anti nanog  (Millipore)


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    Anti NANOG
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    Structured Review

    Millipore anti nanog
    Anti NANOG

    https://www.bioz.com/result/anti nanog/product/Millipore
    Average 94 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    anti nanog - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "Continuous exposure of pancreatic cancer cells to dietary bioactive agents does not induce drug resistance unlike chemotherapy"

    Article Title: Continuous exposure of pancreatic cancer cells to dietary bioactive agents does not induce drug resistance unlike chemotherapy

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.157

    Continuous exposure to gemcitabine increases tumorigenicity but continuous exposure to sulforaphane or quercetin reduces it. ( a ) BxPC-3, Bx-GEM, Bx-Q and Bx-SF cells were seeded at a low density (2000 cells/well) in 6-well plates. After 2 weeks, cells were Coomassie-stained and colonies containing more than 50 cells were counted under a dissecting microscope. The survival fraction and representative photographs of colonies (first generation) are presented on the left. For second-generation colony formation, an equal amount of living cells from first-generation colonies were collected and 2000 cells per well were re-seeded. The colony formation was analyzed as described above and is presented on the right. ( b ) Cells were cultured to 90% confluence before the cell layer was scratched with the tip of a 10- μ l pipette. Closure of the wounded region was evaluated 24 h after scratching by microscopy at × 100 magnification. For quantification of the scratched area, the percentage of the gap area was evaluated and calculated by TScratch software (diagram below photographs). ( c ) Cells were seeded in 6-well plates, followed by exposure to NH Osteo-Diff medium to induce osteocytic differentiation. Fourteen days later, the cells were stained with BCIP/NBT substrate for alkaline phosphatases, expressed by cells differentiated into osteocytes, which appear dark. Representative images at × 200 magnification are shown. ( d ) Proteins were harvested and the expression of EpCAM, Nanog, Twist2 and E-cadherin was measured by western blot analysis. β -Actin was used as a loading control. Three independent experiments were performed at least in triplicates and the data are presented as means ±S.D. * P
    Figure Legend Snippet: Continuous exposure to gemcitabine increases tumorigenicity but continuous exposure to sulforaphane or quercetin reduces it. ( a ) BxPC-3, Bx-GEM, Bx-Q and Bx-SF cells were seeded at a low density (2000 cells/well) in 6-well plates. After 2 weeks, cells were Coomassie-stained and colonies containing more than 50 cells were counted under a dissecting microscope. The survival fraction and representative photographs of colonies (first generation) are presented on the left. For second-generation colony formation, an equal amount of living cells from first-generation colonies were collected and 2000 cells per well were re-seeded. The colony formation was analyzed as described above and is presented on the right. ( b ) Cells were cultured to 90% confluence before the cell layer was scratched with the tip of a 10- μ l pipette. Closure of the wounded region was evaluated 24 h after scratching by microscopy at × 100 magnification. For quantification of the scratched area, the percentage of the gap area was evaluated and calculated by TScratch software (diagram below photographs). ( c ) Cells were seeded in 6-well plates, followed by exposure to NH Osteo-Diff medium to induce osteocytic differentiation. Fourteen days later, the cells were stained with BCIP/NBT substrate for alkaline phosphatases, expressed by cells differentiated into osteocytes, which appear dark. Representative images at × 200 magnification are shown. ( d ) Proteins were harvested and the expression of EpCAM, Nanog, Twist2 and E-cadherin was measured by western blot analysis. β -Actin was used as a loading control. Three independent experiments were performed at least in triplicates and the data are presented as means ±S.D. * P

    Techniques Used: Staining, Microscopy, Cell Culture, Transferring, Software, Expressing, Western Blot

    2) Product Images from "Characterization of Bovine Induced Pluripotent Stem Cells by Lentiviral Transduction of Reprogramming Factor Fusion Proteins"

    Article Title: Characterization of Bovine Induced Pluripotent Stem Cells by Lentiviral Transduction of Reprogramming Factor Fusion Proteins

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.3723

    Expression of stem cell marker genes and methylation status in the promoter regions of Oct4 and Nanog. BEF1 and BEF2 represented different Un-infected bovine embryonic fibroblast lines; BiPS1 and BiPS2 represented different 10 th passage bovine iPS cell lines. (A) The exogenous and endogenous gene expression was analyzed by RT-PCR. (B) The endogenous Oct4 and Nanog expression was analyzed by quantitative RT-PCR. (C) The exogenous and endogenous Oct4 expression was analyzed by western blotting. Exogenous Oct4 protein (70KDa) was composed of Oct4 protein (43KDa) and EGFP (27KDa). (D) Bisulfite sequencing analysis of the Nanog and Oct4 promoters is shown. White and black circles indicate unmethylated and methylated CpG, respectively.
    Figure Legend Snippet: Expression of stem cell marker genes and methylation status in the promoter regions of Oct4 and Nanog. BEF1 and BEF2 represented different Un-infected bovine embryonic fibroblast lines; BiPS1 and BiPS2 represented different 10 th passage bovine iPS cell lines. (A) The exogenous and endogenous gene expression was analyzed by RT-PCR. (B) The endogenous Oct4 and Nanog expression was analyzed by quantitative RT-PCR. (C) The exogenous and endogenous Oct4 expression was analyzed by western blotting. Exogenous Oct4 protein (70KDa) was composed of Oct4 protein (43KDa) and EGFP (27KDa). (D) Bisulfite sequencing analysis of the Nanog and Oct4 promoters is shown. White and black circles indicate unmethylated and methylated CpG, respectively.

    Techniques Used: Expressing, Marker, Methylation, Infection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Methylation Sequencing

    Expression of pluripotent markers in the 10 th passage bovine iPS cells. scale bar: 100 μm. (A1) Bovine iPS cells were used as pluripotent marker Oct4 analysis. (A2) The immunofluorescence staining of marker Oct4 is shown. (A3) EGFP expression is shown. (A4) The merge is shown. (B1) Bovine iPS cells were used as pluripotent marker Nanog analysis. (B2) The immunofluorescence staining of marker Nanog is shown. (B3) EGFP expression is shown. (B4) The merge is shown. (C1) Bovine iPS cells were used as pluripotent marker SSEA1 analysis. (C2) The immunofluorescence staining of marker SSEA1 is shown. (C3) EGFP expression is shown. (C4) The merge is shown.
    Figure Legend Snippet: Expression of pluripotent markers in the 10 th passage bovine iPS cells. scale bar: 100 μm. (A1) Bovine iPS cells were used as pluripotent marker Oct4 analysis. (A2) The immunofluorescence staining of marker Oct4 is shown. (A3) EGFP expression is shown. (A4) The merge is shown. (B1) Bovine iPS cells were used as pluripotent marker Nanog analysis. (B2) The immunofluorescence staining of marker Nanog is shown. (B3) EGFP expression is shown. (B4) The merge is shown. (C1) Bovine iPS cells were used as pluripotent marker SSEA1 analysis. (C2) The immunofluorescence staining of marker SSEA1 is shown. (C3) EGFP expression is shown. (C4) The merge is shown.

    Techniques Used: Expressing, Marker, Immunofluorescence, Staining

    3) Product Images from "Differentiation defect in neural crest-derived smooth muscle cells in patients with aortopathy associated with bicuspid aortic valves"

    Article Title: Differentiation defect in neural crest-derived smooth muscle cells in patients with aortopathy associated with bicuspid aortic valves

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2016.06.045

    iPSCs from BAV/TAA patient are pluripotent and can differentiate to NCSCs. (a) 3-D reconstruction of a Computed Tomography (CT) scan of the aorta in a BAV/TAA patient. (b) Morphology of PBMCs and iPSCs from BAV/TAA. (c) Karyotyping of iPSCs from BAV/TAA. (d) Immunostaining of OCT4, SOX2, NANOG, SSEA4, TRA-1-60 and TRA-1-81 in the BAV/TAA iPSC colonies. The scale bars represent 50 μm. (e) H E staining of teratomas derived from the BAV/TAA iPSCs injected into the flanks of NOD/SCID mice. The scale bars represent 50 μm. (f) Morphological change of NCSCs from day 0 to day 10 in cell culture. The scale bars represent 500 μm. (g) Relative gene expression of NCSC marker genes SLUG , SOX9 , PAX3 in iPSCs, NCSCs and PMCs assessed by quantitative PCR (qPCR). Each group contains two cell lines. Experiments were repeated three times. (h) Immunofluorescence staining of NCSC markers HNK1 and P75 of the NCSCs. The scale bars represent 100 μm. (i) Flow cytometry of HNK1 and P75 of the NCSCs. PBMCs: peripheral blood mononuclear cell. iPSCs: induced pluripotent stem cells. NCSCs: neural crest stem cells. PMCs: paraxial mesoderm cells. H E: hematoxylin and eosin. BAV/TAA: bicuspid aortic valve/ thoracic aortic aneurysm. Ctrl: Control. Data is represented as mean ± SEM. See also Fig. S1.
    Figure Legend Snippet: iPSCs from BAV/TAA patient are pluripotent and can differentiate to NCSCs. (a) 3-D reconstruction of a Computed Tomography (CT) scan of the aorta in a BAV/TAA patient. (b) Morphology of PBMCs and iPSCs from BAV/TAA. (c) Karyotyping of iPSCs from BAV/TAA. (d) Immunostaining of OCT4, SOX2, NANOG, SSEA4, TRA-1-60 and TRA-1-81 in the BAV/TAA iPSC colonies. The scale bars represent 50 μm. (e) H E staining of teratomas derived from the BAV/TAA iPSCs injected into the flanks of NOD/SCID mice. The scale bars represent 50 μm. (f) Morphological change of NCSCs from day 0 to day 10 in cell culture. The scale bars represent 500 μm. (g) Relative gene expression of NCSC marker genes SLUG , SOX9 , PAX3 in iPSCs, NCSCs and PMCs assessed by quantitative PCR (qPCR). Each group contains two cell lines. Experiments were repeated three times. (h) Immunofluorescence staining of NCSC markers HNK1 and P75 of the NCSCs. The scale bars represent 100 μm. (i) Flow cytometry of HNK1 and P75 of the NCSCs. PBMCs: peripheral blood mononuclear cell. iPSCs: induced pluripotent stem cells. NCSCs: neural crest stem cells. PMCs: paraxial mesoderm cells. H E: hematoxylin and eosin. BAV/TAA: bicuspid aortic valve/ thoracic aortic aneurysm. Ctrl: Control. Data is represented as mean ± SEM. See also Fig. S1.

