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Thermo Fisher nanog
Modulating <t>NANOG</t> expression did not affect in vitro cell proliferation and colony formation ( A ) Cell viability was measured over 3 days in Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either <t>Renilla</t> luciferase or NANOG . ( B ) Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either Renilla luciferase or NANOG were grown for 2 weeks before being counted by a colony counter. Only colonies greater than 50 μm in diameter were counted as positive. Error bars, S.D.
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1) Product Images from "NANOG regulates epithelial–mesenchymal transition and chemoresistance in ovarian cancer"

Article Title: NANOG regulates epithelial–mesenchymal transition and chemoresistance in ovarian cancer

Journal: Bioscience Reports

doi: 10.1042/BSR20160247

Modulating NANOG expression did not affect in vitro cell proliferation and colony formation ( A ) Cell viability was measured over 3 days in Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either Renilla luciferase or NANOG . ( B ) Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either Renilla luciferase or NANOG were grown for 2 weeks before being counted by a colony counter. Only colonies greater than 50 μm in diameter were counted as positive. Error bars, S.D.
Figure Legend Snippet: Modulating NANOG expression did not affect in vitro cell proliferation and colony formation ( A ) Cell viability was measured over 3 days in Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either Renilla luciferase or NANOG . ( B ) Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either Renilla luciferase or NANOG were grown for 2 weeks before being counted by a colony counter. Only colonies greater than 50 μm in diameter were counted as positive. Error bars, S.D.

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

NANOG modulation changes susceptibility to cisplatin treatment ( A ) Moody cells were either untransfected or transiently transfected with firefly luciferase or NANOG for 12 h. ( B ) SKOV-3 cells were either untransfected or transiently transfected with shRNA targeting Renilla luciferase or NANOG construct for 12 h. The cells were then treated with indicated doses of cisplatin for 72 h. Cell viability was assessed by the MTT assay.
Figure Legend Snippet: NANOG modulation changes susceptibility to cisplatin treatment ( A ) Moody cells were either untransfected or transiently transfected with firefly luciferase or NANOG for 12 h. ( B ) SKOV-3 cells were either untransfected or transiently transfected with shRNA targeting Renilla luciferase or NANOG construct for 12 h. The cells were then treated with indicated doses of cisplatin for 72 h. Cell viability was assessed by the MTT assay.

Techniques Used: Transfection, Luciferase, shRNA, Construct, MTT Assay

2) Product Images from "Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells"

Article Title: Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells

Journal:

doi: 10.1089/scd.2008.0335

Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were
Figure Legend Snippet: Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were

Techniques Used: Over Expression

Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (
Figure Legend Snippet: Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (

Techniques Used: Over Expression

Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Immunostaining, Staining

Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Staining

Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,
Figure Legend Snippet: Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,

Techniques Used: Expressing

3) Product Images from "Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells"

Article Title: Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells

Journal:

doi: 10.1089/scd.2008.0335

Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were
Figure Legend Snippet: Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were

Techniques Used: Over Expression

Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (
Figure Legend Snippet: Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (

Techniques Used: Over Expression

Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Immunostaining, Staining

Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Staining

Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,
Figure Legend Snippet: Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,

Techniques Used: Expressing

4) Product Images from "First steps to define murine amniotic fluid stem cell microenvironment"

Article Title: First steps to define murine amniotic fluid stem cell microenvironment

Journal: Scientific Reports

doi: 10.1038/srep37080

Characterization of cKit + cells isolated from the murine AF and AM. ( A ) Amniocentesis procedure, from the removal of the fetal membrane to the collection of the AF. ( B ) The percentage of AFS (cKit + ) cells at different embryonic stages. Mean is represented by bar. ( C ) Total number of AFS cells per embryo equivalent (EE). Mean is represented by bar. ( D ) Frequency of AFS cells positive for the expression of each gene ( cKit , cMyc , Klf4 , Nanog , Oct4 , Sca1 , Sox2 ) at different embryonic stages evaluated by single cell PCR (E11.5: 49 cells (n = 7 embryos); E12.5: 38 cells (n = 9 embryos); E13.5: 42 cells (n = 31 embryos); E14.5: 52 cells (n = 10 embryos)). ( E ) Representative immunostaining showing some EdU + cells among the cKit + cells isolated from AF (n = 7 embryos). ( F ) Flow cytometry analysis highlighted the presence of AFS cells positive for the markers CD44, CD90, CD105 and Sca1 (n = 31 embryos). ( G ) Representative immunostaining showing cKit + cells in the AM (n = 3 embryos). ( H ) Percentage of cKit + cells isolated from AM. ( I ) EdU + cells among the cKit + cells isolated from AM (n = 7 embryos). Scale bars = 100 μm. ( L ) Frequency of E13.5 ckit + AM cells positive for the expression of each gene ( cKit , cMyc , Klf4 , Nanog , Oct4 , Sca1 , Sox2 ) studied by single cell PCR (50 cells (n = 18 embryos)).
Figure Legend Snippet: Characterization of cKit + cells isolated from the murine AF and AM. ( A ) Amniocentesis procedure, from the removal of the fetal membrane to the collection of the AF. ( B ) The percentage of AFS (cKit + ) cells at different embryonic stages. Mean is represented by bar. ( C ) Total number of AFS cells per embryo equivalent (EE). Mean is represented by bar. ( D ) Frequency of AFS cells positive for the expression of each gene ( cKit , cMyc , Klf4 , Nanog , Oct4 , Sca1 , Sox2 ) at different embryonic stages evaluated by single cell PCR (E11.5: 49 cells (n = 7 embryos); E12.5: 38 cells (n = 9 embryos); E13.5: 42 cells (n = 31 embryos); E14.5: 52 cells (n = 10 embryos)). ( E ) Representative immunostaining showing some EdU + cells among the cKit + cells isolated from AF (n = 7 embryos). ( F ) Flow cytometry analysis highlighted the presence of AFS cells positive for the markers CD44, CD90, CD105 and Sca1 (n = 31 embryos). ( G ) Representative immunostaining showing cKit + cells in the AM (n = 3 embryos). ( H ) Percentage of cKit + cells isolated from AM. ( I ) EdU + cells among the cKit + cells isolated from AM (n = 7 embryos). Scale bars = 100 μm. ( L ) Frequency of E13.5 ckit + AM cells positive for the expression of each gene ( cKit , cMyc , Klf4 , Nanog , Oct4 , Sca1 , Sox2 ) studied by single cell PCR (50 cells (n = 18 embryos)).

Techniques Used: Isolation, Expressing, Polymerase Chain Reaction, Immunostaining, Flow Cytometry, Cytometry

Characterization of YFP + ESC after IUT and in vivo teratoma formation. ( A ) PCR analysis of pluripotency markers ( cMyc , Klf4 , Nanog , Oct4 , Sox2 ) on YFP + ESC populations before IUT (black bars) and after IUT obtained from AF (dark grey) and AM (light grey) (n = 34 embryos). (*p
Figure Legend Snippet: Characterization of YFP + ESC after IUT and in vivo teratoma formation. ( A ) PCR analysis of pluripotency markers ( cMyc , Klf4 , Nanog , Oct4 , Sox2 ) on YFP + ESC populations before IUT (black bars) and after IUT obtained from AF (dark grey) and AM (light grey) (n = 34 embryos). (*p

Techniques Used: In Vivo, Polymerase Chain Reaction

5) Product Images from "Excluding Oct4 from Yamanaka Cocktail Unleashes the Developmental Potential of iPSCs"

Article Title: Excluding Oct4 from Yamanaka Cocktail Unleashes the Developmental Potential of iPSCs

Journal: Cell Stem Cell

doi: 10.1016/j.stem.2019.10.002

SKM Can Activate Pluripotency Program in Oct4 -KO MEFs (A and B) Time course expression plots of indicated fibroblast (A) and MET (B) genes during OSKM and SKM reprogramming. Only Epcam + and GFP + sorted samples are shown for d6 and d9, respectively. (C) Time course expression of MEF- and ESC-specific genes ( Chronis et al., 2017 ). Only differentially expressed genes (DEGs) with FC ≥ 4 were plotted. (D) Time course expression plots of indicated genes during OSKM and SKM reprogramming. (E) Time course expression of Oct4, Nanog (ChIP-Atlas), and Sall4 ( Lim et al., 2008 ) targets in ESCs. Only top peaks (macs score ≥ 200, on Chip-Atlas) within 5 kb of DEGs (FC ≥ 4 in iPSCs versus MEFs) were plotted. (F) qPCR gene expression analysis of Nr5a2 , Nanog , and Sall4 after short hairpin RNA (shRNA)-driven KD during reprogramming after 2 dpi with OSKM. ActB was used as a reference gene. Error bars represent SD; n = 3. (G) Reprogramming of Oct4-GFP MEFs, expressing Nr5a2 , Nanog , Sall4 , or control shRNAs with OSKM or SKM. GFP + colonies were counted after 7 and 14 dpi. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t test. (H) Strategy for generating Pou5f1 (Oct4)-KO MEFs. (I) PCR genotyping confirming homozygous deletion of Oct4 in 3 MEF lines. (J) Immunofluorescence imaging of Oct4 F/F and Oct4 Δ/Δ MEFs reprogrammed with SKM, on 7 dpi. (K) qPCR gene expression analysis of Epcam + Oct4 F/F and Oct4 Δ/Δ MEFs reprogrammed with OSKM or SKM on 7 dpi. Error bars represent SD; n = 3.
Figure Legend Snippet: SKM Can Activate Pluripotency Program in Oct4 -KO MEFs (A and B) Time course expression plots of indicated fibroblast (A) and MET (B) genes during OSKM and SKM reprogramming. Only Epcam + and GFP + sorted samples are shown for d6 and d9, respectively. (C) Time course expression of MEF- and ESC-specific genes ( Chronis et al., 2017 ). Only differentially expressed genes (DEGs) with FC ≥ 4 were plotted. (D) Time course expression plots of indicated genes during OSKM and SKM reprogramming. (E) Time course expression of Oct4, Nanog (ChIP-Atlas), and Sall4 ( Lim et al., 2008 ) targets in ESCs. Only top peaks (macs score ≥ 200, on Chip-Atlas) within 5 kb of DEGs (FC ≥ 4 in iPSCs versus MEFs) were plotted. (F) qPCR gene expression analysis of Nr5a2 , Nanog , and Sall4 after short hairpin RNA (shRNA)-driven KD during reprogramming after 2 dpi with OSKM. ActB was used as a reference gene. Error bars represent SD; n = 3. (G) Reprogramming of Oct4-GFP MEFs, expressing Nr5a2 , Nanog , Sall4 , or control shRNAs with OSKM or SKM. GFP + colonies were counted after 7 and 14 dpi. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t test. (H) Strategy for generating Pou5f1 (Oct4)-KO MEFs. (I) PCR genotyping confirming homozygous deletion of Oct4 in 3 MEF lines. (J) Immunofluorescence imaging of Oct4 F/F and Oct4 Δ/Δ MEFs reprogrammed with SKM, on 7 dpi. (K) qPCR gene expression analysis of Epcam + Oct4 F/F and Oct4 Δ/Δ MEFs reprogrammed with OSKM or SKM on 7 dpi. Error bars represent SD; n = 3.

