA4 and AMH immunostaining of wild-type ovarian tissues grafted into intact male (XY-host), intact female (XX-host) and castrated male (XY-cast-host) hosts treated with or without testosterone (T), or dihydrotestosterone (DHT). The lower magnified images of GATA4-positive gonadal areas are shown in upper plates in A and B. AMH-positive healthy primary, secondary, and antral follicles were detected in ovaries grafted into female hosts (lower plate in B). (C) Bar graphs indicate the relative numbers of normal healthy follicles (left) and degenerating follicles (right). The data are expressed as means ± SEM (*p
Figure Legend Snippet: Repression of initial follicular growth in ovarian grafts under androgen-excess host conditions. (A, B) Anti-GATA4 and AMH immunostaining of wild-type ovarian tissues grafted into intact male (XY-host), intact female (XX-host) and castrated male (XY-cast-host) hosts treated with or without testosterone (T), or dihydrotestosterone (DHT). The lower magnified images of GATA4-positive gonadal areas are shown in upper plates in A and B. AMH-positive healthy primary, secondary, and antral follicles were detected in ovaries grafted into female hosts (lower plate in B). (C) Bar graphs indicate the relative numbers of normal healthy follicles (left) and degenerating follicles (right). The data are expressed as means ± SEM (*p

Techniques Used: Immunostaining

Ectopic appearance of SOX9-posititve Sertoli-like cells in ovarian grafts under androgen-excess host conditions. (A–D) Anti-GATA4, SOX9, FOXL2, DMRT1, or GDNF immunostaining of wild-type ovarian tissues grafted into XY-, XX-, and XY-cast-hosts treated with or without T or DHT on days 20 post-transplantation. The lower magnified images of GATA4-positive gonadal areas are shown in upper plates in a. Normal follicular structures were mostly absent, and tubular structures containing ectopic SOX9-positive cells were evident near the edge of ovaries grafted into XY-host and T/DHT-treated XX- and XY-cast-hosts on day 20 post-transplantation (arrows in A). The ectopic SOX9-positive cells are FOXL2-negative (B), and they are found in the DMRT1-positive/GDNF-positive tubular region (C, D). In c, the inset shows high-magnification images of tubular structures indicated by arrows. (E) Bar graphs indicate the SOX9-positive cells per gonadal area (mm 2 ). The data are expressed as means ± SEM (*p
Figure Legend Snippet: Ectopic appearance of SOX9-posititve Sertoli-like cells in ovarian grafts under androgen-excess host conditions. (A–D) Anti-GATA4, SOX9, FOXL2, DMRT1, or GDNF immunostaining of wild-type ovarian tissues grafted into XY-, XX-, and XY-cast-hosts treated with or without T or DHT on days 20 post-transplantation. The lower magnified images of GATA4-positive gonadal areas are shown in upper plates in a. Normal follicular structures were mostly absent, and tubular structures containing ectopic SOX9-positive cells were evident near the edge of ovaries grafted into XY-host and T/DHT-treated XX- and XY-cast-hosts on day 20 post-transplantation (arrows in A). The ectopic SOX9-positive cells are FOXL2-negative (B), and they are found in the DMRT1-positive/GDNF-positive tubular region (C, D). In c, the inset shows high-magnification images of tubular structures indicated by arrows. (E) Bar graphs indicate the SOX9-positive cells per gonadal area (mm 2 ). The data are expressed as means ± SEM (*p

Techniques Used: Immunostaining, Transplantation Assay

Sox8 expression in AMH-positive and -negative degenerating follicles and masculinized phenotypes of the Sox8 -null and Amh -null ovaries transplanted into male host mice. (A, B) In situ hybridization using a Sox8 antisense probe and anti-AMH immunostaining of two serial ovarian tissue sections grafted into XY-host on days 10 (A; The most left panels are lower and the others are higher magnified images) and 20 (B) post-transplantation. Sox8 -positive signals (yellow arrowheads) were detected in the granulosa cells of AMH-negative and -positive degenerating follicles on day 10 post-transplantation, as well as in tubular structures, including SOX9-positive Sertoli-like cells on day 20 post-transplantation. (C–F) Anti-GATA4, AMH, SOX9, or GDNF immunostaining of wild-type, Sox8 -null ( Sox8 -/- ), or Amh -null ( Amh -/- ) ovarian tissues grafted into XY-host on day 20 post-transplantation. The gonadal area is identified by GATA4 staining (C). Degenerating follicles were frequently seen in either Sox8 -null or Amh -null grafted ovaries even at this stage (C, D). In contrast, the tubular structure formation (arrows or broken outlines) and ectopic appearance of SOX9-positive Sertoli-like cells in GDNF-positive tubular structures are found in all three types of grafted ovaries (E, F). (G) Bar graphs indicate the relative numbers of normal healthy follicles (left), degenerating follicles (center), and SOX9-positive cells (right) per gonadal area (mm 2 ) in wild-type, Sox8 -null, or Amh -null grafts in male host mice. The Sox8 -null or Amh -null ovarian explants exhibited a large number of degenerating follicles even on day 20 post-transplantation, but no changes in the number of SOX9-positive cells were detected among the three genotypes. The data are expressed as means ± SEM (*p
Figure Legend Snippet: Sox8 expression in AMH-positive and -negative degenerating follicles and masculinized phenotypes of the Sox8 -null and Amh -null ovaries transplanted into male host mice. (A, B) In situ hybridization using a Sox8 antisense probe and anti-AMH immunostaining of two serial ovarian tissue sections grafted into XY-host on days 10 (A; The most left panels are lower and the others are higher magnified images) and 20 (B) post-transplantation. Sox8 -positive signals (yellow arrowheads) were detected in the granulosa cells of AMH-negative and -positive degenerating follicles on day 10 post-transplantation, as well as in tubular structures, including SOX9-positive Sertoli-like cells on day 20 post-transplantation. (C–F) Anti-GATA4, AMH, SOX9, or GDNF immunostaining of wild-type, Sox8 -null ( Sox8 -/- ), or Amh -null ( Amh -/- ) ovarian tissues grafted into XY-host on day 20 post-transplantation. The gonadal area is identified by GATA4 staining (C). Degenerating follicles were frequently seen in either Sox8 -null or Amh -null grafted ovaries even at this stage (C, D). In contrast, the tubular structure formation (arrows or broken outlines) and ectopic appearance of SOX9-positive Sertoli-like cells in GDNF-positive tubular structures are found in all three types of grafted ovaries (E, F). (G) Bar graphs indicate the relative numbers of normal healthy follicles (left), degenerating follicles (center), and SOX9-positive cells (right) per gonadal area (mm 2 ) in wild-type, Sox8 -null, or Amh -null grafts in male host mice. The Sox8 -null or Amh -null ovarian explants exhibited a large number of degenerating follicles even on day 20 post-transplantation, but no changes in the number of SOX9-positive cells were detected among the three genotypes. The data are expressed as means ± SEM (*p