    Techniques Used: Computed Tomography, Immunostaining, Staining, Derivative Assay, Injection, Mouse Assay, Cell Culture, Expressing, Marker, Real-time Polymerase Chain Reaction, Immunofluorescence, Flow Cytometry, Cytometry

    4) Product Images from "miR-125b Regulates the Early Steps of ESC Differentiation through Dies1 in a TGF-Independent Manner"

    Article Title: miR-125b Regulates the Early Steps of ESC Differentiation through Dies1 in a TGF-Independent Manner

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms140713482

    Effects of miR-125 ectopic expression in vitro and in vivo . ( A ) Analysis of stemness markers (Oct3/4, Nanog, Klf2 and Klf5) in undifferentiated ESCs transfected with pre-miR-125b or with control pre-miR (pre-miR-ctrl). The fold change is calculated by assigning the arbitrary value, one, to the amount found in cells transfected with control pre-miRNA; ( B ) Immunofluorescence analysis of four-day differentiated SFEBs upon miR-125b overexpression. Markers of pluripotency (Oct3/4, Nanog) and neuroectoderm (Sox1) are shown. Scale bar: 50 μm; ( C ) qPCR analysis of stemness (Oct3/4 and Nanog) and neuroectodermal (Pax6) markers in differentiating ESCs upon miR-125b overexpression. The fold change is calculated by assigning the arbitrary value, one, to the time point showing the highest amount of the indicated mRNA; ( D ) The level of active ERK (P-ERK) were analyzed by Western blot in cells transfected with pre-miR-125b or the control pre-miR after four days of differentiation—the experiment shown in the Figure is representative of two independent experiments; ( E ) The level of the epiblast marker, Fgf5, was measured by qPCR in undifferentiated ESCs and during differentiation upon pre-miR transfection. The epiblast markers, Cerberus and Dnmt3b, were measured at four days of SFEB differentiation in the cells transfected with the indicated pre-miR. The fold change is calculated as indicated in ( C ); ( F ) ChIP-qPCR analysis was performed on chromatin from ESCs transfected with the indicated pre-miR and induced differentiation for four days as SFEBs. The graphs show the methylation state of histone H3 on the promoters of pluripotency (Nanog and Klf2) and epiblast (Fgf5) markers. Data are expressed as fold enrichment relative to the control; ( G ) Immunodeficient mice were injected with ESCs transfected with the pre-miR-125b (right side) and ctrl pre-miR (left side) after three days of differentiation in vitro (left panel). Teratomas generated by ESCs overexpressing miR-125b were explanted after one month, and the tissues were analyzed after eosin-hematoxylin staining (right panels) ( * p
    Figure Legend Snippet: Effects of miR-125 ectopic expression in vitro and in vivo . ( A ) Analysis of stemness markers (Oct3/4, Nanog, Klf2 and Klf5) in undifferentiated ESCs transfected with pre-miR-125b or with control pre-miR (pre-miR-ctrl). The fold change is calculated by assigning the arbitrary value, one, to the amount found in cells transfected with control pre-miRNA; ( B ) Immunofluorescence analysis of four-day differentiated SFEBs upon miR-125b overexpression. Markers of pluripotency (Oct3/4, Nanog) and neuroectoderm (Sox1) are shown. Scale bar: 50 μm; ( C ) qPCR analysis of stemness (Oct3/4 and Nanog) and neuroectodermal (Pax6) markers in differentiating ESCs upon miR-125b overexpression. The fold change is calculated by assigning the arbitrary value, one, to the time point showing the highest amount of the indicated mRNA; ( D ) The level of active ERK (P-ERK) were analyzed by Western blot in cells transfected with pre-miR-125b or the control pre-miR after four days of differentiation—the experiment shown in the Figure is representative of two independent experiments; ( E ) The level of the epiblast marker, Fgf5, was measured by qPCR in undifferentiated ESCs and during differentiation upon pre-miR transfection. The epiblast markers, Cerberus and Dnmt3b, were measured at four days of SFEB differentiation in the cells transfected with the indicated pre-miR. The fold change is calculated as indicated in ( C ); ( F ) ChIP-qPCR analysis was performed on chromatin from ESCs transfected with the indicated pre-miR and induced differentiation for four days as SFEBs. The graphs show the methylation state of histone H3 on the promoters of pluripotency (Nanog and Klf2) and epiblast (Fgf5) markers. Data are expressed as fold enrichment relative to the control; ( G ) Immunodeficient mice were injected with ESCs transfected with the pre-miR-125b (right side) and ctrl pre-miR (left side) after three days of differentiation in vitro (left panel). Teratomas generated by ESCs overexpressing miR-125b were explanted after one month, and the tissues were analyzed after eosin-hematoxylin staining (right panels) ( * p

    Techniques Used: Expressing, In Vitro, In Vivo, Transfection, Immunofluorescence, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Marker, Chromatin Immunoprecipitation, Methylation, Mouse Assay, Injection, Generated, Staining

    5) Product Images from "5-Aminoimidazole-4-carboxyamide Ribonucleoside Induces G1/S Arrest and Nanog Downregulation via p53 and Enhances Erythroid Differentiation"

    Article Title: 5-Aminoimidazole-4-carboxyamide Ribonucleoside Induces G1/S Arrest and Nanog Downregulation via p53 and Enhances Erythroid Differentiation

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.778

    AICAR treatment down-regulates stability of Nanog protein. (A) R1 mES cells were preincubated with or without AICAR (0.5 mM) for 6h, and then cycloheximide (CHX, 50mg/ml) for indicated hours. Lysates were analyzed by immunoblotting for Nanog and β-tubulin
    Figure Legend Snippet: AICAR treatment down-regulates stability of Nanog protein. (A) R1 mES cells were preincubated with or without AICAR (0.5 mM) for 6h, and then cycloheximide (CHX, 50mg/ml) for indicated hours. Lysates were analyzed by immunoblotting for Nanog and β-tubulin

    Techniques Used:

    AICAR suppresses Nanog expression in and proliferation of H9 hES cells. (A) H9 hES cells were treated with or without AICAR (0.5 mM) for 1d. Cell lysates were analyzed for p53, Nanog, Oct4, and with β-tubulin as a loading control, by immunoblotting.
    Figure Legend Snippet: AICAR suppresses Nanog expression in and proliferation of H9 hES cells. (A) H9 hES cells were treated with or without AICAR (0.5 mM) for 1d. Cell lysates were analyzed for p53, Nanog, Oct4, and with β-tubulin as a loading control, by immunoblotting.

    Techniques Used: Expressing

    6) Product Images from "Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells"

    Article Title: Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130332

    hESCs growing eiMEFs expressed stem cell markers. A, Immunostaining showed pluripotent markers of NANOG, OCT4, SSEA4 and TRA-1-81 expressed in hESCs cultured on eiMEFs. Scar bar, 100 μm. B, FACS assay showed that during consecutive incubation (day 7 (passage 46), day 14 (passage 47), day 21 (passage 48) and day 28 (passage 49), high percentage of cells positive for OCT4 (98.3%±2.55%, 94.3%±2.20%, 96.2%±2.55%, 97.0%±2.95%), NANOG (97.5%±2.15%, 94.4%±1.65%, 95.5%±1.98%, 98.0%±1.95%), SSEA4 (, 97.9%±2.40%, 93.4%±2.10%, 97.4%±2.54%, 97.4%±2.36%) and TRA-1-81 (98.6%±3.12%, 96.1%±1.85%, 96.3%±2.35%, 97.6%±2.88%) stablely expressed in hESCs cultured eiMEFs.
    Figure Legend Snippet: hESCs growing eiMEFs expressed stem cell markers. A, Immunostaining showed pluripotent markers of NANOG, OCT4, SSEA4 and TRA-1-81 expressed in hESCs cultured on eiMEFs. Scar bar, 100 μm. B, FACS assay showed that during consecutive incubation (day 7 (passage 46), day 14 (passage 47), day 21 (passage 48) and day 28 (passage 49), high percentage of cells positive for OCT4 (98.3%±2.55%, 94.3%±2.20%, 96.2%±2.55%, 97.0%±2.95%), NANOG (97.5%±2.15%, 94.4%±1.65%, 95.5%±1.98%, 98.0%±1.95%), SSEA4 (, 97.9%±2.40%, 93.4%±2.10%, 97.4%±2.54%, 97.4%±2.36%) and TRA-1-81 (98.6%±3.12%, 96.1%±1.85%, 96.3%±2.35%, 97.6%±2.88%) stablely expressed in hESCs cultured eiMEFs.