Techniques Used: Expressing, Chromatin Immunoprecipitation, Magnetic Cell Separation, Real-time Polymerase Chain Reaction, shRNA, Polymerase Chain Reaction, Immunofluorescence, Imaging

Sox2, Klf4, and cMyc Can Reprogram to Pluripotency in the Absence of Exogenous POU Factor (A and C) Scheme of KSM and SKM polycistronic vectors derived from OKSM (A) and OSKM (C) reprogramming cassettes. (B and D) Generation of iPSCs with tetO-KSM (B) and SKM (D) vectors, respectively. Phase-contrast and fluorescence microscopy images of primary colonies and passaged clonal lines of KSM and SKM iPSCs (scale bars represent 250 μm). (E) PCR genotyping verifying tetO-KSM and tetO-SKM transgenes in KSM and SKM iPSC lines, respectively, while confirming the absence of Oct4 or Brn4 integration. (F) Bisulfite sequencing analysis of DNA methylation in Oct4 , Nanog , and Col1a1 promoters in MEFs and KSM and SKM iPSC lines. (G) H E staining of teratoma sections with representation of three germ layers (ectoderm: keratinizing epithelium; mesoderm: smooth muscles; endoderm: cuboidal and respiratory epithelium). (H) An adult chimeric mouse generated from SKM iPSC line. (I) Bright-field and GFP merged images of the gonads from E13.5 KSM and SKM iPSC chimeric embryos. (J) Schematic representation of the time course reprogramming experiment. (K) Time course reprogramming experiment of Oct4-GFP MEFs using polycistronic vectors. 10 3 transduced MEFs were plated on feeders and induced with dox for the indicated number of days. GFP + colonies were counted on 10 dpi. Error bars represent SD; n = 3. (L) Western blot analysis of MEFs after transduction of polycistronic vectors, 1 dpi.
Figure Legend Snippet: Sox2, Klf4, and cMyc Can Reprogram to Pluripotency in the Absence of Exogenous POU Factor (A and C) Scheme of KSM and SKM polycistronic vectors derived from OKSM (A) and OSKM (C) reprogramming cassettes. (B and D) Generation of iPSCs with tetO-KSM (B) and SKM (D) vectors, respectively. Phase-contrast and fluorescence microscopy images of primary colonies and passaged clonal lines of KSM and SKM iPSCs (scale bars represent 250 μm). (E) PCR genotyping verifying tetO-KSM and tetO-SKM transgenes in KSM and SKM iPSC lines, respectively, while confirming the absence of Oct4 or Brn4 integration. (F) Bisulfite sequencing analysis of DNA methylation in Oct4 , Nanog , and Col1a1 promoters in MEFs and KSM and SKM iPSC lines. (G) H E staining of teratoma sections with representation of three germ layers (ectoderm: keratinizing epithelium; mesoderm: smooth muscles; endoderm: cuboidal and respiratory epithelium). (H) An adult chimeric mouse generated from SKM iPSC line. (I) Bright-field and GFP merged images of the gonads from E13.5 KSM and SKM iPSC chimeric embryos. (J) Schematic representation of the time course reprogramming experiment. (K) Time course reprogramming experiment of Oct4-GFP MEFs using polycistronic vectors. 10 3 transduced MEFs were plated on feeders and induced with dox for the indicated number of days. GFP + colonies were counted on 10 dpi. Error bars represent SD; n = 3. (L) Western blot analysis of MEFs after transduction of polycistronic vectors, 1 dpi.

Techniques Used: Derivative Assay, Fluorescence, Microscopy, Polymerase Chain Reaction, Methylation Sequencing, DNA Methylation Assay, Staining, Generated, Western Blot, Transduction

6) Product Images from "Functional Evidence that the Self-Renewal Gene NANOG Regulates Human Tumor Development"

Article Title: Functional Evidence that the Self-Renewal Gene NANOG Regulates Human Tumor Development

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.29

Nanog knockdown inhibits MCF7 cell clonogenic growth, reduces proliferation and alters differentiation (A) MCF7 clonal growth. 100 MCF7 cells infected with the indicated vectors were plated (n = 3) in 6-well plates. 21 d post-plating, cells were trypsinized, pooled and counted. (B) BrdU incorporation assays. MCF7 cells infected with the indicated lentiviruses were cultured overnight on glass coverslips and pulsed with 5 µM BrdU for 4 h. Fixed cells were processed for BrdU immunostaining (red), markedly reduced in Nanog-shRNA transduced cells. (C) Functional ‘rescue’ experiments. MCF7 cells were infected with the indicated vectors (LL, LL3.7; N, Nanog-shRNA; TRC, TRC-shRNA; MOI 20) and 24 h later, transfected with either pPyCAG or pPyCAG-PN8 (i.e., NANOGP8 ). 48-h later cells were pulsed with BrdU (4 h) and processed for BrdU staining. A total of 500–1,000 cells were counted by three individuals and the bars represent the mean ± S.D. *P
Figure Legend Snippet: Nanog knockdown inhibits MCF7 cell clonogenic growth, reduces proliferation and alters differentiation (A) MCF7 clonal growth. 100 MCF7 cells infected with the indicated vectors were plated (n = 3) in 6-well plates. 21 d post-plating, cells were trypsinized, pooled and counted. (B) BrdU incorporation assays. MCF7 cells infected with the indicated lentiviruses were cultured overnight on glass coverslips and pulsed with 5 µM BrdU for 4 h. Fixed cells were processed for BrdU immunostaining (red), markedly reduced in Nanog-shRNA transduced cells. (C) Functional ‘rescue’ experiments. MCF7 cells were infected with the indicated vectors (LL, LL3.7; N, Nanog-shRNA; TRC, TRC-shRNA; MOI 20) and 24 h later, transfected with either pPyCAG or pPyCAG-PN8 (i.e., NANOGP8 ). 48-h later cells were pulsed with BrdU (4 h) and processed for BrdU staining. A total of 500–1,000 cells were counted by three individuals and the bars represent the mean ± S.D. *P

Techniques Used: Infection, BrdU Incorporation Assay, Cell Culture, Immunostaining, shRNA, Functional Assay, Transfection, BrdU Staining

NANOG mRNA in cancer cells results from NANOGP8 (A) Schematic of human NANOG1 gene structure. PCR primers and shRNA vector target locations are indicated in blue and red, respectively. Translational start ATG and stop TGA codons are indicated. E, exon; TSS, transcriptional start site; UTR, untranslated region. (B-C) RT-PCR using LDF1-LDR1. ‘No RT’, no reverse transcription control. RNA/cDNA template was derived from cultured cancer cells (B) or primary HPCa samples (C). (D) Sequencing analysis. Shown are the nucleotide (nt; and predicted aa) differences between cancer cell-derived NANOG and published NANOG1 and NANOGP8 sequences. The five conserved nts consistent with expression from the NANOGP8 locus are indicated in blue. The single conserved predicted aa change is indicated in red. (E) Differential RT-PCR. F2/R2 primers are specific for NANOG1 whereas F2/R3 primers generate a 467-bp NANOG1 product and a 446-bp NANOGP8 product (arrow). The asterisks indicate two smaller amplicons. (F) NANOG1 in cancer cells is silenced. PC3 cells were treated (72 h) with AzaC and/or TSA (500 nM each), or vehicle. RT-PCR analysis of extracted RNA (non-treated hESC RNA for comparison) using the F2/R2 primers ( NANOG1 specific) vs. F2/R3 ‘universal’ NANOG primers. p16 INK4a (hypermethylated in PCa) amplification is used as AzaC control. (G) Cancer cell NANOGP8 cDNAs encode NANOG proteins that can be detected by an anti-NANOG1 antibody. Cancer cell NANOGP8 cDNAs cloned in pET28b(−) were either uninduced (U) or induced (I) with IPTG and supernatant (S) or insoluble pellet (P) was used in Western blotting using the mAb. The arrow points to the ~42 kD NANOG band. (H) Peptide sequences obtained by MALDI-TOF from MCF7 NANOGP8 cDNA. (I-J) Western blotting using whole cell lysate (WCL; I) or NE (J) and eBio mAb.
Figure Legend Snippet: NANOG mRNA in cancer cells results from NANOGP8 (A) Schematic of human NANOG1 gene structure. PCR primers and shRNA vector target locations are indicated in blue and red, respectively. Translational start ATG and stop TGA codons are indicated. E, exon; TSS, transcriptional start site; UTR, untranslated region. (B-C) RT-PCR using LDF1-LDR1. ‘No RT’, no reverse transcription control. RNA/cDNA template was derived from cultured cancer cells (B) or primary HPCa samples (C). (D) Sequencing analysis. Shown are the nucleotide (nt; and predicted aa) differences between cancer cell-derived NANOG and published NANOG1 and NANOGP8 sequences. The five conserved nts consistent with expression from the NANOGP8 locus are indicated in blue. The single conserved predicted aa change is indicated in red. (E) Differential RT-PCR. F2/R2 primers are specific for NANOG1 whereas F2/R3 primers generate a 467-bp NANOG1 product and a 446-bp NANOGP8 product (arrow). The asterisks indicate two smaller amplicons. (F) NANOG1 in cancer cells is silenced. PC3 cells were treated (72 h) with AzaC and/or TSA (500 nM each), or vehicle. RT-PCR analysis of extracted RNA (non-treated hESC RNA for comparison) using the F2/R2 primers ( NANOG1 specific) vs. F2/R3 ‘universal’ NANOG primers. p16 INK4a (hypermethylated in PCa) amplification is used as AzaC control. (G) Cancer cell NANOGP8 cDNAs encode NANOG proteins that can be detected by an anti-NANOG1 antibody. Cancer cell NANOGP8 cDNAs cloned in pET28b(−) were either uninduced (U) or induced (I) with IPTG and supernatant (S) or insoluble pellet (P) was used in Western blotting using the mAb. The arrow points to the ~42 kD NANOG band. (H) Peptide sequences obtained by MALDI-TOF from MCF7 NANOGP8 cDNA. (I-J) Western blotting using whole cell lysate (WCL; I) or NE (J) and eBio mAb.

