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  • 99
    Thermo Fisher gene exp nanog hs02387400 g1
    Mechanisms of aspartic acid effects against UVA-induced attenuation of stemness of stem cells. UVA irradiation induces production of PGE 2 and its downstream molecule, cAMP through activation of AP-1 and NF-κB. cAMP molecule sequentially reduces expression of HIF-1α gene through CREB activation, consequently downregulating expression of stemness genes such as <t>NANOG,</t> SOX2, and OCT4. In the UVA irradiation-induced signaling pathway, aspartic acid attenuates UVA-induced effects on expression of stemness genes by inhibiting AP-1, which is upstream of PGE 2 production. AA: aspartic acid
    Gene Exp Nanog Hs02387400 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore nanog
    Old passage E14 mESCs show differentiation defects and loss of methylation at ICRs. ( a ) Schematic representation of embryoid body (EB) differentiation protocol of YP- and OP- mESCs. ( b ) Representative bright field images showing EBs at day 4 (D4) and 9 (D9) obtained from both YP- and OP- E14 mESCs. Scale bar is 400 μm. ( c ) Quantitative real-time PCR showing the expression profiles of differentiation genes ( Nkx2.5, Gata6, Otx2 ) and pluripotency genes ( Rex1, Oct4, <t>Nanog</t> ) in YP- and OP- E14 mESCs (ESC) and during EB differentiation at day 6 (D6) and day 12 (D12). ( d ) Schematic representation of neural differentiation protocol of YP- and OP- mESCs. ( e ) Quantitative real-time PCR showing the expression profiles of Sox1 at day 3 (D3) of N2B27 + retinoic acid (RA) treatment in YP- and OP-E14 mESCs (ESC). ( f ) Representative immunofluorescence images showing Nestin (left panels) and III <t>β-tubulin</t> (TUJ1, right panels) protein expression in YP- and OP- mESCs at day 8 (D8) of neural differentiation. ( g ) Quantitative real-time PCR experiment showing the expression profiles of Pax6 and Fgf5 at day 8 (D8) of N2B27 + retinoic acid (RA) treatment in YP- and OP- E14 mESCs (ESC). (c , e , g ) The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and the results are means of n = 3 independent experiments ± SE ( c , e ) and means of n = 3 technical replicated for SD ( g ). ( c , e ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p
    Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nanog  (Abcam)
    96
    Abcam nanog
    Morphological, protein and gene expression changes in HFFs induced by hESC extract treatment. (A) Morphology of HFFs treated with its own extract (a) or hESC extract on day 3 (b), day 10 (c) and passage 2 (d). (B) <t>OCT4</t> expression was induced in hESC extract-treated HFFs on day 7. (C) LAMIN A/C was demolished in hESC extract-treated HFFs on day 7. (D) Imunoblotting analysis of <t>NANOG,</t> OCT4 and LAMIN A/C expression on day 7. Lanes from 1 to 6 were loaded with proteins (2 µg) from HFFs, hESCs, hESC extract and HFF extract-treated HFFs, water and hESC extracts. (E) Expression of OCT4 , NANOG , SOX2 , KLF4 and C-MYC were upregulated in hESC extract-treated HFFs after 7 days of hESC extract treatment. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., *** P
    Nanog, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 2698 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc nanog
    Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) <t>NANOG</t> and <t>OCT-4</t> were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.