    Techniques Used: Immunostaining, Cell Culture, FACS, Incubation

    7) Product Images from "Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells *"

    Article Title: Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.077156

    Dies1 suppression maintains ESCs in undifferentiated state in the absence of LIF. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. This marker of ESC undifferentiated state was clearly observed in more than 50% of the colonies of Dies1 KD cells, although only about 10% of the NS shRNA transfected colonies were AP-positive. The experiments were done in triplicate. *, p ≤ 0.001. B , Dies1 KD ESCs grown 13 days in neural differentiation medium still expressed Oct3/4 and Nanog, although NS-transfected cells are completely negative for these master genes of stemness. Scale bars , 250 μm. C , mRNA levels of Oct3/4 and Nanog were measured in cells cultured as in A . The two mRNAs were strongly reduced in NS shRNA-transfected cells ( black bars ), although they were clearly detectable at 7 and 13 days after the induction of differentiation in Dies1 KD cells ( gray bars ). D , expression of a Dies1 cDNA resistant to the shRNA used to silence endogenous Dies1 rescued the phenotype of Dies1 KD ESCs. In cells expressing recombinant Dies1 ( gray bars ), Oct3/4 mRNA was expressed at the same levels of controls ( black bars ). E , as in D , Oct3/4 protein levels are rescued by Dies1 overexpression.
    Figure Legend Snippet: Dies1 suppression maintains ESCs in undifferentiated state in the absence of LIF. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. This marker of ESC undifferentiated state was clearly observed in more than 50% of the colonies of Dies1 KD cells, although only about 10% of the NS shRNA transfected colonies were AP-positive. The experiments were done in triplicate. *, p ≤ 0.001. B , Dies1 KD ESCs grown 13 days in neural differentiation medium still expressed Oct3/4 and Nanog, although NS-transfected cells are completely negative for these master genes of stemness. Scale bars , 250 μm. C , mRNA levels of Oct3/4 and Nanog were measured in cells cultured as in A . The two mRNAs were strongly reduced in NS shRNA-transfected cells ( black bars ), although they were clearly detectable at 7 and 13 days after the induction of differentiation in Dies1 KD cells ( gray bars ). D , expression of a Dies1 cDNA resistant to the shRNA used to silence endogenous Dies1 rescued the phenotype of Dies1 KD ESCs. In cells expressing recombinant Dies1 ( gray bars ), Oct3/4 mRNA was expressed at the same levels of controls ( black bars ). E , as in D , Oct3/4 protein levels are rescued by Dies1 overexpression.

    Techniques Used: Stable Transfection, Transfection, Staining, Marker, shRNA, Cell Culture, Expressing, Recombinant, Over Expression

    Inhibition of Nodal/Activin signaling pathway masks the effects of Dies1 silencing in ESCs. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. Cells were treated with SB-431542 or with DMSO as a control. % of colonies AP-positive was calculated in triplicate experiments. *, p ≤ 0.001. B , Western blot analysis of Oct3/4 and Nanog in the cells treated as described in A .
    Figure Legend Snippet: Inhibition of Nodal/Activin signaling pathway masks the effects of Dies1 silencing in ESCs. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. Cells were treated with SB-431542 or with DMSO as a control. % of colonies AP-positive was calculated in triplicate experiments. *, p ≤ 0.001. B , Western blot analysis of Oct3/4 and Nanog in the cells treated as described in A .

    Techniques Used: Inhibition, Stable Transfection, Transfection, Staining, Western Blot

    8) Product Images from "Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells"

    Article Title: Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109588

    Derivation of iPSCs using an inducible lentiviral system. ( A ) Schematic representation of iPSC generation. ( B ) RT-PCR analysis of exogenous Yamanaka factors, ES pluripotent markers in fully and partially reprogrammed iPSCs (iPS-Dox6d, iPS-Dox12d). Parental MEFs and ESCs were used as negative and positive controls. ( C ) Immunofluorescence analysis of pluripotent markers, Nanog and SSEA-1 (Red). Oct4-GFP + cells (Green) represented fully reprogramming iPSCs. Scale bar 50 µm. ( D ) Western blot analysis confirmed the pluripotent properties of iPSCs expressing Oct4, Nanog and Sox2. Parental MEF and ES cells were used as negative and positive controls. ( E ) Teratoma formation of iPSCs transplanted into immunodeficient mice. After 4 weeks, iPSC-derived tumors were sectioned and stained with hematoxylin and eosin. Shown were neural (ectoderm), muscle (mesoderm) and glandular (endoderm) tissues from left to right. Scale bar 100 µm. ( F ) Karyotyping analysis showing normal karyotyping of iPSCs after passage 10.
    Figure Legend Snippet: Derivation of iPSCs using an inducible lentiviral system. ( A ) Schematic representation of iPSC generation. ( B ) RT-PCR analysis of exogenous Yamanaka factors, ES pluripotent markers in fully and partially reprogrammed iPSCs (iPS-Dox6d, iPS-Dox12d). Parental MEFs and ESCs were used as negative and positive controls. ( C ) Immunofluorescence analysis of pluripotent markers, Nanog and SSEA-1 (Red). Oct4-GFP + cells (Green) represented fully reprogramming iPSCs. Scale bar 50 µm. ( D ) Western blot analysis confirmed the pluripotent properties of iPSCs expressing Oct4, Nanog and Sox2. Parental MEF and ES cells were used as negative and positive controls. ( E ) Teratoma formation of iPSCs transplanted into immunodeficient mice. After 4 weeks, iPSC-derived tumors were sectioned and stained with hematoxylin and eosin. Shown were neural (ectoderm), muscle (mesoderm) and glandular (endoderm) tissues from left to right. Scale bar 100 µm. ( F ) Karyotyping analysis showing normal karyotyping of iPSCs after passage 10.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Expressing, Mouse Assay, Derivative Assay, Staining

    9) Product Images from "Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells"

    Article Title: Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2011.219

    Immunostaining. Fourteen days after embryoid body (EB) formation, cells were subjected to immunostaining with Oct3/4, Nanog, SSEA-4 and TRA-1-60 antibodies. Cells cultured without Activin A were negative for all of the antibodies, while cells were positive
    Figure Legend Snippet: Immunostaining. Fourteen days after embryoid body (EB) formation, cells were subjected to immunostaining with Oct3/4, Nanog, SSEA-4 and TRA-1-60 antibodies. Cells cultured without Activin A were negative for all of the antibodies, while cells were positive

    Techniques Used: Immunostaining, Cell Culture

    10) Product Images from "Identification and characterization of epithelial cells derived from human ovarian follicular fluid"

    Article Title: Identification and characterization of epithelial cells derived from human ovarian follicular fluid

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0004-6

    Real-time PCR analysis of human skin fibroblast, human ovarian cortex, epithelial cells cultured for 1 week, and epithelial cells cultured for 1 month. (A) Gene expression of transcripts for pluripotency markers showed higher expression levels for OCT4, Nanog, TERT, and Sox-2 in earlier epithelial cells, and decreased after 1 month of culturing. Human ovarian cortex also showed the presence of transcripts for pluripotency markers. (B) Transcripts for germ cell markers DAZL, STELLA, BLIMP1, STRA8, VAZA, GDF9, SCP3, ZPA, and ZPC in epithelial cells increased significantly after culturing for 1 month. Comparatively, human ovarian cortex also showed different expression levels of transcripts for germ cell markers. Human skin fibroblast cells served as negative controls, and 18s RNA as a house-keeping control gene was detected in all samples. Data represent mean ± standard error of three independent experiments, * P
    Figure Legend Snippet: Real-time PCR analysis of human skin fibroblast, human ovarian cortex, epithelial cells cultured for 1 week, and epithelial cells cultured for 1 month. (A) Gene expression of transcripts for pluripotency markers showed higher expression levels for OCT4, Nanog, TERT, and Sox-2 in earlier epithelial cells, and decreased after 1 month of culturing. Human ovarian cortex also showed the presence of transcripts for pluripotency markers. (B) Transcripts for germ cell markers DAZL, STELLA, BLIMP1, STRA8, VAZA, GDF9, SCP3, ZPA, and ZPC in epithelial cells increased significantly after culturing for 1 month. Comparatively, human ovarian cortex also showed different expression levels of transcripts for germ cell markers. Human skin fibroblast cells served as negative controls, and 18s RNA as a house-keeping control gene was detected in all samples. Data represent mean ± standard error of three independent experiments, * P

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

    Stem cell characteristics of follicular fluid-derived epithelial cells. (A) Typical cell growth curve of epithelial cells after seeding 2.5 × 10 4 cells in each well of 24-well culture plates, compared with granulosa cells. (B) Cell-surface expression of DDX4 in epithelial cells was detected by fluorescence-activated cell sorting analysis after 14 days of propagation. (C), (D), (E), (F) Assessment of epithelial cells proliferation by dual detection of DDX4 expression (green) and bromodeoxyuridine (BrdU) incorporation (red) in vitro cultures. (G), (H) Part of the epithelial cells was OCT4-positive or NANOG-positive. (I) Double staining of OCT4 and cytokeratin 18 in epithelial cells. Scale bars: 100 μm (C, D, E, F, G, H) and 20 μm (I) . DAPI, 4′,6-diamidino-2-phenylindole.
    Figure Legend Snippet: Stem cell characteristics of follicular fluid-derived epithelial cells. (A) Typical cell growth curve of epithelial cells after seeding 2.5 × 10 4 cells in each well of 24-well culture plates, compared with granulosa cells. (B) Cell-surface expression of DDX4 in epithelial cells was detected by fluorescence-activated cell sorting analysis after 14 days of propagation. (C), (D), (E), (F) Assessment of epithelial cells proliferation by dual detection of DDX4 expression (green) and bromodeoxyuridine (BrdU) incorporation (red) in vitro cultures. (G), (H) Part of the epithelial cells was OCT4-positive or NANOG-positive. (I) Double staining of OCT4 and cytokeratin 18 in epithelial cells. Scale bars: 100 μm (C, D, E, F, G, H) and 20 μm (I) . DAPI, 4′,6-diamidino-2-phenylindole.