Techniques Used: Polymerase Chain Reaction, shRNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Cell Culture, Sequencing, Expressing, Amplification, Clone Assay, Western Blot

7) Product Images from "Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers"

Article Title: Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers

Journal: Molecular Cancer

doi: 10.1186/1476-4598-6-12

Expression of stem cell related markers and KIT mutations in dygerminomas . A . Immunohistochemical staining for OCT-3/4, KIT, NANOG, and AP-2γ in dysgerminomas. Scale bar = 25 μm. B . Examples of KIT mutation analysis, with control sequence from normal blood, and three of the mutated KIT (codon 816) sequences from dysgerminomas.
Figure Legend Snippet: Expression of stem cell related markers and KIT mutations in dygerminomas . A . Immunohistochemical staining for OCT-3/4, KIT, NANOG, and AP-2γ in dysgerminomas. Scale bar = 25 μm. B . Examples of KIT mutation analysis, with control sequence from normal blood, and three of the mutated KIT (codon 816) sequences from dysgerminomas.

Techniques Used: Expressing, Immunohistochemistry, Staining, Mutagenesis, Sequencing

8) Product Images from "Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia"

Article Title: Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia

Journal: Stem Cells International

doi: 10.1155/2017/7492914

Immunophenotyping of cells obtained after induction of differentiation, by flow cytometry, showing the markers evaluated and the percentage of positive cells for each marker. On day 0, immunophenotyping of pluripotent cells prior to induction of differentiation showed the high percentage of pluripotency markers, OCT4, SOX2, and NANOG. After 4 days of induction of differentiation, immunophenotyping of the cells showed decreasing pluripotency markers. Analyses are carried out in FACSCalibur.
Figure Legend Snippet: Immunophenotyping of cells obtained after induction of differentiation, by flow cytometry, showing the markers evaluated and the percentage of positive cells for each marker. On day 0, immunophenotyping of pluripotent cells prior to induction of differentiation showed the high percentage of pluripotency markers, OCT4, SOX2, and NANOG. After 4 days of induction of differentiation, immunophenotyping of the cells showed decreasing pluripotency markers. Analyses are carried out in FACSCalibur.

Techniques Used: Flow Cytometry, Cytometry, Marker

Flow cytometry analyses for pluripotency markers OCT4, SOX2, NANOG and SSEA-4 of the iPSC PBscd lines generated.
Figure Legend Snippet: Flow cytometry analyses for pluripotency markers OCT4, SOX2, NANOG and SSEA-4 of the iPSC PBscd lines generated.

Techniques Used: Flow Cytometry, Cytometry, Generated

Characterization of iPSC PBscd01 and confirmation of pluripotency. (a) Flow cytometry analyses of OCT4, NANOG, and SOX2 markers in iPSC PBscd01 and OCT4, NANOG, and SSEA-4 markers in the parental cell (MNCscd01). (b) Expression of endogenous pluripotency markers confirmed by qPCR after seven passages. Quantification of the relative expression of OCT4, SOX2, and NANOG genes. Individual reactions of qPCR were normalized against internal controls (GAPDH and β -actin) and plotted against the level of parental cell expression (MNC PBscd01). (c) Immunostaining of the colonies of iPSC PBscd01 showing the expression of pluripotency markers OCT4, SOX2, and NANOG. Nuclei were stained with DAPI (blue). Confocal microscopy.
Figure Legend Snippet: Characterization of iPSC PBscd01 and confirmation of pluripotency. (a) Flow cytometry analyses of OCT4, NANOG, and SOX2 markers in iPSC PBscd01 and OCT4, NANOG, and SSEA-4 markers in the parental cell (MNCscd01). (b) Expression of endogenous pluripotency markers confirmed by qPCR after seven passages. Quantification of the relative expression of OCT4, SOX2, and NANOG genes. Individual reactions of qPCR were normalized against internal controls (GAPDH and β -actin) and plotted against the level of parental cell expression (MNC PBscd01). (c) Immunostaining of the colonies of iPSC PBscd01 showing the expression of pluripotency markers OCT4, SOX2, and NANOG. Nuclei were stained with DAPI (blue). Confocal microscopy.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Staining, Confocal Microscopy

9) Product Images from "Modeling signaling‐dependent pluripotency with Boolean logic to predict cell fate transitions"

Article Title: Modeling signaling‐dependent pluripotency with Boolean logic to predict cell fate transitions

Journal: Molecular Systems Biology

doi: 10.15252/msb.20177952

LIF stabilizes the pluripotent population while 2i up‐regulates OSN ; related to Fig 4 Summation of frequencies (0–1) of O/S/N‐positive cells assessed by high content screening at 2 days after medium replacement ( n = 6 for Oct4 and Sox2, n = 4 for Nanog; the error bars represent s.d.). Asterisk indicates the significant difference for total OSN assessed by unpaired, two‐sided student t ‐test (** P
Figure Legend Snippet: LIF stabilizes the pluripotent population while 2i up‐regulates OSN ; related to Fig 4 Summation of frequencies (0–1) of O/S/N‐positive cells assessed by high content screening at 2 days after medium replacement ( n = 6 for Oct4 and Sox2, n = 4 for Nanog; the error bars represent s.d.). Asterisk indicates the significant difference for total OSN assessed by unpaired, two‐sided student t ‐test (** P

Techniques Used: High Content Screening

Comparison of predicted and experimentally observed data on gene expression patterns in distinct PSC s; related to Fig 3 Predicted population‐averaged expression level (mean of five independent simulations) for each pluripotency‐associated gene in the control LS condition is comparable to the frequency of gene‐expressing cells from the reported single‐cell measurements using RNA‐seq (triangle; Kolodziejczyk et al , 2015 ) and fluidigm‐qPCR (circle; MacArthur et al , 2012 ). The gene expression levels were binarized into ON or OFF for each single cell to calculate frequencies. The error bars for the simulation resutls represent s.d. of five independent simulations, whereas those for the single‐cell RNA‐seq data represent s.d. of two replicates. Comparison of predicted co‐expressions of epiblast‐specific genes (EpiTFs—Eomes, Otx2, and Fgf5) with O/S/N and those measured by qPCR‐based single‐cell mRNA data. Oct4 and Nanog are likely to be co‐expressed with EpiTFs, whereas Sox2 is not, as it shows a negative correlation with EpiTFs (i.e., EpiTF expression in Sox2—cells is higher) in both our simulation and in published, single‐cell expression data (Hayashi et al , 2008 ; MacArthur et al , 2012 ). P ‐value was calculated by Fisher's exact test. The error bars represent s.d. of five independent simulations. Simulation of distinct PSCs recapitulates population‐level gene expression measurements. All expression data are scaled relative to respective gene expression in control LS conditions. The hierarchical clustering with AU (approximately unbiased) P ‐value and BP (bootstrap probability) value based on multiscale bootstrap resampling was carried out with the pvclust package in R. The results are displayed in the pinwheel view in Fig 3 C, where upper and lower limits are set to +5 and −10, respectively, in the simulation to avoid singularities caused by null expression. The experimental data were taken from indicated GEO entries, and RNA‐seq data for EpiSCs were kindly provided by Dr. Ronald Mckay. Cell lines used are 129 (GSE15603 and GSE62155), J1 (GSE58735), ESF175/1, ESF58/2, ESF122 (GSE7902) cell lines, or mESCs were derived from C57BL/6J strain blastocysts (GSE53275). In silico GOF/LOF study in EpiSC (bF+A) or mESC (LS) conditions. All SCCs above ten profiles and sustainability > 0.7 are shown, and the gene expression levels of each component in each SCC are color‐coded between blue (0.0) to yellow (1.0). The population‐averaged expression levels based on the GOF/LOF results were shown in the PCA metrics in Fig 3 D. Predictions (left) and measurements (right) of population‐averaged expression levels of OSN in EpiSC conditions (bF+A) increased in response to extrinsic manipulation of BMP4. BMP4 was set as continuous‐ON (EpiSC + BMP4) and random (EpiSC). The frequencies reported for Oct4, Sox2, and Nanog‐positive cells, assessed by Cellomics high content screening, represent the mean and s.d. of four replicates. Asterisk indicates the significant difference ( P
Figure Legend Snippet: Comparison of predicted and experimentally observed data on gene expression patterns in distinct PSC s; related to Fig 3 Predicted population‐averaged expression level (mean of five independent simulations) for each pluripotency‐associated gene in the control LS condition is comparable to the frequency of gene‐expressing cells from the reported single‐cell measurements using RNA‐seq (triangle; Kolodziejczyk et al , 2015 ) and fluidigm‐qPCR (circle; MacArthur et al , 2012 ). The gene expression levels were binarized into ON or OFF for each single cell to calculate frequencies. The error bars for the simulation resutls represent s.d. of five independent simulations, whereas those for the single‐cell RNA‐seq data represent s.d. of two replicates. Comparison of predicted co‐expressions of epiblast‐specific genes (EpiTFs—Eomes, Otx2, and Fgf5) with O/S/N and those measured by qPCR‐based single‐cell mRNA data. Oct4 and Nanog are likely to be co‐expressed with EpiTFs, whereas Sox2 is not, as it shows a negative correlation with EpiTFs (i.e., EpiTF expression in Sox2—cells is higher) in both our simulation and in published, single‐cell expression data (Hayashi et al , 2008 ; MacArthur et al , 2012 ). P ‐value was calculated by Fisher's exact test. The error bars represent s.d. of five independent simulations. Simulation of distinct PSCs recapitulates population‐level gene expression measurements. All expression data are scaled relative to respective gene expression in control LS conditions. The hierarchical clustering with AU (approximately unbiased) P ‐value and BP (bootstrap probability) value based on multiscale bootstrap resampling was carried out with the pvclust package in R. The results are displayed in the pinwheel view in Fig 3 C, where upper and lower limits are set to +5 and −10, respectively, in the simulation to avoid singularities caused by null expression. The experimental data were taken from indicated GEO entries, and RNA‐seq data for EpiSCs were kindly provided by Dr. Ronald Mckay. Cell lines used are 129 (GSE15603 and GSE62155), J1 (GSE58735), ESF175/1, ESF58/2, ESF122 (GSE7902) cell lines, or mESCs were derived from C57BL/6J strain blastocysts (GSE53275). In silico GOF/LOF study in EpiSC (bF+A) or mESC (LS) conditions. All SCCs above ten profiles and sustainability > 0.7 are shown, and the gene expression levels of each component in each SCC are color‐coded between blue (0.0) to yellow (1.0). The population‐averaged expression levels based on the GOF/LOF results were shown in the PCA metrics in Fig 3 D. Predictions (left) and measurements (right) of population‐averaged expression levels of OSN in EpiSC conditions (bF+A) increased in response to extrinsic manipulation of BMP4. BMP4 was set as continuous‐ON (EpiSC + BMP4) and random (EpiSC). The frequencies reported for Oct4, Sox2, and Nanog‐positive cells, assessed by Cellomics high content screening, represent the mean and s.d. of four replicates. Asterisk indicates the significant difference ( P

Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay, In Silico, High Content Screening

10) Product Images from "Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells"

Article Title: Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells

Journal: Tissue Engineering. Part C, Methods

doi: 10.1089/ten.tec.2015.0038

Results of immunostaining for E-cadherin and vinculin in hiPSC cultured for 5 days on mesh. Immunofluorescence images showing E-cadherin and Nanog expression (A) , and vinculin and Nanog expression (B) by hiPSC on a partially filled mesh. Merged images
Figure Legend Snippet: Results of immunostaining for E-cadherin and vinculin in hiPSC cultured for 5 days on mesh. Immunofluorescence images showing E-cadherin and Nanog expression (A) , and vinculin and Nanog expression (B) by hiPSC on a partially filled mesh. Merged images

Techniques Used: Immunostaining, Cell Culture, Immunofluorescence, Expressing

11) Product Images from "An extended transcriptional network for pluripotency of embryonic stem cells"

Article Title: An extended transcriptional network for pluripotency of embryonic stem cells

Journal:

doi: 10.1016/j.cell.2008.02.039

Chromosomal view of Nanog occupancy detected by bioChIP-chip and conventional ChIP-chip
Figure Legend Snippet: Chromosomal view of Nanog occupancy detected by bioChIP-chip and conventional ChIP-chip

Techniques Used: Chromatin Immunoprecipitation

12) Product Images from "An extended transcriptional network for pluripotency of embryonic stem cells"

Article Title: An extended transcriptional network for pluripotency of embryonic stem cells

Journal:

doi: 10.1016/j.cell.2008.02.039

Chromosomal view of Nanog occupancy detected by bioChIP-chip and conventional ChIP-chip
Figure Legend Snippet: Chromosomal view of Nanog occupancy detected by bioChIP-chip and conventional ChIP-chip

Techniques Used: Chromatin Immunoprecipitation

13) Product Images from "Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells"

Article Title: Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells

Journal:

doi: 10.1089/scd.2008.0335

Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were
Figure Legend Snippet: Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were

Techniques Used: Over Expression

Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (
Figure Legend Snippet: Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (

Techniques Used: Over Expression

Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Immunostaining, Staining

Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Staining

Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,
Figure Legend Snippet: Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,

Techniques Used: Expressing

14) Product Images from "3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension"

Article Title: 3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12946

Released actin cytoskeleton promotes H3K9 demethylation and Nanog expression. Cultured MSC s at passage 6 in culture medium ( DMEM +10% FBS ) containing cytochalasin D at the indicated concentrations, and 0.1% DMSO as control for 60 hrs. ( A ) Fluorescence images of the F‐actin (green) and G‐actin (red) of MSC s. The cells get rounded with the addition of 2.5 and 5.0 μM cytochalasin D and presented different F‐actin pattern of apical and bottom layer (scale bar, 50 μm). Real‐time PCR was used to analyse the expression of Nanog ( B ) and Suv39h1 ( D ). Nuclear faction of MSC s was immunoblotted for Nanog ( C ), Suv39h1, H3K9me3 ( E ) and histone H3 (nuclear loading control). H3K9me3 ( F ) and Suv39h1 ( G ) Ch IP ‐ qPCR assay was performed to evaluate the accumulation on Nanog. ** P
Figure Legend Snippet: Released actin cytoskeleton promotes H3K9 demethylation and Nanog expression. Cultured MSC s at passage 6 in culture medium ( DMEM +10% FBS ) containing cytochalasin D at the indicated concentrations, and 0.1% DMSO as control for 60 hrs. ( A ) Fluorescence images of the F‐actin (green) and G‐actin (red) of MSC s. The cells get rounded with the addition of 2.5 and 5.0 μM cytochalasin D and presented different F‐actin pattern of apical and bottom layer (scale bar, 50 μm). Real‐time PCR was used to analyse the expression of Nanog ( B ) and Suv39h1 ( D ). Nuclear faction of MSC s was immunoblotted for Nanog ( C ), Suv39h1, H3K9me3 ( E ) and histone H3 (nuclear loading control). H3K9me3 ( F ) and Suv39h1 ( G ) Ch IP ‐ qPCR assay was performed to evaluate the accumulation on Nanog. ** P

Techniques Used: Expressing, Cell Culture, Fluorescence, Real-time Polymerase Chain Reaction

15) Product Images from "Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions"

Article Title: Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions

Journal: Scientific Reports

doi: 10.1038/srep29311

Expression of pluripotency related markers and a sphere culture of BEAS-2B under normoxia. ( a ) HIF-2α expression was detected in the nucleus of BEAS-2B under normoxia by western blotting. Beta tubulin and TATA binding protein (TBP) were used as protein markers for the cytosol and nucleus fraction, respectively. ( b ) Western blotting for Oct-4. ( c ) For knockdown of HIF-2α, BEAS-2B cells were transfected with shRNA targeting HIF-2α and sh-Luc was used as control. ( d ) Western blotting of Nanog and a culture of BEAS-2B containing floating spheres. Representative result from two or three experiments was presented.
Figure Legend Snippet: Expression of pluripotency related markers and a sphere culture of BEAS-2B under normoxia. ( a ) HIF-2α expression was detected in the nucleus of BEAS-2B under normoxia by western blotting. Beta tubulin and TATA binding protein (TBP) were used as protein markers for the cytosol and nucleus fraction, respectively. ( b ) Western blotting for Oct-4. ( c ) For knockdown of HIF-2α, BEAS-2B cells were transfected with shRNA targeting HIF-2α and sh-Luc was used as control. ( d ) Western blotting of Nanog and a culture of BEAS-2B containing floating spheres. Representative result from two or three experiments was presented.

Techniques Used: Expressing, Western Blot, Binding Assay, Transfection, shRNA

16) Product Images from "Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential"

Article Title: Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential

Journal: Stem Cells and Development

doi: 10.1089/scd.2013.0024

Gene expression profiles of all hESC lines upon germ cell-directed differentiation as EBs in the presence of bone morphogenic protein 4 (BMP4) for early primordial germ cell (PGC) markers STELLA, cKIT, FRAGILIS (a–c) , late PGC marker VASA (d) , pluripotency markers OCT4, SOX2, NANOG (e–g) , and the differentiation marker SSEA1 (h) . Activin A-derived hESC lines UGent11-4-ActA and UGent12-3-ActA show significantly higher expression for STELLA and cKIT at day 0 and significant upregulation of VASA at day 7, when FRAGILIS is downregulated. For all hESC lines, pluripotency markers OCT4, SOX2, NANOG were significantly downregulated, and SSEA1 expression increased as differentiation proceeded from day 0 to day 14. STELLA, cKIT: UGent11-4-ActA (a′) compared to standard hESC lines at day 0 (a) , P ≤0.001; UGent12-3-ActA (b′) compared to standard hESC lines at day 0 (b) , P ≤0.001; FRAGILIS: Day 7 UGent12-3-ActA EBs (a′) compared to day 0 and day 3 EBs (a) , P
Figure Legend Snippet: Gene expression profiles of all hESC lines upon germ cell-directed differentiation as EBs in the presence of bone morphogenic protein 4 (BMP4) for early primordial germ cell (PGC) markers STELLA, cKIT, FRAGILIS (a–c) , late PGC marker VASA (d) , pluripotency markers OCT4, SOX2, NANOG (e–g) , and the differentiation marker SSEA1 (h) . Activin A-derived hESC lines UGent11-4-ActA and UGent12-3-ActA show significantly higher expression for STELLA and cKIT at day 0 and significant upregulation of VASA at day 7, when FRAGILIS is downregulated. For all hESC lines, pluripotency markers OCT4, SOX2, NANOG were significantly downregulated, and SSEA1 expression increased as differentiation proceeded from day 0 to day 14. STELLA, cKIT: UGent11-4-ActA (a′) compared to standard hESC lines at day 0 (a) , P ≤0.001; UGent12-3-ActA (b′) compared to standard hESC lines at day 0 (b) , P ≤0.001; FRAGILIS: Day 7 UGent12-3-ActA EBs (a′) compared to day 0 and day 3 EBs (a) , P