Techniques Used: Expressing, Mouse Assay, In Situ Hybridization, Immunostaining, Transplantation Assay, Staining

36) Product Images from "Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency"

Article Title: Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency

Journal: Cell

doi: 10.1016/j.cell.2017.02.005

Interspecies Chimerism of hEPS Cells in E10.5 Mouse Conceptuses (A) Schematic diagram of approximate section planes in hEPS-injected embryos at E10.5. The green and red boxes indicate the sagittal section of brain and heart region respectively. (B) Representative images showing the integration of hEPS-derived cells into mouse E10.5 embryos. Anti-human nuclei (hN) antibody was co-stained with anti-SOX2 (upper panels, the green box in (A)) and anti-GATA4 (lower panels, the red box in (A)) antibodies. The insets are enlargements of the yellow boxes. The pseudo-colors were used. Scale bars, 100 μm. (C) Representative whole-placenta confocal images showing Tdtomato-labeled hEPS derivatives can integrate to the trophoblast layers of the E10.5 chimeric placenta by co-staining with anti-Tdtomato and anti-cytokeratin 8 (CK8) antibodies. Primed hPSCs were injected as controls. Scale bars, 200 μm. The right panels are enlargements of the yellow boxes (scale bars, 20 μm). dec, decidua layer; gc, giant cell layer; sp, spongiotrophoblast layer; laby, labyrinth layer. The pseudo-colors were used. .
Figure Legend Snippet: Interspecies Chimerism of hEPS Cells in E10.5 Mouse Conceptuses (A) Schematic diagram of approximate section planes in hEPS-injected embryos at E10.5. The green and red boxes indicate the sagittal section of brain and heart region respectively. (B) Representative images showing the integration of hEPS-derived cells into mouse E10.5 embryos. Anti-human nuclei (hN) antibody was co-stained with anti-SOX2 (upper panels, the green box in (A)) and anti-GATA4 (lower panels, the red box in (A)) antibodies. The insets are enlargements of the yellow boxes. The pseudo-colors were used. Scale bars, 100 μm. (C) Representative whole-placenta confocal images showing Tdtomato-labeled hEPS derivatives can integrate to the trophoblast layers of the E10.5 chimeric placenta by co-staining with anti-Tdtomato and anti-cytokeratin 8 (CK8) antibodies. Primed hPSCs were injected as controls. Scale bars, 200 μm. The right panels are enlargements of the yellow boxes (scale bars, 20 μm). dec, decidua layer; gc, giant cell layer; sp, spongiotrophoblast layer; laby, labyrinth layer. The pseudo-colors were used. .

Techniques Used: Injection, Derivative Assay, Staining, Labeling

37) Product Images from "Activation of GSK3? by Sirt2 Is Required for Early Lineage Commitment of Mouse Embryonic Stem Cell"

Article Title: Activation of GSK3? by Sirt2 Is Required for Early Lineage Commitment of Mouse Embryonic Stem Cell

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076699

Knockdown of Sirt2 results in the alteration of mouse ESC lineage commitment. ( A ) Phase-contrast images of EB at day 6. All Figures 100×. ( B ) Real-time PCR shows the relative mRNA expression levels of the ectoderm marker genes (Otx2, Sox1 and Pax6), the mesoderm marker genes (Gata6, Mesp2, Mixl1 and T) and the endoderm marker genes (Cxcr4, Gata4, LaminB and Sox17) in control (shpLKO.1) and two Sirt2 stable knockdown cell lines (shSirt2-1 and shSirt2-2) during EB differentiation at day 6. Data were normalized to Gapdh mRNA expression levels and are means ±SEM (n = 3). P
Figure Legend Snippet: Knockdown of Sirt2 results in the alteration of mouse ESC lineage commitment. ( A ) Phase-contrast images of EB at day 6. All Figures 100×. ( B ) Real-time PCR shows the relative mRNA expression levels of the ectoderm marker genes (Otx2, Sox1 and Pax6), the mesoderm marker genes (Gata6, Mesp2, Mixl1 and T) and the endoderm marker genes (Cxcr4, Gata4, LaminB and Sox17) in control (shpLKO.1) and two Sirt2 stable knockdown cell lines (shSirt2-1 and shSirt2-2) during EB differentiation at day 6. Data were normalized to Gapdh mRNA expression levels and are means ±SEM (n = 3). P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Marker

38) Product Images from "Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction"

Article Title: Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction

Journal: Scientific Reports

doi: 10.1038/s41598-018-22830-8

The effect of small molecules acting on GATA4–NKX2-5 transcriptional synergy on the hypertrophic process in vitro in neonatal cardiac myocytes. ( A,B ) The effects of 3i-1000 and 3i-0777 on stretch-induced increase in ANP ( A ) and ( B ) BNP mRNA levels. Cultured neonatal rat cardiomyocytes were stretched cyclically up to 24 h. The compounds were added 1 h prior stretching to the cells. ( C,D ) Effects of the compounds on phenylephrine (PE) induced increase in ANP ( C ) and ( D ) BNP gene expression. Cells were treated for 24 h with PE and the compounds were added 1 h prior to PE. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SD, n = 3. * p
Figure Legend Snippet: The effect of small molecules acting on GATA4–NKX2-5 transcriptional synergy on the hypertrophic process in vitro in neonatal cardiac myocytes. ( A,B ) The effects of 3i-1000 and 3i-0777 on stretch-induced increase in ANP ( A ) and ( B ) BNP mRNA levels. Cultured neonatal rat cardiomyocytes were stretched cyclically up to 24 h. The compounds were added 1 h prior stretching to the cells. ( C,D ) Effects of the compounds on phenylephrine (PE) induced increase in ANP ( C ) and ( D ) BNP gene expression. Cells were treated for 24 h with PE and the compounds were added 1 h prior to PE. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SD, n = 3. * p