    Techniques Used: Derivative Assay, Expressing, Fluorescence, FACS, BrdU Incorporation Assay, In Vitro, Double Staining

    11) Product Images from "Human amniotic epithelial cells can differentiate into granulosa cells and restore folliculogenesis in a mouse model of chemotherapy-induced premature ovarian failure"

    Article Title: Human amniotic epithelial cells can differentiate into granulosa cells and restore folliculogenesis in a mouse model of chemotherapy-induced premature ovarian failure

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt335

    Human amniotic epithelial cells (hAECs) did not express germ-cell-specific genes. (A) Quantitative PCR was used to analyze germ-cell-specific expression in hAECs. CT values were expressed as a percentage of 18S RNA (18S = 100%) and used to calculate mean normalized expression relative to 18S. Results are shown as mean and standard deviation of three experiments. (B) Immunofluorescence analysis of germ-cell-specific genes in hAECs. Note that hAECs expressed OCT4 (red), NANOG (green) and CD117 (green), but did not express DAZL, STELLA or VASA. DAPI staining for nuclei. Scale bars = 50 μm.
    Figure Legend Snippet: Human amniotic epithelial cells (hAECs) did not express germ-cell-specific genes. (A) Quantitative PCR was used to analyze germ-cell-specific expression in hAECs. CT values were expressed as a percentage of 18S RNA (18S = 100%) and used to calculate mean normalized expression relative to 18S. Results are shown as mean and standard deviation of three experiments. (B) Immunofluorescence analysis of germ-cell-specific genes in hAECs. Note that hAECs expressed OCT4 (red), NANOG (green) and CD117 (green), but did not express DAZL, STELLA or VASA. DAPI staining for nuclei. Scale bars = 50 μm.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Immunofluorescence, Staining

    12) Product Images from "A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways"

    Article Title: A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways

    Journal: Scientific Reports

    doi: 10.1038/srep25104

    GoST cells upregulate expression of p27, but retain expression of core pluripotency markers compared to ES cells. ( A ) Real-Time PCR analysis of CDK inhibitor markers. Upon GoST induction, expression of p15 , p16 , p21 and p27 was increased. ( B ) Western blot analysis of p27. Upon GoST induction, expression of p27 was increased. ( C ) Immunofluorescence of p27 (red). DNA was counterstained with DAPI (blue). Upon GoST induction, expression of p27 was increased within cells growing in colonies. Scalebar = 50 μm. ( D ) Real-Time PCR analysis of core pluripotency markers. Upon GoST induction, expression of core pluripotency markers remained mostly unchanged while expression of Nanog and Tbx3 was increased. ( E ) Immunofluorescence of Nanog, Oct4, Sox2 and ALP (red). DNA was counterstained with DAPI (blue). Upon GoST induction, cells continued to express Nanog, Oct4, Sox2 and ALP. Scalebar = 50 μm. ( F ) Western blot analysis of Nanog, Oct4 and Sox2. Upon GoST induction, expression of Nanog, Oct4 and Sox2 showed only a slight decrease and remained at similar levels. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Western blot data were normalized to α-Tubulin and generated from three independent experiments. Error bars correspond to S.E.M. Two stars represent p
    Figure Legend Snippet: GoST cells upregulate expression of p27, but retain expression of core pluripotency markers compared to ES cells. ( A ) Real-Time PCR analysis of CDK inhibitor markers. Upon GoST induction, expression of p15 , p16 , p21 and p27 was increased. ( B ) Western blot analysis of p27. Upon GoST induction, expression of p27 was increased. ( C ) Immunofluorescence of p27 (red). DNA was counterstained with DAPI (blue). Upon GoST induction, expression of p27 was increased within cells growing in colonies. Scalebar = 50 μm. ( D ) Real-Time PCR analysis of core pluripotency markers. Upon GoST induction, expression of core pluripotency markers remained mostly unchanged while expression of Nanog and Tbx3 was increased. ( E ) Immunofluorescence of Nanog, Oct4, Sox2 and ALP (red). DNA was counterstained with DAPI (blue). Upon GoST induction, cells continued to express Nanog, Oct4, Sox2 and ALP. Scalebar = 50 μm. ( F ) Western blot analysis of Nanog, Oct4 and Sox2. Upon GoST induction, expression of Nanog, Oct4 and Sox2 showed only a slight decrease and remained at similar levels. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Western blot data were normalized to α-Tubulin and generated from three independent experiments. Error bars correspond to S.E.M. Two stars represent p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, ALP Assay, Generated

    13) Product Images from "Ectopic expression of GATA6 bypasses requirement for Grb2 in primitive endoderm formation"

    Article Title: Ectopic expression of GATA6 bypasses requirement for Grb2 in primitive endoderm formation

    Journal: Developmental Dynamics

    doi: 10.1002/dvdy.22447

    Failure of primitive endoderm epithelial layer formation in Grb2-null embryos. Embryos enclosed in uterine horns from timed matings of Grb2 (+/−) parents were harvested, fixed, and embedded in paraffin. Adjacent sections were immunostained for Nanog, Dab2, and GATA4. Embryos containing Dab2 and GATA4-positive endoderm cells are assigned as either wildtype or Grb2 heterozygous. Abnormal embryos lacking endoderm markers were presumptive Grb2 (−/−). A : Representative E4.5 implanted embryos from timed matings between Grb2 (+/−) parents are shown. Littermates are compared between a wildtype or Grb2 heterozygous and a presumptive Grb2 (−/−) embryo. B : Representative E5.5 embryos from timed matings between Grb2 (+/−) parents are shown, comparing Dab2 and GATA4 immunostaining of a wildtype or Grb2 heterozygous with a presumptive Grb2 (−/−) embryo. C : Representative cytokeratin 8 immunostaining of E5.5 embryos from timed matings between Grb2 (+/−) parents are shown, comparing a wildtype or Grb2 heterozygous with a presumptive Grb2 (−/−) embryo. D : Representative wildtype or Grb2 heterozygous and a presumptive Grb2 (−/−) E5.5 embryos are shown, of immunofluorescence staining of Dab2, cytokeratin 8 (CK), Nanog, and DAPI. E : Representative Dab2 immunostaining of E4.5 and E5.5 implanted, presumptive GATA6-null embryos from timed matings between GATA6 (+/−) parents are shown for comparison. Scale bars are provided next to the images.
    Figure Legend Snippet: Failure of primitive endoderm epithelial layer formation in Grb2-null embryos. Embryos enclosed in uterine horns from timed matings of Grb2 (+/−) parents were harvested, fixed, and embedded in paraffin. Adjacent sections were immunostained for Nanog, Dab2, and GATA4. Embryos containing Dab2 and GATA4-positive endoderm cells are assigned as either wildtype or Grb2 heterozygous. Abnormal embryos lacking endoderm markers were presumptive Grb2 (−/−). A : Representative E4.5 implanted embryos from timed matings between Grb2 (+/−) parents are shown. Littermates are compared between a wildtype or Grb2 heterozygous and a presumptive Grb2 (−/−) embryo. B : Representative E5.5 embryos from timed matings between Grb2 (+/−) parents are shown, comparing Dab2 and GATA4 immunostaining of a wildtype or Grb2 heterozygous with a presumptive Grb2 (−/−) embryo. C : Representative cytokeratin 8 immunostaining of E5.5 embryos from timed matings between Grb2 (+/−) parents are shown, comparing a wildtype or Grb2 heterozygous with a presumptive Grb2 (−/−) embryo. D : Representative wildtype or Grb2 heterozygous and a presumptive Grb2 (−/−) E5.5 embryos are shown, of immunofluorescence staining of Dab2, cytokeratin 8 (CK), Nanog, and DAPI. E : Representative Dab2 immunostaining of E4.5 and E5.5 implanted, presumptive GATA6-null embryos from timed matings between GATA6 (+/−) parents are shown for comparison. Scale bars are provided next to the images.

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Reduced proliferation of Grb2-null embryos. E4.5 and E5.5 embryos enclosed in uterine horns from timed matings of Grb2 (+/−) parents were harvested, fixed, and embedded in paraffin. Adjacent sections were stained with markers for Nanog, Dab2, and GATA4 to assign genotypes to be either wildtype or Grb2 heterozygous, or presumptive Grb2 (−/−). Examples of adjacent sections of a wildtype and a Grb2-null embryo stained with either ki67 or activated/cleaved caspase-3 are shown: A : E4.5; B : E5.5.
    Figure Legend Snippet: Reduced proliferation of Grb2-null embryos. E4.5 and E5.5 embryos enclosed in uterine horns from timed matings of Grb2 (+/−) parents were harvested, fixed, and embedded in paraffin. Adjacent sections were stained with markers for Nanog, Dab2, and GATA4 to assign genotypes to be either wildtype or Grb2 heterozygous, or presumptive Grb2 (−/−). Examples of adjacent sections of a wildtype and a Grb2-null embryo stained with either ki67 or activated/cleaved caspase-3 are shown: A : E4.5; B : E5.5.

