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Transcription coactivator Cited1 acts as an inducer of trophoblast-like state from mouse embryonic stem cells through the activation of BMP signaling 
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a Typical morphological changes and AKP staining in E14T ESCs after overexpressing Cited1 for 2 days. Scale bar: 100 μm. b Results of qRT-PCR analysis of expression levels of trophoblast markers in ESCs overexpressing Cited1 , <t>Cdx2</t> , or Gata3 for 3 days. The average mRNA level in cells transfected with the control vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. c , d Results of qRT-PCR analysis of expression levels of three germ layer markers ( c ) and pluripotency-associated markers ( d ) in ESCs overexpressing Cited1 for 3 days. The average mRNA level in cells transfected with the control vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. e qRT-PCR analysis of expression levels of trophoblast markers after transfection of plasmids as indicated in ESCs over a time course. The average mRNA level in cells transfected with the control vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). ** p < 0.01, *** p < 0.001. f , g Immunofluorescence staining of ESCs after transfection of Cited1 for 6 days. Samples were stained with anti-Oct4 antibody (red) ( e ) and anti-Krt7 antibody (red) ( f ), respectively. DAPI staining highlights the nuclei (blue). Scale bar: 50 μm. h Flow cytometry density plots for Krt7 expression in ESCs after transfection of Cited1 for 6 days. Cells were fully dispersed, fixed, and immunostained for Krt7. For the negative control (NC), cells were exposed only to secondary antibody without prior exposure to the primary Krt7 antibody. i The statistical analysis of flow cytometry data for percentages of cells positive for Krt7 expression in ESCs overexpressing Cited1 or an empty vector for 6 days. Data are shown as mean ± SD ( n = 3). *** p < 0.001
Transcription coactivator Cited1 acts as an inducer of trophoblast-like state from mouse embryonic stem cells through the activation of BMP signaling 
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a A heatmap of differentially expressed genes (DEGs) induced by Cited1 overexpression in ESCs (fold change > 2 and p < 0.05). Green and red values represent fold changes for down- and upregulation, respectively. b The numbers of up- and downregulated genes induced by Cited1 overexpression in ESCs (fold change > 2 and p < 0.05). c qRT-PCR analysis to validate the expression changes of DEGs identified by the microarray assay in ESCs 48 h after Cited1 overexpression. The average mRNA level in control cells transfected with an empty vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. d Significantly enriched GO terms of the top 60 DEGs induced by Cited1 overexpression at day 2 compared with those from empty vector pPy overexpression. e A venn diagram showing intersections of 3 sets of DEGs induced by Cited1 overexpression (pink), Gata3 overexpression (blue), or <t>Cdx2</t> overexpression (green), with gene numbers indicated. Out of 696 DEGs induced by Cited1 , 462 DEGs were shared with those induced by Gata3 or Cdx2 . f The GSEA using ordered gene expression levels from Cited1 -overexpressing cells over control ESCs ( X -axis) with gene sets indicated. Cdx2 OE (368 genes), and Gata3 OE (384 genes) in ESCs, top 1% of upregulated genes upon Oct4 KD (204 genes), TSC-specific (313 genes) and ESC-specific (218 genes) gene sets
Transcription coactivator Cited1 acts as an inducer of trophoblast-like state from mouse embryonic stem cells through the activation of BMP signaling Cell Death & Disease, 2018 Sep 11
"a Typical morphological changes and AKP staining in E14T ESCs after overexpressing Cited1 for 2 days. Scale bar: 100 μm. b Results of qRT-PCR analysis of expression levels of trophoblast markers in ESCs overexpressing Cited1 , <t>Cdx2</t> , or Gata3 for 3 days. The average mRNA level in cells transfected with the control vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. c , d Results of qRT-PCR analysis of expression levels of three germ layer markers ( c ) and pluripotency-associated markers ( d ) in ESCs overexpressing Cited1 for 3 days. The average mRNA level in cells transfected with the control vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. e qRT-PCR analysis of expression levels of trophoblast markers after transfection of plasmids as indicated in ESCs over a time course. The average mRNA level in cells transfected with the control vector pPy was set at 1.0. Data are shown as mean ± SD ( n = 3). ** p < 0.01, *** p < 0.001. f , g Immunofluorescence staining of ESCs after transfection of Cited1 for 6 days. Samples were stained with anti-Oct4 antibody (red) ( e ) and anti-Krt7 antibody (red) ( f ), respectively. DAPI staining highlights the nuclei (blue). Scale bar: 50 μm. h Flow cytometry density plots for Krt7 expression in ESCs after transfection of Cited1 for 6 days. Cells were fully dispersed, fixed, and immunostained for Krt7. For the negative control (NC), cells were exposed only to secondary antibody without prior exposure to the primary Krt7 antibody. i The statistical analysis of flow cytometry data for percentages of cells positive for Krt7 expression in ESCs overexpressing Cited1 or an empty vector for 6 days. Data are shown as mean ± SD ( n = 3). *** p < 0.001 "
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