Techniques Used: In Vitro, Aqueous Normal-phase Chromatography, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

The effect of small molecule 3i-1000 on GATA4 protein levels and phosphorylation in vitro in neonatal rat cardiomyocytes. ( A ) Compound 3i-1000 at concentration of 50 µM had no influence on baseline levels of nuclear GATA4 or Ser-105 phosphorylation of GATA4 (pGATA4) protein. ( B–E . * p
Figure Legend Snippet: The effect of small molecule 3i-1000 on GATA4 protein levels and phosphorylation in vitro in neonatal rat cardiomyocytes. ( A ) Compound 3i-1000 at concentration of 50 µM had no influence on baseline levels of nuclear GATA4 or Ser-105 phosphorylation of GATA4 (pGATA4) protein. ( B–E . * p

Techniques Used: In Vitro, Concentration Assay

The effect of small molecule 3i-1000 on GATA–NKX2-5 interaction in cell-based reporter gene assay. ( A ) COS-1 cells were transfected with a reporter construct containing three high-activation binding sites for NKX2-5 together with protein expression vectors for GATA4 and NKX2-5. The cells were lysed, and the reporter gene activity was measured by a luminometer. The small molecule 3i-1000 inhibited GATA4–NKX2-5 transcriptional synergy in luciferase reporter assay at the concentration of 5 µM. The results are an average of three parallel samples ± SD. ** p
Figure Legend Snippet: The effect of small molecule 3i-1000 on GATA–NKX2-5 interaction in cell-based reporter gene assay. ( A ) COS-1 cells were transfected with a reporter construct containing three high-activation binding sites for NKX2-5 together with protein expression vectors for GATA4 and NKX2-5. The cells were lysed, and the reporter gene activity was measured by a luminometer. The small molecule 3i-1000 inhibited GATA4–NKX2-5 transcriptional synergy in luciferase reporter assay at the concentration of 5 µM. The results are an average of three parallel samples ± SD. ** p

Techniques Used: Reporter Gene Assay, Transfection, Construct, Activation Assay, Binding Assay, Expressing, Activity Assay, Luciferase, Reporter Assay, Concentration Assay

39) Product Images from "Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction"

Article Title: Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction

Journal: Scientific Reports

doi: 10.1038/s41598-018-22830-8

The effect of small molecules acting on GATA4–NKX2-5 transcriptional synergy on the hypertrophic process in vitro in neonatal cardiac myocytes. ( A,B ) The effects of 3i-1000 and 3i-0777 on stretch-induced increase in ANP ( A ) and ( B ) BNP mRNA levels. Cultured neonatal rat cardiomyocytes were stretched cyclically up to 24 h. The compounds were added 1 h prior stretching to the cells. ( C,D ) Effects of the compounds on phenylephrine (PE) induced increase in ANP ( C ) and ( D ) BNP gene expression. Cells were treated for 24 h with PE and the compounds were added 1 h prior to PE. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SD, n = 3. * p
Figure Legend Snippet: The effect of small molecules acting on GATA4–NKX2-5 transcriptional synergy on the hypertrophic process in vitro in neonatal cardiac myocytes. ( A,B ) The effects of 3i-1000 and 3i-0777 on stretch-induced increase in ANP ( A ) and ( B ) BNP mRNA levels. Cultured neonatal rat cardiomyocytes were stretched cyclically up to 24 h. The compounds were added 1 h prior stretching to the cells. ( C,D ) Effects of the compounds on phenylephrine (PE) induced increase in ANP ( C ) and ( D ) BNP gene expression. Cells were treated for 24 h with PE and the compounds were added 1 h prior to PE. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SD, n = 3. * p

Techniques Used: In Vitro, Aqueous Normal-phase Chromatography, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

The effect of small molecule 3i-1000 on GATA4 protein levels and phosphorylation in vitro in neonatal rat cardiomyocytes. ( A ) Compound 3i-1000 at concentration of 50 µM had no influence on baseline levels of nuclear GATA4 or Ser-105 phosphorylation of GATA4 (pGATA4) protein. ( B–E ) Compound 3i-1000 (50 µM) inhibited the elevation of GATA4 and phospho-GATA4 protein levels produced with PE. The experiment was repeated three times, and the results presented here are an average of three parallel samples ± SD. The original whole blot images are presented in Supplementary Figure S1 . * p
Figure Legend Snippet: The effect of small molecule 3i-1000 on GATA4 protein levels and phosphorylation in vitro in neonatal rat cardiomyocytes. ( A ) Compound 3i-1000 at concentration of 50 µM had no influence on baseline levels of nuclear GATA4 or Ser-105 phosphorylation of GATA4 (pGATA4) protein. ( B–E ) Compound 3i-1000 (50 µM) inhibited the elevation of GATA4 and phospho-GATA4 protein levels produced with PE. The experiment was repeated three times, and the results presented here are an average of three parallel samples ± SD. The original whole blot images are presented in Supplementary Figure S1 . * p

Techniques Used: In Vitro, Concentration Assay, Produced

The effect of small molecule 3i-1000 on GATA–NKX2-5 interaction in cell-based reporter gene assay. ( A ) COS-1 cells were transfected with a reporter construct containing three high-activation binding sites for NKX2-5 together with protein expression vectors for GATA4 and NKX2-5. The cells were lysed, and the reporter gene activity was measured by a luminometer. The small molecule 3i-1000 inhibited GATA4–NKX2-5 transcriptional synergy in luciferase reporter assay at the concentration of 5 µM. The results are an average of three parallel samples ± SD. ** p
Figure Legend Snippet: The effect of small molecule 3i-1000 on GATA–NKX2-5 interaction in cell-based reporter gene assay. ( A ) COS-1 cells were transfected with a reporter construct containing three high-activation binding sites for NKX2-5 together with protein expression vectors for GATA4 and NKX2-5. The cells were lysed, and the reporter gene activity was measured by a luminometer. The small molecule 3i-1000 inhibited GATA4–NKX2-5 transcriptional synergy in luciferase reporter assay at the concentration of 5 µM. The results are an average of three parallel samples ± SD. ** p