    Techniques Used: Staining

    Retinoic acid induces differentiation of ES cells in cultures. Following withdrawal of LIF from medium or addition of retinoic acid (1 μM) for 4 days in culture, the differentiation of RW4 wildtype and Grb2 (−/−) ES cells was monitored by the loss of pluripotent markers Oct3/4 and Nanog, and the induction of endoderm lineage markers Dab2, GATA4, and GATA6, by both immunofluorescence microscopy and Western blots. A: Double immunostaining of Oct3/4 and Nanog was performed in RW4 wildtype and Grb2 (−/−) Es cells with or without treatment with retinoic acid. DAPI staining reveals the nuclei. B : The loss of Oct3/4 and Nanog expression following retinoic acid treatment was assayed by Western blots. C : Double immunostaining of Dab2 and GATA4, Dab2 and GATA6, or Dab2 and Nanog, was performed in RW4 wildtype and Grb2 (−/−) ES cells treated with retinoic acid. DAPI staining reveals the nuclei. D : The induction of extra-embryonic endoderm markers Dab2, laminin, GATA6, and GATA4, and trophectoderm marker cytokeratin 8, was assayed by Western blots. E : The activation of MAPK was assayed by Western using phospho-specific antibodies to phosphorylated ERK1/2.
    Figure Legend Snippet: Retinoic acid induces differentiation of ES cells in cultures. Following withdrawal of LIF from medium or addition of retinoic acid (1 μM) for 4 days in culture, the differentiation of RW4 wildtype and Grb2 (−/−) ES cells was monitored by the loss of pluripotent markers Oct3/4 and Nanog, and the induction of endoderm lineage markers Dab2, GATA4, and GATA6, by both immunofluorescence microscopy and Western blots. A: Double immunostaining of Oct3/4 and Nanog was performed in RW4 wildtype and Grb2 (−/−) Es cells with or without treatment with retinoic acid. DAPI staining reveals the nuclei. B : The loss of Oct3/4 and Nanog expression following retinoic acid treatment was assayed by Western blots. C : Double immunostaining of Dab2 and GATA4, Dab2 and GATA6, or Dab2 and Nanog, was performed in RW4 wildtype and Grb2 (−/−) ES cells treated with retinoic acid. DAPI staining reveals the nuclei. D : The induction of extra-embryonic endoderm markers Dab2, laminin, GATA6, and GATA4, and trophectoderm marker cytokeratin 8, was assayed by Western blots. E : The activation of MAPK was assayed by Western using phospho-specific antibodies to phosphorylated ERK1/2.

    Techniques Used: Immunofluorescence, Microscopy, Western Blot, Double Immunostaining, Staining, Expressing, Marker, Activation Assay

    Grb2-null ES cells are unable to form primitive endoderm in embryoid bodies. RW4 wildtype and Grb2 (−/−) ES cells were allowed to aggregate in non-adherent culture flasks to form embryoid bodies both in the presence and absence of 1 μM retinoic acid. A : Representative day 5 embryoid bodies viewed in bright field under a microscope show an outer layer for wildtype embryoid bodies and absence of an outer layer in Grb2 (−/−) cell aggregates. B : Embryoid bodies were harvested on day 5 and processed for histology. Immunofluorescence microscopy shows the presence of an outer endoderm layer stained with endoderm markers Dab2 (green) and GATA4 (red) in RW4 but not in Grb2 (−/−) embryoid bodies. Nuclei were stained with DAPI. C : The embryoid bodies were stained with Nanog and Oct3/4. D : Western blot was used to determine the time course of MAPK activation during the formation of embryoid bodies. E : Embryoid bodies were harvested on day 5 of the cultures and were assayed for MAPK activation by Western blots.
    Figure Legend Snippet: Grb2-null ES cells are unable to form primitive endoderm in embryoid bodies. RW4 wildtype and Grb2 (−/−) ES cells were allowed to aggregate in non-adherent culture flasks to form embryoid bodies both in the presence and absence of 1 μM retinoic acid. A : Representative day 5 embryoid bodies viewed in bright field under a microscope show an outer layer for wildtype embryoid bodies and absence of an outer layer in Grb2 (−/−) cell aggregates. B : Embryoid bodies were harvested on day 5 and processed for histology. Immunofluorescence microscopy shows the presence of an outer endoderm layer stained with endoderm markers Dab2 (green) and GATA4 (red) in RW4 but not in Grb2 (−/−) embryoid bodies. Nuclei were stained with DAPI. C : The embryoid bodies were stained with Nanog and Oct3/4. D : Western blot was used to determine the time course of MAPK activation during the formation of embryoid bodies. E : Embryoid bodies were harvested on day 5 of the cultures and were assayed for MAPK activation by Western blots.

    Techniques Used: Microscopy, Immunofluorescence, Staining, Western Blot, Activation Assay

    Suppression of Oct3/4 or Nanog in Grb2-null ES cells does not induce endoderm differentiation. RW4 wildtype and Grb2 (−/−) ES cells were treated with siRNA to Nanog, Oct3/4, or scrambled controls for 3 days in LIF-containing ES cell culture medium. The cells were then collected and subjected to Western blot analysis to determine cell differentiation and lineages. A : The ES cells were treated with control or siRNA to Nanog. Western blot was performed for endoderm markers, laminin, Dab2, GATA6, GATA4; and pluripotent markers Nanog and Oct3/4. B : The cells were treated with control or siRNA to Oct3/4. Western blots were performed for the endoderm markers: laminin, Dab2, GATA6, GATA4; the trophectoderm marker cytokeratin 8; and the pluripotent markers Nanog and Oct3/4. C : MAPK activation was assayed in Nanog siRNA treated and control cells by Western blot using phospho-specific antibodies.
    Figure Legend Snippet: Suppression of Oct3/4 or Nanog in Grb2-null ES cells does not induce endoderm differentiation. RW4 wildtype and Grb2 (−/−) ES cells were treated with siRNA to Nanog, Oct3/4, or scrambled controls for 3 days in LIF-containing ES cell culture medium. The cells were then collected and subjected to Western blot analysis to determine cell differentiation and lineages. A : The ES cells were treated with control or siRNA to Nanog. Western blot was performed for endoderm markers, laminin, Dab2, GATA6, GATA4; and pluripotent markers Nanog and Oct3/4. B : The cells were treated with control or siRNA to Oct3/4. Western blots were performed for the endoderm markers: laminin, Dab2, GATA6, GATA4; the trophectoderm marker cytokeratin 8; and the pluripotent markers Nanog and Oct3/4. C : MAPK activation was assayed in Nanog siRNA treated and control cells by Western blot using phospho-specific antibodies.

    Techniques Used: Cell Culture, Western Blot, Cell Differentiation, Marker, Activation Assay

    14) Product Images from "CtBP-interacting BTB Zinc Finger Protein (CIBZ) Promotes Proliferation and G1/S Transition in Embryonic Stem Cells via Nanog *"

    Article Title: CtBP-interacting BTB Zinc Finger Protein (CIBZ) Promotes Proliferation and G1/S Transition in Embryonic Stem Cells via Nanog *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.333856

    Constitutive overexpression of Nanog in ESCs rescues the compromised proliferation by siRNA-mediated CIBZ knockdown. A , Western blotting analysis of CIBZ knockdown in vector ESC line and Nanog-overexpressing ESC lines (#1 and #2). CIBZ siRNA or scrambled
    Figure Legend Snippet: Constitutive overexpression of Nanog in ESCs rescues the compromised proliferation by siRNA-mediated CIBZ knockdown. A , Western blotting analysis of CIBZ knockdown in vector ESC line and Nanog-overexpressing ESC lines (#1 and #2). CIBZ siRNA or scrambled

    Techniques Used: Over Expression, Western Blot, Plasmid Preparation

    15) Product Images from "The Pluripotency Factor NANOG Binds to GLI Proteins and Represses Hedgehog-mediated Transcription *"

    Article Title: The Pluripotency Factor NANOG Binds to GLI Proteins and Represses Hedgehog-mediated Transcription *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.714857

    GLI1 and GLI3 bind to NANOG. A , endogenous NANOG (∼42k Da), but not OCT4 (∼38kDa), co-immunoprecipitated with both FLAG-tagged GLI activator (GLI1 FLAG ; ∼122 kDa) and GLI repressor (GLI3R FLAG ; ∼84 kDa) using GLI1 FLAG - and
    Figure Legend Snippet: GLI1 and GLI3 bind to NANOG. A , endogenous NANOG (∼42k Da), but not OCT4 (∼38kDa), co-immunoprecipitated with both FLAG-tagged GLI activator (GLI1 FLAG ; ∼122 kDa) and GLI repressor (GLI3R FLAG ; ∼84 kDa) using GLI1 FLAG - and

    Techniques Used: Immunoprecipitation

    16) Product Images from "A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways"