    Nanog, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1688 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology nanog
    Assessment of pluripotent marker expression and characterization of young and old BM-MSCs: (A) Passage 3 BM-MSCs from young and old rats were harvested and labeled with a PE-coupled antibody against CD45 and CD31, and FITC-coupled antibodies against CD105, CD90 and CD73 or immunoglobin isotype control IgGs and analyzed with FACS. Histograms show counts for specific antibody staining profiles (blue). (B) Immunocytochemical analysis demonstrates that old BM-MSCs express the pluripotent maker Oct-4 but fail to express <t>Sox-2</t> and <t>NANOG.</t> (C) qPCR analysis for the relative expression of Oct-4, Sox-2 and NANOG in young and old BM-MSCs. We failed to detect Sox-2 and NANOG in old BM-MSCs. Columns show the combined mean ΔΔC q values of young (n = 4) and old rats (n = 4) for each marker. Data represent relative expression of transcripts normalized relative to GAPDH and expressed as the Mean ± SEM for three biologically independent experiments (n = 3). The '‡' sign indicates a significant difference between the indicated group and any passage 3 groups from younger donors ( p
    Nanog, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti nanog
    Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of <t>OCT4</t> and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , <t>NANOG</t> , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.
    Anti Nanog, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems human nanog antibody
    Human iPSCs expanded in a bioreactor have typical hiPSC morphology and express <t>hPSC-associated</t> markers post-harvest. ( A ) Phase contrast images of iPSCs expanded in a bioreactor plated either as single cells after cell release from MCs (left panel), or as clusters of cells on MCs (right panel). Magnification, 100×; Scale bar: 100 µm; ( B ) Immunofluorescence staining of OCT3/4, <t>NANOG,</t> SSEA-4, TRA-1-60 and TRA-1-81 of iPSCs expanded in a bioreactor and plated onto 2D cell culture plates. Representative images from two different cell lines are shown. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers OCT3/4, SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of RTiPSC3B and LiPSC18R cells expanded in a bioreactor. Data obtained from either three independent runs (RTiPSC3B) or two independent runs (LiPSC18R). Data plotted are mean ± standard deviation (SD); ( D ) Pluripotency of RTiPSC3B cells expanded in a bioreactor demonstrated by embryoid body (EB) formation followed by immunofluorescence staining for germ layer-specific markers. Magnification, 100× (ectoderm, endoderm), 200× (mesoderm). ( E ) Representative immunofluorescence staining for lineage-specific markers RTiPSC3B and LiPSC18R cell lines. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)
    Human Nanog Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 414 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam rabbit anti nanog
    Isolation and characterization of pES-269 cells with commonly used markers. ( A ) pES-269 cells isolation, Alkaline phosphatase (AKP) staining and immunofluorescence staining using <t>anti-OCT4,</t> <t>-NANOG</t> and -TRA-1-60 antibodies; ( B ) RT-PCR assays for the expression of pluripotency-associated markers in pES-269 cells; ( C ) Immunostaining of differentiated cells from embryoid bodies (EBs) formed by pES-269 cells, using antibodies against β-tubulin (ectoderm), SMA (mesoderm), and AFP (endoderm); ( D ) Karyotype analysis of pES-269 cells; ( E ) Genome-wide SNP analysis of pES-269 cells using an Affymetrix 250 K SNP Array. A homozygous AA allele maps to approximately +1, and a homozygous BB allele maps to approximately −1, with the heterozygote mapping to approximately 0.
    Rabbit Anti Nanog, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti nanog
    Characterization of iPS cells for their pluripotency. Number 1 represents the cell line UTA.04602.WT and 2 the line UTA.04607.WT. A : iPS cells formed typical colonies for pluripotent stem cells that are rather compact and round in shape. B : The iPS cell colonies typically had well defined edges and distinct cell borders, and the iPS cells had a high nucleus to cytoplasm -ratio and a large nucleoli characteristic for stem cells. C : Endogenous pluripotency gene expression was studied using RT-PCR. <t>Nanog,</t> Oct4, Sox2 and Rex1 were all expressed at mRNA level in the iPS cells. β-actin and GAPDH were used as housekeeping control genes for both endogenous and exogenous markers. D : The expression of pluripotency genes was also studied at the protein level by immunocytochemical staining. The iPS cell expressed several markers for the pluripotent state: Nanog, Oct4, Sox2, SSEA4, TRA-1-60, and TRA-1-81 (all in red). Pictures in the left panel are from the line UTA.04602.WT and the ones on the right side are from UTA.04607.WT. Blue in all pictures indicates the DAPI staining of nuclei. E : Using RT-PCR, it was shown that all the transgenes were silenced in the iPS cells. Negative control is marked with “-” and positive control with “+”. F : Embryoid body (EB) -assay was used to define the pluripotency of the iPS cells in vitro . Markers for all three germ layers were detected from the EBs formed from both cell lines. Alpha - fetoprotein (AFP) was used as a marker for endoderm, kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR-2) was used as a marker for mesoderm and nestin was used as an ectoderm marker. GAPDH was used as an endogenous control gene.