Techniques Used: Reporter Gene Assay, Transfection, Construct, Activation Assay, Binding Assay, Expressing, Activity Assay, Luciferase, Reporter Assay, Concentration Assay

40) Product Images from "RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects"

Article Title: RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.031534

RERE regulates the expression of GATA4 in the AV canal. (A,B) GATA4 was visualized with anti-GATA4 antibodies on sections through the AV canals of wild-type embryos and Rere om/eyes3 embryos. The immunoreactivity of GATA4 in the wild-type AV endocardial cushions was stronger than that observed in Rere om/eyes3 AV endocardial cushions. (C,D) In contrast, the levels of GATA4 expression were comparable in the AV canals of Rere flox/flox and Rere flox/flox ;Tie2-Cre embryos. These results suggest that decreased expression of RERE in cells other than the endocardium play a role in regulating the expression of GATA4 in the AV canal. Arrows and dashed lines indicate the AV endocardial cushions. Representative images are shown from the analysis of sections from three embryos of each genotype. Scale bars: 100 µm.
Figure Legend Snippet: RERE regulates the expression of GATA4 in the AV canal. (A,B) GATA4 was visualized with anti-GATA4 antibodies on sections through the AV canals of wild-type embryos and Rere om/eyes3 embryos. The immunoreactivity of GATA4 in the wild-type AV endocardial cushions was stronger than that observed in Rere om/eyes3 AV endocardial cushions. (C,D) In contrast, the levels of GATA4 expression were comparable in the AV canals of Rere flox/flox and Rere flox/flox ;Tie2-Cre embryos. These results suggest that decreased expression of RERE in cells other than the endocardium play a role in regulating the expression of GATA4 in the AV canal. Arrows and dashed lines indicate the AV endocardial cushions. Representative images are shown from the analysis of sections from three embryos of each genotype. Scale bars: 100 µm.

Techniques Used: Expressing

RERE and GATA are co-expressed in the AV canal, and Rere and Gata4 interact genetically in the development of CHD. (A-C) Sections of wild-type embryos were prepared at E10.5 and stained with anti-RERE and anti-GATA4 antibodies. RERE (red) and GATA4 (green) were colocalized in the endocardium and mesenchymal cells of the AV endocardial cushions. Dashed lines indicate the boundary between the AV endocardial cushions and the myocardium; white arrows indicate the endocardium of the AV cushion. Representative images are presented from sections obtained from four wild-type embryos. Atr, atrium; Vt, ventricle. Scale bars: 100 µm. (D,E) Gata4 +/− and Rere −/eyes3 embryos on a mixed B6/129S6 background have normal ventricular septums at E15.5. (F) E15.5 Rere −/eyes3 ; Gata4 +/− embryos on a mixed B6/129S6 background have several types of CHD including perimembranous VSDs (arrow). Scale bars: 200 µm.
Figure Legend Snippet: RERE and GATA are co-expressed in the AV canal, and Rere and Gata4 interact genetically in the development of CHD. (A-C) Sections of wild-type embryos were prepared at E10.5 and stained with anti-RERE and anti-GATA4 antibodies. RERE (red) and GATA4 (green) were colocalized in the endocardium and mesenchymal cells of the AV endocardial cushions. Dashed lines indicate the boundary between the AV endocardial cushions and the myocardium; white arrows indicate the endocardium of the AV cushion. Representative images are presented from sections obtained from four wild-type embryos. Atr, atrium; Vt, ventricle. Scale bars: 100 µm. (D,E) Gata4 +/− and Rere −/eyes3 embryos on a mixed B6/129S6 background have normal ventricular septums at E15.5. (F) E15.5 Rere −/eyes3 ; Gata4 +/− embryos on a mixed B6/129S6 background have several types of CHD including perimembranous VSDs (arrow). Scale bars: 200 µm.

Techniques Used: Staining

RERE regulates the transcription of Gata4 . (A) RT-qPCR analyses were performed using mRNA extracted from the hearts of wild-type and Rere om/eyes3 embryos at E10.5. The relative amounts of Gata4 and Erbb3 transcripts were significantly reduced in the hearts of Rere om/eyes3 embryos compared with those of wild-type embryos. Three independent RT-qPCR analyses were performed using mRNA prepared from the hearts of three different littermates. (B) A firefly luciferase reporter gene fused with a previously described 5 kb promoter of Gata4 ) was transfected into HEK293 T cells with/without an RERE -expressing vector. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. Luciferase activity driven by the 5 kb Gata4 promoter was increased by overexpression of RERE. Experiments were performed in triplicate. (C) A 5kb-Gata4-Luc plasmid was transfected into NIH3T3 cells with a nontargeting siRNA pool or an Rere siRNA pool. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. NIH3T3 cells transfected with the Rere siRNA pool showed decreased luciferase activity when compared with those transfected with a nontargeting siRNA pool. Luciferase experiments were performed in triplicate. In all graphs, data are mean±s.d. Unpaired two-tailed Student's t -test was used to determine P -values (* P
Figure Legend Snippet: RERE regulates the transcription of Gata4 . (A) RT-qPCR analyses were performed using mRNA extracted from the hearts of wild-type and Rere om/eyes3 embryos at E10.5. The relative amounts of Gata4 and Erbb3 transcripts were significantly reduced in the hearts of Rere om/eyes3 embryos compared with those of wild-type embryos. Three independent RT-qPCR analyses were performed using mRNA prepared from the hearts of three different littermates. (B) A firefly luciferase reporter gene fused with a previously described 5 kb promoter of Gata4 ) was transfected into HEK293 T cells with/without an RERE -expressing vector. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. Luciferase activity driven by the 5 kb Gata4 promoter was increased by overexpression of RERE. Experiments were performed in triplicate. (C) A 5kb-Gata4-Luc plasmid was transfected into NIH3T3 cells with a nontargeting siRNA pool or an Rere siRNA pool. Luciferase activity was normalized by cotransfection with a Renillar luciferase plasmid. NIH3T3 cells transfected with the Rere siRNA pool showed decreased luciferase activity when compared with those transfected with a nontargeting siRNA pool. Luciferase experiments were performed in triplicate. In all graphs, data are mean±s.d. Unpaired two-tailed Student's t -test was used to determine P -values (* P