    Article Title: A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways

    Journal: Scientific Reports

    doi: 10.1038/srep25104

    GoST cells upregulate expression of p27, but retain expression of core pluripotency markers compared to ES cells. ( A ) Real-Time PCR analysis of CDK inhibitor markers. Upon GoST induction, expression of p15 , p16 , p21 and p27 was increased. ( B ) Western blot analysis of p27. Upon GoST induction, expression of p27 was increased. ( C ) Immunofluorescence of p27 (red). DNA was counterstained with DAPI (blue). Upon GoST induction, expression of p27 was increased within cells growing in colonies. Scalebar = 50 μm. ( D ) Real-Time PCR analysis of core pluripotency markers. Upon GoST induction, expression of core pluripotency markers remained mostly unchanged while expression of Nanog and Tbx3 was increased. ( E ) Immunofluorescence of Nanog, Oct4, Sox2 and ALP (red). DNA was counterstained with DAPI (blue). Upon GoST induction, cells continued to express Nanog, Oct4, Sox2 and ALP. Scalebar = 50 μm. ( F ) Western blot analysis of Nanog, Oct4 and Sox2. Upon GoST induction, expression of Nanog, Oct4 and Sox2 showed only a slight decrease and remained at similar levels. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Western blot data were normalized to α-Tubulin and generated from three independent experiments. Error bars correspond to S.E.M. Two stars represent p
    Figure Legend Snippet: GoST cells upregulate expression of p27, but retain expression of core pluripotency markers compared to ES cells. ( A ) Real-Time PCR analysis of CDK inhibitor markers. Upon GoST induction, expression of p15 , p16 , p21 and p27 was increased. ( B ) Western blot analysis of p27. Upon GoST induction, expression of p27 was increased. ( C ) Immunofluorescence of p27 (red). DNA was counterstained with DAPI (blue). Upon GoST induction, expression of p27 was increased within cells growing in colonies. Scalebar = 50 μm. ( D ) Real-Time PCR analysis of core pluripotency markers. Upon GoST induction, expression of core pluripotency markers remained mostly unchanged while expression of Nanog and Tbx3 was increased. ( E ) Immunofluorescence of Nanog, Oct4, Sox2 and ALP (red). DNA was counterstained with DAPI (blue). Upon GoST induction, cells continued to express Nanog, Oct4, Sox2 and ALP. Scalebar = 50 μm. ( F ) Western blot analysis of Nanog, Oct4 and Sox2. Upon GoST induction, expression of Nanog, Oct4 and Sox2 showed only a slight decrease and remained at similar levels. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Western blot data were normalized to α-Tubulin and generated from three independent experiments. Error bars correspond to S.E.M. Two stars represent p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, ALP Assay, Generated

    17) Product Images from "A Chemical Platform for Improved Induction of Human iPS Cells"

    Article Title: A Chemical Platform for Improved Induction of Human iPS Cells

    Journal: Nature methods

    doi: 10.1038/nmeth.1393

    Compound treatment for seven days is sufficient to induce pluripotent stem cells from human fibroblasts transduced with the four reprogramming factors ( a ) Timeline for human iPSC induction using combined SB431542 and PD0325901 treatment along with 4TFs. Treatment began with cell re-seeding at day 7 after 4TF transduction and was maintained for 7 days. ( b ) Staining for ALP + colonies that emerged in the untreated (left) or 2 compound-treated (right) cultures within seven days. ( c ) RT-PCR showing elevated endogenous mRNA expression of pluripotency markers OCT4 and NANOG in 2 compound-treated cultures. ( d ) Tra-1-81 staining at day 14 without (left) or with (right) 2 compound treatment. ( e ) The numbers of NANOG + colonies at day 14 under different treatment conditions are plotted. ( f ) Typical staining for hESC-specific markers (NANOG and SSEA4) exhibited by D14 iPSCs. Scale bars, 50 µm in ( d f )
    Figure Legend Snippet: Compound treatment for seven days is sufficient to induce pluripotent stem cells from human fibroblasts transduced with the four reprogramming factors ( a ) Timeline for human iPSC induction using combined SB431542 and PD0325901 treatment along with 4TFs. Treatment began with cell re-seeding at day 7 after 4TF transduction and was maintained for 7 days. ( b ) Staining for ALP + colonies that emerged in the untreated (left) or 2 compound-treated (right) cultures within seven days. ( c ) RT-PCR showing elevated endogenous mRNA expression of pluripotency markers OCT4 and NANOG in 2 compound-treated cultures. ( d ) Tra-1-81 staining at day 14 without (left) or with (right) 2 compound treatment. ( e ) The numbers of NANOG + colonies at day 14 under different treatment conditions are plotted. ( f ) Typical staining for hESC-specific markers (NANOG and SSEA4) exhibited by D14 iPSCs. Scale bars, 50 µm in ( d f )

    Techniques Used: Transduction, Staining, ALP Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

    18) Product Images from "Abrogation of E-Cadherin-Mediated Cellular Aggregation Allows Proliferation of Pluripotent Mouse Embryonic Stem Cells in Shake Flask Bioreactors"

    Article Title: Abrogation of E-Cadherin-Mediated Cellular Aggregation Allows Proliferation of Pluripotent Mouse Embryonic Stem Cells in Shake Flask Bioreactors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012921

    EcadAb ES cells grown in shake flasks maintain an undifferentiated phenotype. EcadAb ES cells grown in shake flasks for 15 d or wtES cells grown under standard adherent conditions at an equivalent passage number were transferred to gelatin-treated plates and allowed to adhere overnight prior to analysis. (A) Phase contrast images show that both (i) wt and (ii) EcadAb ES cells exhibit typical ES cellular morphology. (B) Assessment of SSEA-1 expression by FACS analysis (n = 3) shows that in (i) wt and (ii) EcadAb ES cells, ∼95% of cells are SSEA-1 positive (green profile). Isotype control antibody (purple closed population). (C) RT-PCR analysis was performed on (i) wt and (ii) EcadAb ES cells to determine expression of pluripotency markers and (D) differentiation markers. Immunofluorescent analysis of (E) Oct3/4, (F) Nanog, (G) E-cad and DAPI in (i) wtES cells and (ii) in EcadAb ES cells. All images captured at ×20 magnification.
    Figure Legend Snippet: EcadAb ES cells grown in shake flasks maintain an undifferentiated phenotype. EcadAb ES cells grown in shake flasks for 15 d or wtES cells grown under standard adherent conditions at an equivalent passage number were transferred to gelatin-treated plates and allowed to adhere overnight prior to analysis. (A) Phase contrast images show that both (i) wt and (ii) EcadAb ES cells exhibit typical ES cellular morphology. (B) Assessment of SSEA-1 expression by FACS analysis (n = 3) shows that in (i) wt and (ii) EcadAb ES cells, ∼95% of cells are SSEA-1 positive (green profile). Isotype control antibody (purple closed population). (C) RT-PCR analysis was performed on (i) wt and (ii) EcadAb ES cells to determine expression of pluripotency markers and (D) differentiation markers. Immunofluorescent analysis of (E) Oct3/4, (F) Nanog, (G) E-cad and DAPI in (i) wtES cells and (ii) in EcadAb ES cells. All images captured at ×20 magnification.

    Techniques Used: Expressing, FACS, Reverse Transcription Polymerase Chain Reaction

    19) Product Images from "Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst"

    Article Title: Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst

    Journal:

    doi: 10.1242/dev.021519

    Localisation of Nanog, Gata4 and GFP in Pdgfra H2B-GFP/+ blastocysts
    Figure Legend Snippet: Localisation of Nanog, Gata4 and GFP in Pdgfra H2B-GFP/+ blastocysts

    Techniques Used:

    20) Product Images from "Zfp281 Functions as a Transcriptional Repressor for Pluripotency of Mouse Embryonic Stem Cells"

    Article Title: Zfp281 Functions as a Transcriptional Repressor for Pluripotency of Mouse Embryonic Stem Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.736

    Abnormal in vitro differentiation of Zfp281 deficient embryonic stem cells (ESCs). (A): Morphology of embryoid bodies (EBs) during the time course of differentiation of wild-type and null ESCs. (B): Analysis of the sizes of wild-type (3+/+) and Zfp281 null (3.34−/−) EBs at day 6. Eleven each of randomly selected wild-type and null EBs were scored and their relative diameters from images taken under identical magnification were calculated. The data were presented with the average sizes of the wild-type and null EBs, and the error bars denote SD. (C): Quantitative reverse transcriptase-polymerase chain reaction for relative expression levels of pluripotency genes ( Nanog and Oct4 ) during the time course of EB differentiation. For both genes, their expression levels in ESCs (at day 0) were arbitrarily set one and their relative expression levels during EB differentiation were normalized to the mRNA levels at day 0. Note that the actual overall expression levels of both genes are higher in 3.34 Null ESCs (red bars) than in three WT ESCs (blue bars) (Supporting Information Fig. S5B). Abbreviations: EB, embryoid body; WT, wild-type.
    Figure Legend Snippet: Abnormal in vitro differentiation of Zfp281 deficient embryonic stem cells (ESCs). (A): Morphology of embryoid bodies (EBs) during the time course of differentiation of wild-type and null ESCs. (B): Analysis of the sizes of wild-type (3+/+) and Zfp281 null (3.34−/−) EBs at day 6. Eleven each of randomly selected wild-type and null EBs were scored and their relative diameters from images taken under identical magnification were calculated. The data were presented with the average sizes of the wild-type and null EBs, and the error bars denote SD. (C): Quantitative reverse transcriptase-polymerase chain reaction for relative expression levels of pluripotency genes ( Nanog and Oct4 ) during the time course of EB differentiation. For both genes, their expression levels in ESCs (at day 0) were arbitrarily set one and their relative expression levels during EB differentiation were normalized to the mRNA levels at day 0. Note that the actual overall expression levels of both genes are higher in 3.34 Null ESCs (red bars) than in three WT ESCs (blue bars) (Supporting Information Fig. S5B). Abbreviations: EB, embryoid body; WT, wild-type.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Expressing