Techniques Used: Expressing, Pyrolysis Gas Chromatography, Marker, Derivative Assay

Pluripotency assays on both Activin A-derived and standard human embryonic stem cell (hESC) lines. (a) Immunofluorescence staining for pluripotency markers OCT4 and NANOG, nuclear staining by DAPI and merged images of all hESC lines when maintained in their undifferentiated state. Scale bar, 50 μm. (b) Alkaline phosphatase live staining for all hESC lines in their undifferentiated state. Scale bar, 50 μm. (c) Karyograms for XX hESC lines UGent11-4-ActA, UGent11-5 and UGent11-6 and XY hESC lines UGent12-3-ActA and UGent11-7. All hESC lines were karyotypically normal. (d) Embryoid body (EB) differentiation assay of all hESC lines. (i) Gene expression profile of germ layer markers in day 14 EBs. Fold change values are analyzed compared to OCT4 gene expression in day 14 EBs. For all hESC lines, after 14 days of differentiation as EBs, ectoderm marker NESTIN and endoderm markers GATA4 and GATA6 upregulated significantly compared to OCT4, P
Figure Legend Snippet: Pluripotency assays on both Activin A-derived and standard human embryonic stem cell (hESC) lines. (a) Immunofluorescence staining for pluripotency markers OCT4 and NANOG, nuclear staining by DAPI and merged images of all hESC lines when maintained in their undifferentiated state. Scale bar, 50 μm. (b) Alkaline phosphatase live staining for all hESC lines in their undifferentiated state. Scale bar, 50 μm. (c) Karyograms for XX hESC lines UGent11-4-ActA, UGent11-5 and UGent11-6 and XY hESC lines UGent12-3-ActA and UGent11-7. All hESC lines were karyotypically normal. (d) Embryoid body (EB) differentiation assay of all hESC lines. (i) Gene expression profile of germ layer markers in day 14 EBs. Fold change values are analyzed compared to OCT4 gene expression in day 14 EBs. For all hESC lines, after 14 days of differentiation as EBs, ectoderm marker NESTIN and endoderm markers GATA4 and GATA6 upregulated significantly compared to OCT4, P

Techniques Used: Derivative Assay, Immunofluorescence, Staining, Differentiation Assay, Expressing, Marker

17) Product Images from "Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells"

Article Title: Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

Journal: Cell Cycle

doi: 10.1080/15384101.2015.1014144

DLK activity is upregulated upon differentiation. ( A ) The mRNA expression levels of DLK increased upon differentiation. DLK, Nanog, and Oct4 transcripts from D3 mouse ES cells and mouse EBs (6th and 12th days) were quantified by real-time quantitative
Figure Legend Snippet: DLK activity is upregulated upon differentiation. ( A ) The mRNA expression levels of DLK increased upon differentiation. DLK, Nanog, and Oct4 transcripts from D3 mouse ES cells and mouse EBs (6th and 12th days) were quantified by real-time quantitative

Techniques Used: Activity Assay, Expressing

Overexpression of DLK reduces mouse ES cell numbers and the expression of Nanog. ( A ) The protein expression amounts in DLK overexpression mouse ES cells were similar in Day 15 and Day 20 EBs. DLK protein was detected in DLK overexpressing mouse ES cells
Figure Legend Snippet: Overexpression of DLK reduces mouse ES cell numbers and the expression of Nanog. ( A ) The protein expression amounts in DLK overexpression mouse ES cells were similar in Day 15 and Day 20 EBs. DLK protein was detected in DLK overexpressing mouse ES cells

Techniques Used: Over Expression, Expressing

18) Product Images from "Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning"

Article Title: Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118165

Characterization of ntES cells by real time RT-PCR analysis. Expressions of Oct4, Nanog, Sox2, and Rex01genes in (A) PES1 at passages 8, (B) PES3 at passages 15 and (C) pig fibroblast cells.
Figure Legend Snippet: Characterization of ntES cells by real time RT-PCR analysis. Expressions of Oct4, Nanog, Sox2, and Rex01genes in (A) PES1 at passages 8, (B) PES3 at passages 15 and (C) pig fibroblast cells.

Techniques Used: Quantitative RT-PCR

Immunofluorescence staining of pluripotency markers in ntES cell colonies derived from aggregated cloned embryos. Expressions of pluripotency markers (Oct4, Nanog and Sox2) are shown (red) in PES3 at passage 15 (left panel) and PES1 at passage 10 (right panel). Nuclei are stained with DAPI (blue). Scale bars = 100 μm.
Figure Legend Snippet: Immunofluorescence staining of pluripotency markers in ntES cell colonies derived from aggregated cloned embryos. Expressions of pluripotency markers (Oct4, Nanog and Sox2) are shown (red) in PES3 at passage 15 (left panel) and PES1 at passage 10 (right panel). Nuclei are stained with DAPI (blue). Scale bars = 100 μm.

Techniques Used: Immunofluorescence, Staining, Derivative Assay, Clone Assay

19) Product Images from "Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males"

Article Title: Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

Journal: Cell Stem Cell

doi: 10.1016/j.stem.2018.05.001

Males Undergo Transient XCI (A) RNA FISH for Xist (red) in male 2iL ESCs at 1.5 days of differentiation in FA using a strand-specific probe. White arrowheads indicate Xist signals. Quantification of the different Xist RNA patterns is shown. (B) Immuno-RNA FISH for Xist (red) and H3K27me3 or H3K27ac (green) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist cloud. (C) RNA FISH for Xist (red) and Rnf12 , Nexmif , or Huwe1 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. (D) Quantification of RNA FISH patterns for the X-linked genes Rnf12 , Nexmif , or Huwe1 and Xist as shown in (C). Gray indicates the presence of Rnf12 / Nexmif / Huwe1 signal, and pink indicates the absence of Rnf12 / Nexmif / Huwe1 signal. (E) RNA FISH for Xist (red) in female 2iL ESCs at 1.5 days of differentiation in FA using ss probe. Quantification of different Xist RNA patterns is shown. (F) RNA FISH for Xist (red) and Rnf12 or Huwe1 (grayscale) in female 2iL ESCs at 1.5 days of differentiation in FA. (G) Quantification of RNA FISH patterns for X-linked genes Rnf12 or Huwe1 and Xist as shown in (F). Dark gray indicates biallelic Rnf12 / Huwe1 signal, light gray indicates monoallelic Rnf12 / Huwe1 signal, and pink indicates the absence of Rnf12 / Huwe1 signal. (H) Immuno-RNA FISH for Xist (red), NANOG (green), and OCT4 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist clouds. (I) Percentage of NANOG- and OCT4-expressing cells in the population (left) and in cells exhibiting Xist cloud (right) as shown in (H). Fisher’s exact test was used for statistical analysis. ESC lines used were XY1 and XX1. Scale bar represents 5 μm.
Figure Legend Snippet: Males Undergo Transient XCI (A) RNA FISH for Xist (red) in male 2iL ESCs at 1.5 days of differentiation in FA using a strand-specific probe. White arrowheads indicate Xist signals. Quantification of the different Xist RNA patterns is shown. (B) Immuno-RNA FISH for Xist (red) and H3K27me3 or H3K27ac (green) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist cloud. (C) RNA FISH for Xist (red) and Rnf12 , Nexmif , or Huwe1 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. (D) Quantification of RNA FISH patterns for the X-linked genes Rnf12 , Nexmif , or Huwe1 and Xist as shown in (C). Gray indicates the presence of Rnf12 / Nexmif / Huwe1 signal, and pink indicates the absence of Rnf12 / Nexmif / Huwe1 signal. (E) RNA FISH for Xist (red) in female 2iL ESCs at 1.5 days of differentiation in FA using ss probe. Quantification of different Xist RNA patterns is shown. (F) RNA FISH for Xist (red) and Rnf12 or Huwe1 (grayscale) in female 2iL ESCs at 1.5 days of differentiation in FA. (G) Quantification of RNA FISH patterns for X-linked genes Rnf12 or Huwe1 and Xist as shown in (F). Dark gray indicates biallelic Rnf12 / Huwe1 signal, light gray indicates monoallelic Rnf12 / Huwe1 signal, and pink indicates the absence of Rnf12 / Huwe1 signal. (H) Immuno-RNA FISH for Xist (red), NANOG (green), and OCT4 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist clouds. (I) Percentage of NANOG- and OCT4-expressing cells in the population (left) and in cells exhibiting Xist cloud (right) as shown in (H). Fisher’s exact test was used for statistical analysis. ESC lines used were XY1 and XX1. Scale bar represents 5 μm.

Techniques Used: Fluorescence In Situ Hybridization, Expressing

20) Product Images from "DRP1-mediated regulation of mitochondrial dynamics determines the apoptotic response upon embryonic differentiation"

Article Title: DRP1-mediated regulation of mitochondrial dynamics determines the apoptotic response upon embryonic differentiation

Journal: bioRxiv

doi: 10.1101/835751

Drp1 deletion facilitates early apoptotic events. A. ATP-b and NANOG immunostaining showing mitochondrial morphology in wild type and Drp1 −/− ESCs. B . Quantitative RT-PCR showing gene expression levels of naïve and primed pluripotency markers in wild-type and Drp1 −/− ESCs. Gene expression normalized against Gapdh . C . % of cells with MOMP detected by TMRM staining in wild-type and Drp1 −/− ESCs untreated or treated with 1µM sodium arsenite for 16h or D 1µM Thapsigargin for 16h. Data normalized against wild-type cells. E . % cells with MOMP in ESCs and EpiSCs treated with with either the BMI (0.5µM), BID (2.5µM) or control (1µM) peptides for the indicated amounts of time. Average of 3 (B, C), 4 (D) or 7 (E) independent experiments +/-SEM (C, D) or +/-SD (E) is shown. 2-way ANOVA with Šidák correction *p
Figure Legend Snippet: Drp1 deletion facilitates early apoptotic events. A. ATP-b and NANOG immunostaining showing mitochondrial morphology in wild type and Drp1 −/− ESCs. B . Quantitative RT-PCR showing gene expression levels of naïve and primed pluripotency markers in wild-type and Drp1 −/− ESCs. Gene expression normalized against Gapdh . C . % of cells with MOMP detected by TMRM staining in wild-type and Drp1 −/− ESCs untreated or treated with 1µM sodium arsenite for 16h or D 1µM Thapsigargin for 16h. Data normalized against wild-type cells. E . % cells with MOMP in ESCs and EpiSCs treated with with either the BMI (0.5µM), BID (2.5µM) or control (1µM) peptides for the indicated amounts of time. Average of 3 (B, C), 4 (D) or 7 (E) independent experiments +/-SEM (C, D) or +/-SD (E) is shown. 2-way ANOVA with Šidák correction *p