Techniques Used: Quantitative RT-PCR, Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Cotransfection, Over Expression, Two Tailed Test

Related Articles

Immunoprecipitation:

Article Title: A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart
Article Snippet: .. For immunoprecipitation, 2 μg of anti-TCF7L2, anti-IgG (17–10109, Millipore), anti-GATA4 (sc-25310 X, SantaCruz) or anti-H3K27ac (C15410196, Diagenode) was added to the nuclear extracts and incubated O/N at 4°C. .. Antibodies were pulled down using protein-A-sepharose beads followed by washing and DNA extraction.

Article Title: A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation
Article Snippet: .. After centrifugation for 10 min at 4°C, 500 μl of supernatant was subjected to overnight incubation at 4°C with 0.4 μg anti-GATA4 or control goat IgG (Santa Cruz) in immunoprecipitation buffer [20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100]. .. The immunocomplexes were recovered by a 2 h incubation with 20 μl protein G Sepharose beads.

Incubation:

Article Title: miR-202-3p Regulates Sertoli Cell Proliferation, Synthesis Function, and Apoptosis by Targeting LRP6 and Cyclin D1 of Wnt/β-Catenin Signaling
Article Snippet: .. The cells were then incubated with primary antibodies overnight at 4°C, including GATA4 (1:200; Santa Cruz, Dallas, TX, USA), WT1 (1:200; Santa Cruz, Dallas, TX, USA), SOX9 (1:500; Millipore, Bedford, MA, USA), GDNF (1:300; Santa Cruz, Dallas, TX, USA), SCF (1:300; Santa Cruz, Dallas, TX, USA), VIM (1:100; Cell Signaling Technology [CST], Danvers, MA, USA), OCLN (1:200; Abcam, Cambridge, UK), α-SMA (1:200; Abcam, Cambridge, UK), CYP11A1 (1:200; Abcam, Cambridge, UK), VASA (1:100; Santa Cruz, Dallas, TX, USA), and ki-67 (1:200; Santa Cruz, Dallas, TX, USA). .. After extensive washes with PBS, the cells were incubated with the secondary antibody, namely immunoglobulin G (IgG) conjugated with fluorescein isothiocyanate (FITC) (Sigma) or rhodamine-conjugated IgG (Sigma), at a 1:200 dilution for 1 hr at room temperature.

Article Title: A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart
Article Snippet: .. For immunoprecipitation, 2 μg of anti-TCF7L2, anti-IgG (17–10109, Millipore), anti-GATA4 (sc-25310 X, SantaCruz) or anti-H3K27ac (C15410196, Diagenode) was added to the nuclear extracts and incubated O/N at 4°C. .. Antibodies were pulled down using protein-A-sepharose beads followed by washing and DNA extraction.

Article Title: A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation
Article Snippet: .. After centrifugation for 10 min at 4°C, 500 μl of supernatant was subjected to overnight incubation at 4°C with 0.4 μg anti-GATA4 or control goat IgG (Santa Cruz) in immunoprecipitation buffer [20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100]. .. The immunocomplexes were recovered by a 2 h incubation with 20 μl protein G Sepharose beads.

Article Title: Modulation of the Pentose Phosphate Pathway Induces Endodermal Differentiation in Embryonic Stem Cells
Article Snippet: .. For antibody anti-Sox17 and anti-GATA4 after fixation the cells were permeabilized with 0.5% triton X-100 in 1× PBS for 5 min, blocked with 0.1% Triton, 10% BSA and 1× PBS for 1 hr and incubated with primary antibody (goat polyclonal anti-Sox17, 1∶20, R & D; goat polyclonal anti-GATA4, 1∶100, Santa Cruz Biotecnology Inc.) in 0.1% triton, 10% BSA in 1× PBS at 4°C overnight. .. Following primary antibody incubation, cells were rinsed three times in 1× PBS and further incubated with secondary antibodies: either anti-mouse IgG FITC-conjugated (1∶400; Molecular Probe) or anti-rabbit IgG FITC-conjugated (1∶200; Santa Cruz Biotechnology) in 10% normal goat serum and 1× PBS in for 30 min at room temperature; anti-goat Alexa Fluor 594 (1∶400; Invitrogen) in 0.1% triton, 0,1% BSA for 30 min at room temperature.

Centrifugation:

Article Title: A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation
Article Snippet: .. After centrifugation for 10 min at 4°C, 500 μl of supernatant was subjected to overnight incubation at 4°C with 0.4 μg anti-GATA4 or control goat IgG (Santa Cruz) in immunoprecipitation buffer [20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100]. .. The immunocomplexes were recovered by a 2 h incubation with 20 μl protein G Sepharose beads.

Immunostaining:

Article Title: Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells
Article Snippet: .. The immunostaining was performed using the following primary antibodies and reagents: anti-α-Actinin (A7811, Sigma-Aldrich), anti-ANP (sc-20158, Santa Cruz), anti- Troponin-I (ab52862, Abcam), anti-GATA4 (sc-1237, Santa Cruz), anti-Nkx2.5 (sc-8697, Santa Cruz), anti-Titin (HPA007042, Sigma-Aldrich), anti-SERCA2 (MAB2636, Millipore), anti-Connexin43(c6219, Sigma-Aldrich), anti-Na/Ca exchanger(MA3-926) and 4′,6-Diamidino-2-Phenylindole (DAPI, Molecular Probes). .. The secondary antibodies used were anti-rabbit IgG and anti-mouse IgG or IgM conjugated with Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes).