    Dysregulation of pluripotency and lineage specific markers in Zfp281 deficient embryonic stem cells (ESCs). (A): Depiction of the strategy for transgenic rescue of Zfp281 deficient ESCs with the pPyCAG-Zfp281IP expression vector. The two null (−) alleles (with and without the neomycin resistance gene) are shown. (B): Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of expression levels of Zfp281 transcripts in wild-type, heterozygous, null, and rescued ESCs. Note that the rescue level of Zfp281 is approximately 80% of the wild-type level. “3.34−/−R” represents Zfp281 deficient ESCs in which expression of Zfp281 is restored by ectopic expression of transgenic Zfp281 cDNA. Error bars represent SD. (C): Quantitative RT-PCR for relative expression levels of pluripotency markers in ESCs. (D): Quantitative RT-PCR for relative expression levels of endodermal markers in ESCs. (E): Quantitative RT-PCR for relative expression levels of differentiation markers in ESCs. (F): Western blot analysis showing a higher level of Nanog but a relatively smaller increase of Oct4 expression in Zfp281 deficient ESCs. β-Actin was used as a loading control. Western data were scanned and density of target bands was quantified using the ImageJ Software of the NIH. Band density was normalized to that of the β-actin loading control. (G): Heterogeneous expression of Nanog in wild-type, heterozygous, and null ESCs. Note that expression of Nanog in Zfp281 null ESCs is still as heterogeneous as that in wild-type controls (CJ7 and 3+/+).
    Figure Legend Snippet: Dysregulation of pluripotency and lineage specific markers in Zfp281 deficient embryonic stem cells (ESCs). (A): Depiction of the strategy for transgenic rescue of Zfp281 deficient ESCs with the pPyCAG-Zfp281IP expression vector. The two null (−) alleles (with and without the neomycin resistance gene) are shown. (B): Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of expression levels of Zfp281 transcripts in wild-type, heterozygous, null, and rescued ESCs. Note that the rescue level of Zfp281 is approximately 80% of the wild-type level. “3.34−/−R” represents Zfp281 deficient ESCs in which expression of Zfp281 is restored by ectopic expression of transgenic Zfp281 cDNA. Error bars represent SD. (C): Quantitative RT-PCR for relative expression levels of pluripotency markers in ESCs. (D): Quantitative RT-PCR for relative expression levels of endodermal markers in ESCs. (E): Quantitative RT-PCR for relative expression levels of differentiation markers in ESCs. (F): Western blot analysis showing a higher level of Nanog but a relatively smaller increase of Oct4 expression in Zfp281 deficient ESCs. β-Actin was used as a loading control. Western data were scanned and density of target bands was quantified using the ImageJ Software of the NIH. Band density was normalized to that of the β-actin loading control. (G): Heterogeneous expression of Nanog in wild-type, heterozygous, and null ESCs. Note that expression of Nanog in Zfp281 null ESCs is still as heterogeneous as that in wild-type controls (CJ7 and 3+/+).

    Techniques Used: Transgenic Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software

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    Incubation:

    Article Title: Transposon-mediated BAC transgenesis in human ES cells
    Article Snippet: .. Immunostaining and microscopy The incubation with primary antibodies was for 1 h at room temperature with mouse anti-Oct4 (1:50, sc-5279; Santa Cruz), rabbit anti-Nanog (1:30, AB5731; Chemicon) or overnight at +4°C with mouse anti-Pax6 (1:30; Developmental Studies Hybridoma Bank). .. The cells were incubated with secondary antibodies diluted 1:500 (FITC goat anti-rabbit and TRITC goat anti-mouse; Jackson Immunoresearch Laboratories and Alexa633 goat anti-mouse; Molecular Probes, Invitrogen) for 1 h at room temperature.

    Article Title: Characterization of Bovine Induced Pluripotent Stem Cells by Lentiviral Transduction of Reprogramming Factor Fusion Proteins
    Article Snippet: .. The primary antibodies anti-Oct4 in rabbit (Abcam, ab19875), anti-Nanog in goat (R & D, BAF1997), anti-SSEA1 in mouse (Chemicon, MAB4301), anti-SSEA3 in mouse (Chemicon, MAB4303), anti-SSEA4 in mouse (Chemicon, MAB4304), anti-TRA-1-60 (Chemicon, MAB4360) and TRA-1-80 in mouse (Chemicon, MAB4381), anti-α-Actinin (Sarcomeric) in mouse (Sigma, A7811), Anti-α-Fetoprotein (AFP) in mouse (Sigma, A8452), Anti-Neurofilament 200 in rabbit (Sigma, N4142), anti-Nobox (Abcam, ab41521) in rabbit and anti-Vasa (Abcam, ab13840) in rabbit were diluted 1:200, added onto cells and incubated at 4°C overnight. .. Cells were washed with PBS, incubated with CY3-labeled secondary antibody (1:200 dilution) at room temperature for 1 h and then washed with PBS.

    Chromatin Immunoprecipitation:

    Article Title: Nanog induces suppression of senescence through downregulation of p27KIP1 expression
    Article Snippet: .. After dilution, the protein–DNA complexes were immunoprecipitated overnight at 4°C with rotation using primary antibodies against Nanog (AB5731, Millipore) and rabbit control IgG ChIP grade (ab46540, Abcam). .. Immunoprecipitated chromatin was incubated with Protein A/G Plus agarose beads (sc-2003; Santa Cruz Biotechnology) for another 1 h at 4°C and then washed three times with low-salt washing buffer (20 mM Tris-HCl pH 8.0, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA and 0.01% Tween 20) and twice with high-salt washing buffer (20 mM Tris-HCl pH 8.0, 1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA and 0.01% Tween 20).

    Immunostaining:

    Article Title: Transposon-mediated BAC transgenesis in human ES cells
    Article Snippet: .. Immunostaining and microscopy The incubation with primary antibodies was for 1 h at room temperature with mouse anti-Oct4 (1:50, sc-5279; Santa Cruz), rabbit anti-Nanog (1:30, AB5731; Chemicon) or overnight at +4°C with mouse anti-Pax6 (1:30; Developmental Studies Hybridoma Bank). .. The cells were incubated with secondary antibodies diluted 1:500 (FITC goat anti-rabbit and TRITC goat anti-mouse; Jackson Immunoresearch Laboratories and Alexa633 goat anti-mouse; Molecular Probes, Invitrogen) for 1 h at room temperature.

    Microscopy:

    Article Title: Transposon-mediated BAC transgenesis in human ES cells
    Article Snippet: .. Immunostaining and microscopy The incubation with primary antibodies was for 1 h at room temperature with mouse anti-Oct4 (1:50, sc-5279; Santa Cruz), rabbit anti-Nanog (1:30, AB5731; Chemicon) or overnight at +4°C with mouse anti-Pax6 (1:30; Developmental Studies Hybridoma Bank). .. The cells were incubated with secondary antibodies diluted 1:500 (FITC goat anti-rabbit and TRITC goat anti-mouse; Jackson Immunoresearch Laboratories and Alexa633 goat anti-mouse; Molecular Probes, Invitrogen) for 1 h at room temperature.

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  • 94
    Millipore primary antibodies against nanog
    ChIP analysis reveals <t>Nanog</t> - binding sites regulating p27 KIP1 expression. (A) Schematic representation (not drawn to scale) of the p27 genomic locus (RefSeq: NM_009875) highlighting the location of putative Nanog-binding sites designated ‘D’ (primer pairs designated as D1 and D2) and ‘P’ (primer pairs designated as P1 and P2) in the upstream region of the p27 KIP1 gene ( Chen et al., 2008 ; Marson et al., 2008 ). PCR primer pairs were designed for these sites for ChIP analyses (dumbbell shaped; Table S2 ). (B) ChIP analysis reveals that Nanog protein in ESCs binds within the upstream region of the p27 KIP1 gene. Oct4-GiP MEF and Oct4-GiP ESCs were cultured, harvested and processed for ChIP analysis with beads only, IgG and Nanog antibody. The Oct4-GiP MEF cell line was used as a negative control. Input <t>DNA</t> (10%) was used as a control for ChIP. Beads only and IgG served as negative controls. Putative Nanog-binding regions were amplified by the designed primer pairs (D1, D2, P1 and P2). Primer pairs were also designed randomly in the 3′UTR region of the p27 KIP1 gene to serve as a negative (desert) control (Dc). (C) RT-qPCR analysis on the ChIP samples explained in B using primers pairs ‘P’ (P1) and ‘D’ (D1) to amplify the Nanog-binding p27 KIP1 sites. RT-qPCR was also performed on the Dc primer set but no amplification was observed other than the input samples (data not shown). Two independent biological replicates were performed for ChIP analysis. ** P
    Primary Antibodies Against Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primary antibodies against nanog/product/Millipore
    Average 94 stars, based on 2 article reviews
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    primary antibodies against nanog - by Bioz Stars, 2020-07
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    93
    Millipore nanog
    Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, <t>NANOG,</t> PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.
    Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nanog/product/Millipore
    Average 93 stars, based on 72 article reviews
    Price from $9.99 to $1999.99
    nanog - by Bioz Stars, 2020-07
    93/100 stars
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    87
    Millipore mouse anti human nanog homeobox nanog monoclonal antibody monoclonal antibody cat no mabd24a4
    Pluripotentiality of human parthenogenetic embryonic stem cell line-2 (hPES-2). (A) Alkaline phosphatase (AP) staining, (B) embryoid bodies (EBs), (C) pluripotency gene expression, including octamer-binding transcription factor 4 (OCT-4), REX1 [also referred to as zinc-finger protein-42 (Zfp42)], SRY (sex determining region Y)-box 2 (SOX2), <t>Nanog</t> <t>homeobox</t> (NANOG), Lin-28 homolog A (LIN28) and nucleophosmin (β-actin was used as a control), amplified by real-time PCR, and (D) pluripotency immunofluorescent markers, including stage-specific embryonic antigen (SSEA)3, SSEA4, tumor-rejection antigen (TRA)-1-60, TRA-1-81, OCT-4 and NANOG.
    Mouse Anti Human Nanog Homeobox Nanog Monoclonal Antibody Monoclonal Antibody Cat No Mabd24a4, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human nanog homeobox nanog monoclonal antibody monoclonal antibody cat no mabd24a4/product/Millipore
    Average 87 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    mouse anti human nanog homeobox nanog monoclonal antibody monoclonal antibody cat no mabd24a4 - by Bioz Stars, 2020-07
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      Buy from Supplier