Techniques Used: Immunostaining, Quantitative RT-PCR, Expressing, Staining

21) Product Images from "Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states"

Article Title: Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

Journal: eLife

doi: 10.7554/eLife.20487

Validation of inferred cell types and lineage relationships. ( A ) Comparison of clustering configuration (left column) and culture conditions (right column) shows that there is mixing of cells from different culture conditions to the same cluster as well as cells from the same culture conditions being assigned to different clusters. ( B ) Mean and coefficient of variation (c.v.) of percent variance explained by the largest eigenvalues of each cell cluster normalized by the percent variance explained by that of randomly shuffled gene expression data (y-axis) for each cell cluster (x-axis). The last point along the x-axis shows the mean and c.v. of percent variance explained by the largest eigenvalues of each cell cluster normalized by the percent variance explained by that of randomly shuffled data, for all merged pairs of cell clusters, which is significantly higher than that derived from single clusters (maximum p value 0.2675 × 10 − 12 ) . ( C ) A scatter plot of Etv5 (x-axis) and FoxA2 (y-axis) expression from immunofluorescence data (each dot is a mesendodermal cell, as identified by T expression; data not shown) shows that from day 3 to day 4 of mesendodermal differentiation (Materials and methods), Etv5 expression decreases and some cells go on to upregulate FoxA2. ( D ) Histogram of Nanog expression (as measured by immunostaining and flow cytometry) before differentiation (yellow; Lif2i C 0 state) and after two days of differentiation (orange; D2 PD03 C 1 state) shows that Nanog expression is downregulated throughout most of the population during the first two days, similar to the observed changes of Klf4 during this time ( Figure 3D ). We thus use Nanog immunostaining, which produces a better signal compared to Klf4 antibodies, to identify cells that are still remaining in the naïve pluripotent C 0 state. DOI: http://dx.doi.org/10.7554/eLife.20487.013
Figure Legend Snippet: Validation of inferred cell types and lineage relationships. ( A ) Comparison of clustering configuration (left column) and culture conditions (right column) shows that there is mixing of cells from different culture conditions to the same cluster as well as cells from the same culture conditions being assigned to different clusters. ( B ) Mean and coefficient of variation (c.v.) of percent variance explained by the largest eigenvalues of each cell cluster normalized by the percent variance explained by that of randomly shuffled gene expression data (y-axis) for each cell cluster (x-axis). The last point along the x-axis shows the mean and c.v. of percent variance explained by the largest eigenvalues of each cell cluster normalized by the percent variance explained by that of randomly shuffled data, for all merged pairs of cell clusters, which is significantly higher than that derived from single clusters (maximum p value 0.2675 × 10 − 12 ) . ( C ) A scatter plot of Etv5 (x-axis) and FoxA2 (y-axis) expression from immunofluorescence data (each dot is a mesendodermal cell, as identified by T expression; data not shown) shows that from day 3 to day 4 of mesendodermal differentiation (Materials and methods), Etv5 expression decreases and some cells go on to upregulate FoxA2. ( D ) Histogram of Nanog expression (as measured by immunostaining and flow cytometry) before differentiation (yellow; Lif2i C 0 state) and after two days of differentiation (orange; D2 PD03 C 1 state) shows that Nanog expression is downregulated throughout most of the population during the first two days, similar to the observed changes of Klf4 during this time ( Figure 3D ). We thus use Nanog immunostaining, which produces a better signal compared to Klf4 antibodies, to identify cells that are still remaining in the naïve pluripotent C 0 state. DOI: http://dx.doi.org/10.7554/eLife.20487.013

Techniques Used: Expressing, Derivative Assay, Immunofluorescence, Immunostaining, Flow Cytometry, Cytometry

22) Product Images from "Clones of Ectopic Stem Cells in the Regeneration of Muscle Defects In Vivo"

Article Title: Clones of Ectopic Stem Cells in the Regeneration of Muscle Defects In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013547

Myogenic clones in comparison with their parent stem/progenitor cells. A: Cumulative population doubling (PD) of two myogenic clones and their parent stem/progenitor cells showing a lack of statistically significant differences in PD kinetics. B: Expression of CD146, Stro1, CD133, Oct4 and Nanog by the two tested myogenic clones, B6 and C3, as well as their parent stem/progenitor cells. P: cell passage. Strikingly, B6 and C3 were overwhelmingly Oct4+, Nanog+ and Stro1+, and sustained the expression of these markers up to the tested 5 passages. C–H: Representative immunofluorescence of Stro1, Oct4 and Nanog by the two identified myogenic clones (passage 5) merged with DAPI-stain nuclei. Scale bar: 100 µm.
Figure Legend Snippet: Myogenic clones in comparison with their parent stem/progenitor cells. A: Cumulative population doubling (PD) of two myogenic clones and their parent stem/progenitor cells showing a lack of statistically significant differences in PD kinetics. B: Expression of CD146, Stro1, CD133, Oct4 and Nanog by the two tested myogenic clones, B6 and C3, as well as their parent stem/progenitor cells. P: cell passage. Strikingly, B6 and C3 were overwhelmingly Oct4+, Nanog+ and Stro1+, and sustained the expression of these markers up to the tested 5 passages. C–H: Representative immunofluorescence of Stro1, Oct4 and Nanog by the two identified myogenic clones (passage 5) merged with DAPI-stain nuclei. Scale bar: 100 µm.

Techniques Used: Clone Assay, Expressing, Immunofluorescence, Staining

23) Product Images from "Development of Hepatocyte‐like Cell Derived from Human Induced Pluripotent Stem cell as a Host for Clinically Isolated Hepatitis C Virus). Development of hepatocyte‐like cell derived from human induced pluripotent stem cell as a host for clinically isolated hepatitis C virus"

Article Title: Development of Hepatocyte‐like Cell Derived from Human Induced Pluripotent Stem cell as a Host for Clinically Isolated Hepatitis C Virus). Development of hepatocyte‐like cell derived from human induced pluripotent stem cell as a host for clinically isolated hepatitis C virus

Journal: Current Protocols in Stem Cell Biology

doi: 10.1002/cpsc.35

iPSC colonies were live‐stained with TRA‐1‐60 antibody, a pluripotency surface marker. The iPSC colony was grown on MEF for 25 days after transduction ( A ) and live‐stained with TRA‐1‐60 antibody followed by Alexa 488 conjugated goat anti mouse IgG. The TRA‐1‐60‐positive colony was verified under fluorescence microscope ( B ). The dTomato disappeared in iPSCs ( C ). The iPSCs on Geltrex exhibited high nuclease‐to‐cytoplasm ratio with prominent nucleoli ( D ). Immunofluorescent staining for OCT4, SOX2, NANOG, TRA‐160, TRA‐1‐81, and SSEA4 followed by the Alexa fluor 488 conjugated secondary antibody confirmed the identity iPSCs after the reprogramming from MSCs ( E ). Nuclei were localized by DAPI (blue). Scale bars = 100 µM. The expressions of pluripotency markers were determined by RT‐PCR. iPSCs exhibited similar expression profile of pluripotent markers to that of embryonic stem cells (ESCs) ( G ).
Figure Legend Snippet: iPSC colonies were live‐stained with TRA‐1‐60 antibody, a pluripotency surface marker. The iPSC colony was grown on MEF for 25 days after transduction ( A ) and live‐stained with TRA‐1‐60 antibody followed by Alexa 488 conjugated goat anti mouse IgG. The TRA‐1‐60‐positive colony was verified under fluorescence microscope ( B ). The dTomato disappeared in iPSCs ( C ). The iPSCs on Geltrex exhibited high nuclease‐to‐cytoplasm ratio with prominent nucleoli ( D ). Immunofluorescent staining for OCT4, SOX2, NANOG, TRA‐160, TRA‐1‐81, and SSEA4 followed by the Alexa fluor 488 conjugated secondary antibody confirmed the identity iPSCs after the reprogramming from MSCs ( E ). Nuclei were localized by DAPI (blue). Scale bars = 100 µM. The expressions of pluripotency markers were determined by RT‐PCR. iPSCs exhibited similar expression profile of pluripotent markers to that of embryonic stem cells (ESCs) ( G ).