SDS Page:

Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
Article Snippet: .. Total protein (20 μ g) was separated by SDS-PAGE and transferred to PVDF membranes which were subsequently probed with anti-Nanog (Bethyl, Montgomery TX, USA), anti-Oct4 (BD Transduction, San José, CA, USA), anti-Pdx1 (Abcam, Cambridge, UK), anti-Sox17 (Millipore, Billerica, MA, USA), or anti-Gata4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β -actin (Sigma-Aldrich) as loading control. .. Glass coverslips 24-mm in diameter were washed in hydrochloric acid and sonicated in a water bath for 30 min. Coverslips were subsequently rinsed with 50% ethanol and sonicated for 30 min.

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    Santa Cruz Biotechnology goat anti gata4
    Abnormal Sertoli Cell Organization in Dmrt7 Mutant Testes (A) Testes from P14 wild-type and Dmrt7 mutant mice were sectioned and stained with antibody to <t>GATA4.</t> (B) P14 testis sections stained with antibody to GATA1. At P14, GATA4 and GATA1 levels are similar in wild-type and mutant Sertoli cell. (C) Wild-type and mutant testis sections double-stained with antibody to GATA1 (red) and DAPI (blue). Most Sertoli cell nuclei were adjacent to the basal membrane in wild-type, but mutant Sertoli cells were displaced in some tubules (arrowhead). White dotted line indicates position of basal membranes. (D) Testis sections from 10-wk-old wild-type and Sertoli cell-specific Dmrt7 mutant (SC-Dmrt7KO) mice stained with hematoxylin and eosin. Spermatogenesis and spermiogenesis are normal in SC-Dmrt7KO testis. (E) Testis sections from 10-wk-old wild-type and SC-Dmrt7KO mice stained with antibodies to GATA1 (red) and smooth muscle actin (to outline seminiferous tubules; green). Sertoli cell nuclei are positioned normally near the basal membrane in SC-Dmrt7KO mice.
    Goat Anti Gata4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abnormal Sertoli Cell Organization in Dmrt7 Mutant Testes (A) Testes from P14 wild-type and Dmrt7 mutant mice were sectioned and stained with antibody to GATA4. (B) P14 testis sections stained with antibody to GATA1. At P14, GATA4 and GATA1 levels are similar in wild-type and mutant Sertoli cell. (C) Wild-type and mutant testis sections double-stained with antibody to GATA1 (red) and DAPI (blue). Most Sertoli cell nuclei were adjacent to the basal membrane in wild-type, but mutant Sertoli cells were displaced in some tubules (arrowhead). White dotted line indicates position of basal membranes. (D) Testis sections from 10-wk-old wild-type and Sertoli cell-specific Dmrt7 mutant (SC-Dmrt7KO) mice stained with hematoxylin and eosin. Spermatogenesis and spermiogenesis are normal in SC-Dmrt7KO testis. (E) Testis sections from 10-wk-old wild-type and SC-Dmrt7KO mice stained with antibodies to GATA1 (red) and smooth muscle actin (to outline seminiferous tubules; green). Sertoli cell nuclei are positioned normally near the basal membrane in SC-Dmrt7KO mice.

    Journal: PLoS Genetics

    Article Title: A Mammal-Specific Doublesex Homolog Associates with Male Sex Chromatin and Is Required for Male Meiosis

    doi: 10.1371/journal.pgen.0030062

    Figure Lengend Snippet: Abnormal Sertoli Cell Organization in Dmrt7 Mutant Testes (A) Testes from P14 wild-type and Dmrt7 mutant mice were sectioned and stained with antibody to GATA4. (B) P14 testis sections stained with antibody to GATA1. At P14, GATA4 and GATA1 levels are similar in wild-type and mutant Sertoli cell. (C) Wild-type and mutant testis sections double-stained with antibody to GATA1 (red) and DAPI (blue). Most Sertoli cell nuclei were adjacent to the basal membrane in wild-type, but mutant Sertoli cells were displaced in some tubules (arrowhead). White dotted line indicates position of basal membranes. (D) Testis sections from 10-wk-old wild-type and Sertoli cell-specific Dmrt7 mutant (SC-Dmrt7KO) mice stained with hematoxylin and eosin. Spermatogenesis and spermiogenesis are normal in SC-Dmrt7KO testis. (E) Testis sections from 10-wk-old wild-type and SC-Dmrt7KO mice stained with antibodies to GATA1 (red) and smooth muscle actin (to outline seminiferous tubules; green). Sertoli cell nuclei are positioned normally near the basal membrane in SC-Dmrt7KO mice.

    Article Snippet: Other primary antibodies used for immunofluorescence were rat anti-GATA1 (1:200, Santa Cruz Biotechnology, http://www.scbt.com , sc-265), goat anti-GATA4 (1:200, Santa Cruz Biotechnology, sc-1237), rat anti-TRA98 (1:200, gift of H. Tanaka and Y. Nishimune), rat anti-BC7 (1:50, gift of H. Tanaka and Y. Nishimune), rat anti-TRA369 (1:200, gift of H. Tanaka and Y. Nishimune), rabbit anti-RAD51 (1:600 Calbiochem, http://www.calbiochem.com , PC130), mouse anti-GMP-1/SUMO-1 (1:200, Zymed, http://invitrogen.com , 33–2400), rabbit anti-phospho-H2AX (Ser139) (1:200, Upstate, http://www.millipore.com , 01–164), mouse anti-phospho-H2AX (1:200, Upstate, 05–636), mouse anti-SYCP3 (1:200, Abcam, http://www.abcam.com , ab12452), rabbit anti-HP1β (1:100, Abcam, ab10478), rabbit anti-H3-2meK9 (1:100, Upstate, 07–441), rabbit anti-H3-3meK9 (1:200, Upstate, 07–442), rabbit anti-AR (N-20) (1:200, Santa Cruz Biotechnology, sc-816), and mouse anti-αSMA clone 1A4 (1:800, Sigma, http://www.sigmaaldrich.com , A2547).