    Anti Nanog, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti nanog
    Expression of stem cell marker genes and methylation status in the promoter regions of <t>Oct4</t> and <t>Nanog.</t> 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.
    Anti Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gene exp nanog hs04260366 g1
    Embryonic stem cell markers. Immunohistochemistry: A . <t>NANOG;</t> B . OCT4. The majority of nuclei in the NANOG IHC are brown indicating the presence of NANOG. Some, but not all, nuclei are positive for OCT4. Negative (secondary antibody only) and positive (seminoma) controls are presented in the thumbnails below the photomicrographs. C . Immunofluorescence against nucleostemin. Left: anti-nucleostemin antibody, center: DAPI, right: merge. Bar = 10 microns.
    Gene Exp Nanog Hs04260366 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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.
    Nanog, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cosmo Bio nanog
    PAF1c function during early lineage decisions. Our data suggests that PAF1c is required for molecular identity of ICM and TE cells after the first cell fate decision. Loss of function of CTR9 or RTF1 results in increased <t>OCT4</t> and <t>NANOG</t> positive TE cells,
    Nanog, supplied by Cosmo Bio, used in various techniques. Bioz Stars score: 93/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ReproCELL nanog
    Generation of iPSCs and spinal MN differentiation. a iPSCs were generated from healthy control individuals and HMSN-P patients with TFG P285L mutation. Control and HMSN-P patient iPSCs were morphologically identical to human ESCs and expressed the pluripotent stem cell markers <t>NANOG</t> and <t>SSEA4.</t> Nucleus was stained with DAPI. Scale bars = 200 μm. Differentiated MNs were stained with neuronal marker βIII-tubulin, MAP2, and spinal MN marker SMI-32. Glial cells were stained with GFAP. Scale bars = 50 μm. b - e Proportions of control and HMSN-P patient neurons stained positive for βIII-tubulin ( b ), MAP2 ( c ), SMI-32 ( d ), and GFAP ( e ) ( n = 3, n.s. by Student t -test). There were no significant differences between control and HMSN-P groups. Error bars are ± s.e.m., n.s.: not significant
    Nanog, supplied by ReproCELL, used in various techniques. Bioz Stars score: 92/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rabbit anti nanog
    Suppression of MEK/ERK signaling promotes self-renewal and colony morphology of mESCs. (a) PD promotes colony morphology of mESCs. J1 mESCs were treated with 1 μ M PD or equal volume of DMSO for 24 h. Morphological changes were observed and recorded under a phase contrast microscope. Scale bar = 50 μ m. (b) PD influences the expression pluripotent factors. ESCs were treated with or without 1 μ M PD for 24 h; then the expression levels of <t>Nanog</t> , Tfcp2l1 , <t>Klf4</t> , c- Myc , and Egr1 were analyzed by RT-qPCR. Gapdh was used as a normalization control. Error bars indicate mean ± SD of three independent experiments, ∗ p
    Rabbit Anti Nanog, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bethyl rabbit anti nanog antibody affinity purified
    Ythdf1, Ythdf2 and <t>Ythdf3</t> are redundant in ESCs differentiation a) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in KH2 mESCs, showing a protein expression in the cytosolic compartment of the cell. b) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. c) Brightfield and immunostaining of <t>Nanog</t> (green), Esrrb (red) and DAPI (blue), in KO cells (single, triple and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb. d) Teratomas generated by the KO cell line and by WT control. Single-KO cell lines show all germ layers, while triple-KO teratomas as poorly differentiated. e) Immunostaining of triple-KO and WT control with Oct4 (red), Foxa (green), Tuj1 (purple) and DAPI (blue). Triple-KO contains patches of Oct4 staining, unlike WT teratomas. f) Alkaline phosphatase (AP) staining of disassociated teratomas from Triple-KO and WT control, showing a greater AP staining in the triple-KO cells. g) RT-PCR of pluripotent genes (left) and differentiation genes (right), measured in WT and KO EBs, and in WT mESCs as a control. In the triple-KO EBs, pluripotent markers are higher than the control and differentiation markers are lower than the WT control.