Techniques Used: Staining, Marker, Transduction, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing

24) Product Images from "Gatekeeper transcription factors regulate switch between lineage preservation and cell plasticity"

Article Title: Gatekeeper transcription factors regulate switch between lineage preservation and cell plasticity

Journal: bioRxiv

doi: 10.1101/2020.03.19.999433

Dedifferentiation of Rat Embryonic Fibroblasts to dMSCs and dPSCs by transient repression of Snai2 and Prrx1. A,D) Brightfield photomicrographs of REFs transfected with siCntrl, siPrrx1 siSnai2, (Rows 1,2 and 3) that were incubated in MSC media or Rat ESC media for 14 days. IF photomicrographs assess expression of MSC TF Myc (Red) and ESC TFs (Sox2 and Nanog in Red) and DAPI (Blue) in all three groups (Scale bar, 10µm). D) (Column 4) Brightfield photomicrographs of free-floating embryoid bodies on day 8 of suspension culture in differentiation media following transfection of REFs with siCntrl, siPrrx1 siSnai2 and incubation in Rat ESC media for 14 days. B) MSC activity was ascertained using Alkaline Phosphatase Assay. C,E) q-RTPCR analysis to assess increase in relative expression of Myc, Klf4, Sox2 and Nanog compared to housekeeping gene (B2m) in siPrrx1 and siSnai2 treated REFs when incubated in MSC and ESC media respectively for 14 days. F, G and H) IF Confocal microscopy images assess expression of Ectodermal TFs Sox2 (green) and Otx-2 (red), Mesodermal TFs Brachyury (green) and Hand1 (red), and Endodermal TFs Gata-4 (green) and Sox17 (red) of embryoid bodies at Day 8 in the siSnai2 group, nuclear counterstain DAPI (blue).
Figure Legend Snippet: Dedifferentiation of Rat Embryonic Fibroblasts to dMSCs and dPSCs by transient repression of Snai2 and Prrx1. A,D) Brightfield photomicrographs of REFs transfected with siCntrl, siPrrx1 siSnai2, (Rows 1,2 and 3) that were incubated in MSC media or Rat ESC media for 14 days. IF photomicrographs assess expression of MSC TF Myc (Red) and ESC TFs (Sox2 and Nanog in Red) and DAPI (Blue) in all three groups (Scale bar, 10µm). D) (Column 4) Brightfield photomicrographs of free-floating embryoid bodies on day 8 of suspension culture in differentiation media following transfection of REFs with siCntrl, siPrrx1 siSnai2 and incubation in Rat ESC media for 14 days. B) MSC activity was ascertained using Alkaline Phosphatase Assay. C,E) q-RTPCR analysis to assess increase in relative expression of Myc, Klf4, Sox2 and Nanog compared to housekeeping gene (B2m) in siPrrx1 and siSnai2 treated REFs when incubated in MSC and ESC media respectively for 14 days. F, G and H) IF Confocal microscopy images assess expression of Ectodermal TFs Sox2 (green) and Otx-2 (red), Mesodermal TFs Brachyury (green) and Hand1 (red), and Endodermal TFs Gata-4 (green) and Sox17 (red) of embryoid bodies at Day 8 in the siSnai2 group, nuclear counterstain DAPI (blue).

Techniques Used: Transfection, Incubation, Expressing, Activity Assay, ALP Assay, Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy

25) Product Images from "Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells"

Article Title: Effects of Ectopic Nanog and Oct4 Overexpression on Mesenchymal Stem Cells

Journal:

doi: 10.1089/scd.2008.0335

Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were
Figure Legend Snippet: Overexpression of Nanog and Oct4 enhanced colony formation of mesenchymal stem cells (MSCs) compared with no insert control (Dest) MSCs. ( A ) Nanog and Oct4 overexpressing MSCs were seeded at 250, 500, and 1,000 cells in 10 cm dishes, and colonies were

Techniques Used: Over Expression

Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (
Figure Legend Snippet: Characterization of Nanog and Oct4 overexpressing mesenchymal stem cells (MSCs) and effects of their overexpression on proliferation of MSCs, showing Nanog and Oct4 promoted proliferation of MSCs by an average 1.67-fold and 1.51-fold, respectively. (

Techniques Used: Over Expression

Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on chondrogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR, COL2A1 immunostaining, and Alcian blue stain compared to no insert control (Dest) MSCs under chondrogenic medium at Day 28. ( A ) Transcriptional

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Immunostaining, Staining

Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis
Figure Legend Snippet: Effect of Nanog and Oct4 overexpression on adipogenesis of mesenchymal stem cells (MSCs) evaluated by QPCR and oil red stain compared to no insert control (Dest) MSCs under adipogenic medium at Day 14, showing Nanog overexpression slowed down adipogenesis

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Staining

Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,
Figure Legend Snippet: Global gene expression analyses by microarrays. ( A ) Pearson correlation analysis of 11,426 probes was performed to cluster no insert control (Dest) mesenchymal stem cells (MSCs), Nanog, and Oct4 overexpressing MSCs. Red indicates increased expression,

Techniques Used: Expressing

26) Product Images from "Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell-matrix interactions, rationalizing their enhanced functional potency for myocardial repair"

Article Title: Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell-matrix interactions, rationalizing their enhanced functional potency for myocardial repair

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.532

Expression of c-kit, SOX2, and Nanog in cells under cardiosphere and monolayer culture conditions
Figure Legend Snippet: Expression of c-kit, SOX2, and Nanog in cells under cardiosphere and monolayer culture conditions

Techniques Used: Expressing

27) Product Images from "Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males"

Article Title: Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

Journal: Cell Stem Cell

doi: 10.1016/j.stem.2018.05.001

Males Undergo Transient XCI (A) RNA FISH for Xist (red) in male 2iL ESCs at 1.5 days of differentiation in FA using a strand-specific probe. White arrowheads indicate Xist signals. Quantification of the different Xist RNA patterns is shown. (B) Immuno-RNA FISH for Xist (red) and H3K27me3 or H3K27ac (green) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist cloud. (C) RNA FISH for Xist (red) and Rnf12 , Nexmif , or Huwe1 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. (D) Quantification of RNA FISH patterns for the X-linked genes Rnf12 , Nexmif , or Huwe1 and Xist as shown in (C). Gray indicates the presence of Rnf12 / Nexmif / Huwe1 signal, and pink indicates the absence of Rnf12 / Nexmif / Huwe1 signal. (E) RNA FISH for Xist (red) in female 2iL ESCs at 1.5 days of differentiation in FA using ss probe. Quantification of different Xist RNA patterns is shown. (F) RNA FISH for Xist (red) and Rnf12 or Huwe1 (grayscale) in female 2iL ESCs at 1.5 days of differentiation in FA. (G) Quantification of RNA FISH patterns for X-linked genes Rnf12 or Huwe1 and Xist as shown in (F). Dark gray indicates biallelic Rnf12 / Huwe1 signal, light gray indicates monoallelic Rnf12 / Huwe1 signal, and pink indicates the absence of Rnf12 / Huwe1 signal. (H) Immuno-RNA FISH for Xist (red), NANOG (green), and OCT4 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist clouds. (I) Percentage of NANOG- and OCT4-expressing cells in the population (left) and in cells exhibiting Xist cloud (right) as shown in (H). Fisher’s exact test was used for statistical analysis. ESC lines used were XY1 and XX1. Scale bar represents 5 μm.
Figure Legend Snippet: Males Undergo Transient XCI (A) RNA FISH for Xist (red) in male 2iL ESCs at 1.5 days of differentiation in FA using a strand-specific probe. White arrowheads indicate Xist signals. Quantification of the different Xist RNA patterns is shown. (B) Immuno-RNA FISH for Xist (red) and H3K27me3 or H3K27ac (green) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist cloud. (C) RNA FISH for Xist (red) and Rnf12 , Nexmif , or Huwe1 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. (D) Quantification of RNA FISH patterns for the X-linked genes Rnf12 , Nexmif , or Huwe1 and Xist as shown in (C). Gray indicates the presence of Rnf12 / Nexmif / Huwe1 signal, and pink indicates the absence of Rnf12 / Nexmif / Huwe1 signal. (E) RNA FISH for Xist (red) in female 2iL ESCs at 1.5 days of differentiation in FA using ss probe. Quantification of different Xist RNA patterns is shown. (F) RNA FISH for Xist (red) and Rnf12 or Huwe1 (grayscale) in female 2iL ESCs at 1.5 days of differentiation in FA. (G) Quantification of RNA FISH patterns for X-linked genes Rnf12 or Huwe1 and Xist as shown in (F). Dark gray indicates biallelic Rnf12 / Huwe1 signal, light gray indicates monoallelic Rnf12 / Huwe1 signal, and pink indicates the absence of Rnf12 / Huwe1 signal. (H) Immuno-RNA FISH for Xist (red), NANOG (green), and OCT4 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist clouds. (I) Percentage of NANOG- and OCT4-expressing cells in the population (left) and in cells exhibiting Xist cloud (right) as shown in (H). Fisher’s exact test was used for statistical analysis. ESC lines used were XY1 and XX1. Scale bar represents 5 μm.

Techniques Used: Fluorescence In Situ Hybridization, Expressing

Related Articles

Staining:

Article Title: p38 (Mapk14/11) occupies a regulatory node governing entry into primitive endoderm differentiation during preimplantation mouse embryo development
Article Snippet: .. Please note that owing to the non-specific interaction between microinjected OGDBs and the goat-derived anti-Gata4 antibody (sc-1237), any embryos that had been microinjected and were to be co-immunofluorescently stained for Nanog and Gata4 were done so using the following explicit primary antibody combination (hence the reason why two Nanog and Gata4 primary antibodies are listed; in non-microinjected embryos immuno-stained for Nanog and Gata4, the alternative primary antibody combination was used): mouse-derived anti-Nanog (14-5761-80) and rabbit-derived anti-Gata4 (sc-9053). .. Image analysis/cell counting Individual E4.5 (and E4.0) stage blastocyst cell contributions to specific cell lineages (deduced by presence and/or absence of specific immunofluorescent (IF) signal for the relevant marker protein expression), or within inner- or outer-cell populations, were determined in both experimental and control embryos by inspection of confocal micrograph z-sections using Fluoview v. 1.7.a (Olympus), Imaris (Bitplane) and Image J software.

Incubation:

Article Title: The Transcriptional and Epigenomic Foundations of Ground State Pluripotency
Article Snippet: .. Overnight incubation was performed with Oct3/4 (Santa Cruz sc-5279, c-20), Klf4 (R & D Systems AF3158) or Nanog (E-biosciences 14-5761-80) antibody at 4°C. ..

Generated:

Article Title: An Mll4/COMPASS-Lsd1 epigenetic axis governs enhancer function and pluripotency transition in embryonic stem cells
Article Snippet: .. Antibodies The following antibodies were used in this study: anti-H3K4me1 (generated in-house), anti-H3K4me2 (generated in-house), anti-H3K4me3 (generated in-house), anti-H3K27ac (Cell Signaling, 8173), anti-H3 (generated in-house), anti-Mll3 (generated in-house), anti-Mll4 NT (generated in-house), anti-Mll4 CT (generated in-house), anti-Rbbp5 (Bethyl Laboratories, A300-109A), anti-Oct4 (Santa Cruz Biotechnology, 5279), anti-Nanog (eBioscience, 14-5761-80), anti-Lsd1 (Abcam, 17721), and anti-tubulin (Developmental Studies Hybridoma Bank, E7). .. Immunostaining Cells were grown on 1% gelatin–coated glass coverslips, fixed with 4% paraformaldehyde for 20 min, and blocked with 10% FBS solutions before immunofluorescence staining.