    Techniques: Mutagenesis, Mouse Assay, Staining

    Testicular cell types affected by lack of functional insulin. A : Testes sections from 9-week-old wild-type, Akita heterozygote, and homozygote males. GATA4 staining of Akita homozygous testes reveals a retained population of Sertoli cells ( n = 3). B : qRT-PCR analysis of cell types in control and Akita testes suggests increased Sertoli cell and spermatogonia cell populations in homozygous Akita testes, while both spermatocytes and spermatids are virtually absent compared with controls and heterozygotes. Insulin treatment reversed this trend and restored all cell populations in the testes to normal levels. n = 5 for wild-type (WT), Akita heterozygous (het), and Akita homozygous (hom) mice; n = 2 for Akita homozygote + insulin (hom + ins). * P

    Journal: Diabetes

    Article Title: Insulin Rescues Impaired Spermatogenesis via the Hypothalamic-Pituitary-Gonadal Axis in Akita Diabetic Mice and Restores Male Fertility

    doi: 10.2337/db11-1527

    Figure Lengend Snippet: Testicular cell types affected by lack of functional insulin. A : Testes sections from 9-week-old wild-type, Akita heterozygote, and homozygote males. GATA4 staining of Akita homozygous testes reveals a retained population of Sertoli cells ( n = 3). B : qRT-PCR analysis of cell types in control and Akita testes suggests increased Sertoli cell and spermatogonia cell populations in homozygous Akita testes, while both spermatocytes and spermatids are virtually absent compared with controls and heterozygotes. Insulin treatment reversed this trend and restored all cell populations in the testes to normal levels. n = 5 for wild-type (WT), Akita heterozygous (het), and Akita homozygous (hom) mice; n = 2 for Akita homozygote + insulin (hom + ins). * P

    Article Snippet: Paraffin sections were processed for immune-peroxidase staining as described , using goat anti-GATA4 (sc-1237; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:200 dilution and donkey anti-goat biotinylated IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:1,000 dilution.

    Techniques: Functional Assay, Staining, Quantitative RT-PCR, Mouse Assay

    Hypertrophy and GATA4 activation by IGF1 and PE. A and B , IGF1 (10 nmol/liter) and PE (20 μmol/liter) induced similar degrees of hypertrophy. A , Representative cultured cardiomyocytes stained for sarcomeres (desmin, green ) and nuclei (ToPro3,

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription Factor GATA4 Is Activated but Not Required for Insulin-like Growth Factor 1 (IGF1)-induced Cardiac Hypertrophy

    doi: 10.1074/jbc.M111.338749

    Figure Lengend Snippet: Hypertrophy and GATA4 activation by IGF1 and PE. A and B , IGF1 (10 nmol/liter) and PE (20 μmol/liter) induced similar degrees of hypertrophy. A , Representative cultured cardiomyocytes stained for sarcomeres (desmin, green ) and nuclei (ToPro3,

    Article Snippet: For immunoprecipitation, a goat primary antibody against GATA4 (Santa Cruz Biotechnology, sc-1237) was used.

    Techniques: Activation Assay, Cell Culture, Staining

    Role of GATA4 in IGF1- and PE-induced gene expression. A–D , relative mRNA expression of GATA4 downstream genes determined by qRT-PCR in cardiomyocytes infected with AdCon or AdG4i ( n = 7 per group). A , PE (20 μmol/liter) up-regulated expression

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription Factor GATA4 Is Activated but Not Required for Insulin-like Growth Factor 1 (IGF1)-induced Cardiac Hypertrophy

    doi: 10.1074/jbc.M111.338749

    Figure Lengend Snippet: Role of GATA4 in IGF1- and PE-induced gene expression. A–D , relative mRNA expression of GATA4 downstream genes determined by qRT-PCR in cardiomyocytes infected with AdCon or AdG4i ( n = 7 per group). A , PE (20 μmol/liter) up-regulated expression

    Article Snippet: For immunoprecipitation, a goat primary antibody against GATA4 (Santa Cruz Biotechnology, sc-1237) was used.

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Requirement of GATA4 in IGF1 and PE-induced hypertrophy. A–C , AdG4i blocked PE- but not IGF1-induced hypertrophy. A , cultured cardiomyocytes after infection with AdG4i (multiplicity of infection of 15) and stimulation with IGF1 (10 nmol/liter)

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription Factor GATA4 Is Activated but Not Required for Insulin-like Growth Factor 1 (IGF1)-induced Cardiac Hypertrophy

    doi: 10.1074/jbc.M111.338749

    Figure Lengend Snippet: Requirement of GATA4 in IGF1 and PE-induced hypertrophy. A–C , AdG4i blocked PE- but not IGF1-induced hypertrophy. A , cultured cardiomyocytes after infection with AdG4i (multiplicity of infection of 15) and stimulation with IGF1 (10 nmol/liter)

    Article Snippet: For immunoprecipitation, a goat primary antibody against GATA4 (Santa Cruz Biotechnology, sc-1237) was used.

    Techniques: Cell Culture, Infection

    IGF1- and PE-dependent signaling for GATA4 activation. A , Western blot analysis of cytoplasmic and nuclear protein extracts after 3 h of stimulation, the ratio of nuclear to cytoplasmic GATA4 levels increased after IGF1 (10 nmol/liter) and PE (20 μmol/liter)

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription Factor GATA4 Is Activated but Not Required for Insulin-like Growth Factor 1 (IGF1)-induced Cardiac Hypertrophy

    doi: 10.1074/jbc.M111.338749

    Figure Lengend Snippet: IGF1- and PE-dependent signaling for GATA4 activation. A , Western blot analysis of cytoplasmic and nuclear protein extracts after 3 h of stimulation, the ratio of nuclear to cytoplasmic GATA4 levels increased after IGF1 (10 nmol/liter) and PE (20 μmol/liter)

    Article Snippet: For immunoprecipitation, a goat primary antibody against GATA4 (Santa Cruz Biotechnology, sc-1237) was used.