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    ChIP analysis reveals Nanog - binding sites regulating p27 KIP1 expression. (A) Schematic representation (not drawn to scale) of the p27 genomic locus (RefSeq: NM_009875) highlighting the location of putative Nanog-binding sites designated ‘D’ (primer pairs designated as D1 and D2) and ‘P’ (primer pairs designated as P1 and P2) in the upstream region of the p27 KIP1 gene ( Chen et al., 2008 ; Marson et al., 2008 ). PCR primer pairs were designed for these sites for ChIP analyses (dumbbell shaped; Table S2 ). (B) ChIP analysis reveals that Nanog protein in ESCs binds within the upstream region of the p27 KIP1 gene. Oct4-GiP MEF and Oct4-GiP ESCs were cultured, harvested and processed for ChIP analysis with beads only, IgG and Nanog antibody. The Oct4-GiP MEF cell line was used as a negative control. Input DNA (10%) was used as a control for ChIP. Beads only and IgG served as negative controls. Putative Nanog-binding regions were amplified by the designed primer pairs (D1, D2, P1 and P2). Primer pairs were also designed randomly in the 3′UTR region of the p27 KIP1 gene to serve as a negative (desert) control (Dc). (C) RT-qPCR analysis on the ChIP samples explained in B using primers pairs ‘P’ (P1) and ‘D’ (D1) to amplify the Nanog-binding p27 KIP1 sites. RT-qPCR was also performed on the Dc primer set but no amplification was observed other than the input samples (data not shown). Two independent biological replicates were performed for ChIP analysis. ** P

    Journal: Journal of Cell Science

    Article Title: Nanog induces suppression of senescence through downregulation of p27KIP1 expression

    doi: 10.1242/jcs.167932

    Figure Lengend Snippet: ChIP analysis reveals Nanog - binding sites regulating p27 KIP1 expression. (A) Schematic representation (not drawn to scale) of the p27 genomic locus (RefSeq: NM_009875) highlighting the location of putative Nanog-binding sites designated ‘D’ (primer pairs designated as D1 and D2) and ‘P’ (primer pairs designated as P1 and P2) in the upstream region of the p27 KIP1 gene ( Chen et al., 2008 ; Marson et al., 2008 ). PCR primer pairs were designed for these sites for ChIP analyses (dumbbell shaped; Table S2 ). (B) ChIP analysis reveals that Nanog protein in ESCs binds within the upstream region of the p27 KIP1 gene. Oct4-GiP MEF and Oct4-GiP ESCs were cultured, harvested and processed for ChIP analysis with beads only, IgG and Nanog antibody. The Oct4-GiP MEF cell line was used as a negative control. Input DNA (10%) was used as a control for ChIP. Beads only and IgG served as negative controls. Putative Nanog-binding regions were amplified by the designed primer pairs (D1, D2, P1 and P2). Primer pairs were also designed randomly in the 3′UTR region of the p27 KIP1 gene to serve as a negative (desert) control (Dc). (C) RT-qPCR analysis on the ChIP samples explained in B using primers pairs ‘P’ (P1) and ‘D’ (D1) to amplify the Nanog-binding p27 KIP1 sites. RT-qPCR was also performed on the Dc primer set but no amplification was observed other than the input samples (data not shown). Two independent biological replicates were performed for ChIP analysis. ** P

    Article Snippet: After dilution, the protein–DNA complexes were immunoprecipitated overnight at 4°C with rotation using primary antibodies against Nanog (AB5731, Millipore) and rabbit control IgG ChIP grade (ab46540, Abcam).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Expressing, Polymerase Chain Reaction, Cell Culture, Negative Control, Amplification, Quantitative RT-PCR

    Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.

    Journal: Molecular and Cellular Biology

    Article Title: Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation

    doi: 10.1128/MCB.00499-18

    Figure Lengend Snippet: Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.

    Article Snippet: Primary antibodies used were OCT4 (1:100; Millipore), NANOG (1:100; Millipore), CUX1 (1:100; Abcam), glial fibrillary acidic protein (GFAP) (1:250; Dako), TUBB3/TUJ1 (1:1,000; Covance), BRN2 (1:100; Abcam), PAX6 (1:1,000; Developmental Studies Hybridoma Bank [DSHB]), anti-phospho-histone H3 (Ser10) (1:200; Cell Signaling Technology) and were applied for 3 to 4 h at room temperature or overnight at 4°C.

    Techniques: In Vitro, Cell Culture, Immunocytochemistry, Expressing, Marker, Staining, Polymerase Chain Reaction, Sequencing, Negative Control

    Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Generation of Transgenic Rats through Induced Pluripotent Stem Cells *

    doi: 10.1074/jbc.M113.492900

    Figure Lengend Snippet: Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

    Article Snippet: Primary antibodies against the following markers were used: Oct4 (1:100; Santa Cruz Biotechnology), Sox2 (1:100; Santa Cruz Biotechnology), Nanog (1:100; Millipore), SSEA-1 (1:100; Santa Cruz Biotechnology), and Esrrb (1:100; Santa Cruz Biotechnology).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Negative Control, Staining, Positive Control, Polymerase Chain Reaction, Marker, Western Blot, Immunofluorescence, Genomic Sequencing, Methylation

    Pluripotentiality of human parthenogenetic embryonic stem cell line-2 (hPES-2). (A) Alkaline phosphatase (AP) staining, (B) embryoid bodies (EBs), (C) pluripotency gene expression, including octamer-binding transcription factor 4 (OCT-4), REX1 [also referred to as zinc-finger protein-42 (Zfp42)], SRY (sex determining region Y)-box 2 (SOX2), Nanog homeobox (NANOG), Lin-28 homolog A (LIN28) and nucleophosmin (β-actin was used as a control), amplified by real-time PCR, and (D) pluripotency immunofluorescent markers, including stage-specific embryonic antigen (SSEA)3, SSEA4, tumor-rejection antigen (TRA)-1-60, TRA-1-81, OCT-4 and NANOG.

    Journal: International Journal of Molecular Medicine

    Article Title: X chromosome inactivation in human parthenogenetic embryonic stem cells following prolonged passaging

    doi: 10.3892/ijmm.2014.2044

    Figure Lengend Snippet: Pluripotentiality of human parthenogenetic embryonic stem cell line-2 (hPES-2). (A) Alkaline phosphatase (AP) staining, (B) embryoid bodies (EBs), (C) pluripotency gene expression, including octamer-binding transcription factor 4 (OCT-4), REX1 [also referred to as zinc-finger protein-42 (Zfp42)], SRY (sex determining region Y)-box 2 (SOX2), Nanog homeobox (NANOG), Lin-28 homolog A (LIN28) and nucleophosmin (β-actin was used as a control), amplified by real-time PCR, and (D) pluripotency immunofluorescent markers, including stage-specific embryonic antigen (SSEA)3, SSEA4, tumor-rejection antigen (TRA)-1-60, TRA-1-81, OCT-4 and NANOG.

    Article Snippet: Briefly, for immunofluorescence staining, the hPES-2 cell colonies were incubated with the following primary antibodies against stage-specific embryonic antigens: rat anti-human stage-specific embryonic antigen (SSEA)3 monoclonal antibody (Cat. no. LV1528429), rat anti-human SSEA4 monoclonal antibody (Cat. no. LV1488380), mouse anti-human tumor-rejection antigen (TRA)-1-60 monoclonal antibody (Cat. no. LV1541028), mouse anti-human TRA-1-81 monoclonal antibody (Cat. no. LV1580855); mouse anti-human octamer-binding transcription factor 4 (OCT-4) monoclonal antibody (Cat. no. MAB4419A4) and mouse anti-human Nanog homeobox (NANOG) monoclonal antibody (Cat. no. MABD24A4) (all from Millipore) (all were used at 1:100).

    Techniques: Staining, Expressing, Binding Assay, Amplification, Real-time Polymerase Chain Reaction