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  • 99
    Thermo Fisher gene exp nanog mm02019550 s1
    BRG1 knockdown in F9 cells using SMARTpool siRNA and OCT4 target genes. (A) SiRNA-mediated knockdown of Brg1 in F9 cells was achieved with about 90% efficiency. (B) Levels of Oct4 were increased, Sox2 levels were decreased, whereas <t>Nanog</t> levels remained unchanged in F9 cells at 48 h posttransfection. (C) OCT4 target genes Oct11 and Fgf4 were upregulated in BRG1-knockdown ES cells at 48 h posttransfection. (D) Oct11 levels were also increased in BRG1-knockdown F9 cells at 72 h posttransfection.
    Gene Exp Nanog Mm02019550 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nanog sirna
    <t>NANOG</t> regulates colorectal cancer cell proliferation through the control of cyclin D1 expression. (A) Growth curve of HCT116 colorectal cancer cells. NANOG <t>siRNA-transfected</t> cells exhibited decreased cell proliferation compared to the control. (B) Quantitative RT-PCR (*P
    Nanog Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BRG1 knockdown in F9 cells using SMARTpool siRNA and OCT4 target genes. (A) SiRNA-mediated knockdown of Brg1 in F9 cells was achieved with about 90% efficiency. (B) Levels of Oct4 were increased, Sox2 levels were decreased, whereas Nanog levels remained unchanged in F9 cells at 48 h posttransfection. (C) OCT4 target genes Oct11 and Fgf4 were upregulated in BRG1-knockdown ES cells at 48 h posttransfection. (D) Oct11 levels were also increased in BRG1-knockdown F9 cells at 72 h posttransfection.

    Journal: BioResearch Open Access

    Article Title: BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

    doi: 10.1089/biores.2013.0047

    Figure Lengend Snippet: BRG1 knockdown in F9 cells using SMARTpool siRNA and OCT4 target genes. (A) SiRNA-mediated knockdown of Brg1 in F9 cells was achieved with about 90% efficiency. (B) Levels of Oct4 were increased, Sox2 levels were decreased, whereas Nanog levels remained unchanged in F9 cells at 48 h posttransfection. (C) OCT4 target genes Oct11 and Fgf4 were upregulated in BRG1-knockdown ES cells at 48 h posttransfection. (D) Oct11 levels were also increased in BRG1-knockdown F9 cells at 72 h posttransfection.

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction analysis TaqMan Assays-on-Demand was used for the detection of the following genes (TaqMan primer ID): Brg1 (Mm01151948_m1), Oct4 (Mm00658129_gH), Sox2 (Mm00488369_s1), Nanog (Mm02019550_s1), Oct11 (Mm00478284_m1), Fgf4 (Mm00438917_m1), Fgf5 (Mm00438919_m1), Gata4 (Mm00484689_m1), Meox1 (Mm00440285_m1), and T (Mm00436877_m1).

    Techniques:

    Time-course analysis was performed by using shRNA directed against Brg1 . (A) Transfection of shRNA against Brg1 showed up to 90% downregulation of Brg1 in ESD3 cells compared with embryonic stem (ES) cells transfected with control shRNA. (B) Transfection of sh Brg1 led to an increase in Oct4 levels at 24 h posttransfection but a decrease in Oct4 levels at 48 and 72 h posttransfection with sh Brg1 , compared with cells transfected with control shRNA. (C) Nanog levels remained elevated for up to 72 h after knockdown of BRG1. (D) siRNA-mediated BRG1 showed 85% knockdown efficiency. (E) Western blot analysis was carried out at 48 h posttransfection using cell lysates prepared from mouse ES cells. BRG1 protein levels were detected by using polyclonal anti-BRG1 (N15) antibody; β-TUBULIN was used as a loading control. (F) Levels of Oct4 and Nanog were increased and Sox2 levels were decreased in ES cells at 48 h posttransfection.

    Journal: BioResearch Open Access

    Article Title: BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

    doi: 10.1089/biores.2013.0047

    Figure Lengend Snippet: Time-course analysis was performed by using shRNA directed against Brg1 . (A) Transfection of shRNA against Brg1 showed up to 90% downregulation of Brg1 in ESD3 cells compared with embryonic stem (ES) cells transfected with control shRNA. (B) Transfection of sh Brg1 led to an increase in Oct4 levels at 24 h posttransfection but a decrease in Oct4 levels at 48 and 72 h posttransfection with sh Brg1 , compared with cells transfected with control shRNA. (C) Nanog levels remained elevated for up to 72 h after knockdown of BRG1. (D) siRNA-mediated BRG1 showed 85% knockdown efficiency. (E) Western blot analysis was carried out at 48 h posttransfection using cell lysates prepared from mouse ES cells. BRG1 protein levels were detected by using polyclonal anti-BRG1 (N15) antibody; β-TUBULIN was used as a loading control. (F) Levels of Oct4 and Nanog were increased and Sox2 levels were decreased in ES cells at 48 h posttransfection.

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction analysis TaqMan Assays-on-Demand was used for the detection of the following genes (TaqMan primer ID): Brg1 (Mm01151948_m1), Oct4 (Mm00658129_gH), Sox2 (Mm00488369_s1), Nanog (Mm02019550_s1), Oct11 (Mm00478284_m1), Fgf4 (Mm00438917_m1), Fgf5 (Mm00438919_m1), Gata4 (Mm00484689_m1), Meox1 (Mm00440285_m1), and T (Mm00436877_m1).

    Techniques: shRNA, Transfection, Western Blot

    RA stimulates SMC and urothelial markers in pluripotent stem cells cultured on silk scaffolds. [ A, B ] Real time RT-PCR analyses of mRNA transcript levels of pluripotency transcription factors (OCT4, Nanog, REX1), SMC contractile genes (SM-MHC, α-actin, SM22α), and urothelial-associated uroplakins (UP) in ESC [A] or iPS cells [B] cultured on fibronectin-coated Group 2 matrices for 14 d in the presence of RA or maintained as spontaneously differentiating controls (C). D0 = naïve, undifferentiated controls. Levels normalized to GAPDH expression. Mean ± SD per data point. (#) = p ≤0.05, in comparison to D0. (*) = p ≤0.05, in comparison to D0 and C conditions.

    Journal: PLoS ONE

    Article Title: Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells

    doi: 10.1371/journal.pone.0056237

    Figure Lengend Snippet: RA stimulates SMC and urothelial markers in pluripotent stem cells cultured on silk scaffolds. [ A, B ] Real time RT-PCR analyses of mRNA transcript levels of pluripotency transcription factors (OCT4, Nanog, REX1), SMC contractile genes (SM-MHC, α-actin, SM22α), and urothelial-associated uroplakins (UP) in ESC [A] or iPS cells [B] cultured on fibronectin-coated Group 2 matrices for 14 d in the presence of RA or maintained as spontaneously differentiating controls (C). D0 = naïve, undifferentiated controls. Levels normalized to GAPDH expression. Mean ± SD per data point. (#) = p ≤0.05, in comparison to D0. (*) = p ≤0.05, in comparison to D0 and C conditions.

    Article Snippet: Expression kits included: murine: uroplakin (UP) 1A, Mm01176597_g1; UP1B, Mm00769504_m1 UP2, Mm00447665_m1; UP3A, Mm00447665_m1, GAPDH, Mm99999915_g1; OCT4, Mm00658129_gH; Nanog, Mm02019550_s1; REX1, Mm01194090_g1; α-actin, Mm00725412_s1; smooth muscle myosin heavy chain (SM-MHC), Mm00443013_m1; SM22α, Mm00441660_m1; human: α-actin, Hs00426835_g1; SM22α, Hs00162558_m1; GAPDH, Hs99999905_m1.

    Techniques: Cell Culture, Quantitative RT-PCR, Expressing

    Fold change in gene expression (qPCR) of Oct4, Sox2 and Nanog in B16 ( A ) and ID8agg ( B ) cells. P values, unpaired t test. Each bar represents fold increase in stemness gene expression in TIC versus respective total cultures, with P value for same directly

    Journal: Signal transduction and targeted therapy

    Article Title: Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer

    doi: 10.1038/sigtrans.2016.30

    Figure Lengend Snippet: Fold change in gene expression (qPCR) of Oct4, Sox2 and Nanog in B16 ( A ) and ID8agg ( B ) cells. P values, unpaired t test. Each bar represents fold increase in stemness gene expression in TIC versus respective total cultures, with P value for same directly

    Article Snippet: Quantitative PCR (qPCR) was conducted using the 7900HT Real-Time PCR System (Applied Biosystems), amplified with Taqman gene expression assays (Applied Biosystems) for mouse nanog (Mm02019550_s1), sox2 (Mm03053810_s1), pou5f1 (oct4, Mm03053917_g1) and rptor (Mm01242613_m1) according to the manufacturer’s instructions with β-actin (Mm02619580_g1) as the internal control.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    NANOG regulates colorectal cancer cell proliferation through the control of cyclin D1 expression. (A) Growth curve of HCT116 colorectal cancer cells. NANOG siRNA-transfected cells exhibited decreased cell proliferation compared to the control. (B) Quantitative RT-PCR (*P

    Journal: Oncology Reports

    Article Title: E-cadherin regulates proliferation of colorectal cancer stem cells through NANOG

    doi: 10.3892/or.2018.6464

    Figure Lengend Snippet: NANOG regulates colorectal cancer cell proliferation through the control of cyclin D1 expression. (A) Growth curve of HCT116 colorectal cancer cells. NANOG siRNA-transfected cells exhibited decreased cell proliferation compared to the control. (B) Quantitative RT-PCR (*P

    Article Snippet: HCT116 cells were seeded in 35-mm dishes and transfected with control siRNA or NANOG siRNA using Lipofectamine 3000 according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Transfection, Quantitative RT-PCR