    Techniques: Activation Assay, Western Blot

    Role of GATA4 in IGF1-induced hypertrophy in vivo . A , HW/BW ratio was similarly increased in IGFR and G4D-IGFR but decreased in G4D. B , dissociated cardiomyocytes showed hypertrophy in G4D and IGFR and an additive effect of both in G4D-IGFR. C , fractional

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription Factor GATA4 Is Activated but Not Required for Insulin-like Growth Factor 1 (IGF1)-induced Cardiac Hypertrophy

    doi: 10.1074/jbc.M111.338749

    Figure Lengend Snippet: Role of GATA4 in IGF1-induced hypertrophy in vivo . A , HW/BW ratio was similarly increased in IGFR and G4D-IGFR but decreased in G4D. B , dissociated cardiomyocytes showed hypertrophy in G4D and IGFR and an additive effect of both in G4D-IGFR. C , fractional

    Article Snippet: For immunoprecipitation, a goat primary antibody against GATA4 (Santa Cruz Biotechnology, sc-1237) was used.

    Techniques: In Vivo

    Loss-of-function model against Gata4 . A , Western blot for GATA4 protein levels after infection ( p.i. ) with AdG4i (multiplicity of infection of 15) or control virus ( AdCon ) for indicated number of days. AdG4i resulted in almost complete knockdown of GATA4.

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription Factor GATA4 Is Activated but Not Required for Insulin-like Growth Factor 1 (IGF1)-induced Cardiac Hypertrophy

    doi: 10.1074/jbc.M111.338749

    Figure Lengend Snippet: Loss-of-function model against Gata4 . A , Western blot for GATA4 protein levels after infection ( p.i. ) with AdG4i (multiplicity of infection of 15) or control virus ( AdCon ) for indicated number of days. AdG4i resulted in almost complete knockdown of GATA4.

    Article Snippet: For immunoprecipitation, a goat primary antibody against GATA4 (Santa Cruz Biotechnology, sc-1237) was used.

    Techniques: Western Blot, Infection

    Loss of Ptbp1 reduces proliferation of spermatogonia during neonatal period. (A) Immunohistochemical analysis of germ cell distribution in control (top) and a Ptbp1 conditional knockout (cKO) (bottom) mouse during the neonatal period. Sections were stained with anti-TRA98 (a marker of germ cells) and anti-GATA4 (a marker of the Sertoli cells and Leydig cells). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar denotes 100 µm. (B) Ratio of tubules without germ cells. The asterisk depicts a significant difference (** P

    Journal: The Journal of Reproduction and Development

    Article Title: PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia

    doi: 10.1262/jrd.2018-109

    Figure Lengend Snippet: Loss of Ptbp1 reduces proliferation of spermatogonia during neonatal period. (A) Immunohistochemical analysis of germ cell distribution in control (top) and a Ptbp1 conditional knockout (cKO) (bottom) mouse during the neonatal period. Sections were stained with anti-TRA98 (a marker of germ cells) and anti-GATA4 (a marker of the Sertoli cells and Leydig cells). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar denotes 100 µm. (B) Ratio of tubules without germ cells. The asterisk depicts a significant difference (** P

    Article Snippet: The primary antibodies used were as follows: rabbit anti-PLZF (sc-22839, Santa Cruz Biotechnology Inc., Dallas, TX, USA), goat anti-PTBP1 (sc-16547, Santa Cruz Biotechnology), rabbit anti-WT1 (sc-192, Santa Cruz Biotechnology), rabbit anti-MVH (ab13840, Abcam, Cambridge, MA, USA), rat anti-germ cell–specific nuclear antigen (clone TRA98, BioAcademia, Osaka, Japan), rabbit anti-cleaved caspase-3 (CC3) antibody (9664, Cell Signaling Technology), rat anti-haploid sperm cell–specific antigen (clone TRA54, ab92286, Abcam), goat anti-SCP3 (sc-20845, Santa Cruz Biotechnology), and goat anti-GATA4 (sc-1237, Santa Cruz Biotechnology).

    Techniques: Immunohistochemistry, Knock-Out, Staining, Marker

    Loss of Ptbp1 reduces proliferation of spermatogonia during adulthood. (A) Immunohistochemical analysis of testis from 2- and 6-month-old Ptbp1 cKO (bottom) mice and age-matched controls (top). Sections were stained with anti-PLZF (a marker of spermatogonia) and anti-GATA4 (a marker of the Sertoli cells and Leydig cells). Nuclei were stained with DAPI. Scale bar denotes 50 µm. (B) Ratio of tubules without PLZF-positive spermatogonia. The asterisk depicts a significant difference (** P

    Journal: The Journal of Reproduction and Development

    Article Title: PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia

    doi: 10.1262/jrd.2018-109

    Figure Lengend Snippet: Loss of Ptbp1 reduces proliferation of spermatogonia during adulthood. (A) Immunohistochemical analysis of testis from 2- and 6-month-old Ptbp1 cKO (bottom) mice and age-matched controls (top). Sections were stained with anti-PLZF (a marker of spermatogonia) and anti-GATA4 (a marker of the Sertoli cells and Leydig cells). Nuclei were stained with DAPI. Scale bar denotes 50 µm. (B) Ratio of tubules without PLZF-positive spermatogonia. The asterisk depicts a significant difference (** P

    Article Snippet: The primary antibodies used were as follows: rabbit anti-PLZF (sc-22839, Santa Cruz Biotechnology Inc., Dallas, TX, USA), goat anti-PTBP1 (sc-16547, Santa Cruz Biotechnology), rabbit anti-WT1 (sc-192, Santa Cruz Biotechnology), rabbit anti-MVH (ab13840, Abcam, Cambridge, MA, USA), rat anti-germ cell–specific nuclear antigen (clone TRA98, BioAcademia, Osaka, Japan), rabbit anti-cleaved caspase-3 (CC3) antibody (9664, Cell Signaling Technology), rat anti-haploid sperm cell–specific antigen (clone TRA54, ab92286, Abcam), goat anti-SCP3 (sc-20845, Santa Cruz Biotechnology), and goat anti-GATA4 (sc-1237, Santa Cruz Biotechnology).

    Techniques: Immunohistochemistry, Mouse Assay, Staining, Marker