    Rabbit Anti Nanog Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 99/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson nanog
    Depletion of MGP allows for multipotency and osteoinduction in human aortic endothelial cells (HAECs) (A) Expression of multipotent markers <t>Sox2,</t> <t>Nanog</t> and Oct3/4 in HAECs after transfection of scrambled control siRNA (SCR) or MGP siRNA only (lane 1, 2), siRNA transfection with BMP4 treatment (lane 3, 4), siRNA transfection with Noggin treatment (lane 5, 6), or siRNA transfection with BMP4 and Noggin treatment (lane 7, 8), as determined by immunoblotting. (B) Co-expression of CD31 and SSEA-3, or CD31 and SSEA-4 after transfection of scrambled control siRNA or MGP siRNA without additional treatment (left), and with BMP4 treatment (right), as determined by FACS analysis. (C) Expression of multipotent markers Sox2, Nanog and Oct3/4, osteogenic markers Cbfa1, Osterix (OSX), early SMC markers SM22α, α-SM actin (αSMA), and late SMC marker SM-myosin heavy chain (SM-MHC) (top) after transfection of scrambled control siRNA or MGP siRNA and treatment with control (C), BMP2 (B), osteogenic media (O) or both (B+O), as determined by immunoblotting. (D) Staining for alkaline phosphatase (ALP), and mineral (Alizarin Red and Von Kossa staining) in HAECS treated as described in (c). (E) Expression of multipotent markers Sox2, Nanog and Oct3/4, osteogenic markers Cbfa1, OSX, SM22α, αSMA, and SM-MHC (top) after transfection of scrambled control siRNA or MGP siRNA and treatment with control (C), BMP2 (B), high glucose medium (G) or both (B+G), as determined by immunoblotting. (F) Staining for ALP and mineral in HAECS treated as described in (E).
    Nanog, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti nanog antibody chip grade
    The effects of miR-3622a-3p and <t>SALL4</t> on stemness features of CRC cells. a – d Flow cytometric analysis was used to examine the percentage of CD133(+) CRC cells. e – h Sphere formation assay was performed to investigate the stem-cell like properties of stable transfected CRC cells. i , j Expression levels of Sox2, <t>Nanog,</t> Oct4, CD44, and CD133 were determined by qRT-PCR in stable transfected CRC cells. k , l Protein levels of CSC-related biomarkers and pluripotency-related genes were detected by western blot in stable transfected CRC cells. All data are from three independent experiments and are presented as the means ± SD (* p
    Anti Nanog Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp nanog mm02019550 s1
    Fold change in gene expression (qPCR) of Oct4, Sox2 and <t>Nanog</t> 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
    Gene Exp Nanog Mm02019550 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti nanog
    LIF alone supports ESCs self-renew and pluripotency. (A) Experimental outline of the L-ESCs derivation procedures from ESCs. (B) 2i/L-ESCs were switched to L-medium and cultured to passages 3 (p3), p5, p25. Here we use 2i/L-ESCs with GOF/GFP reporter. Scale bars, 100 μm. (C) Karyotyping of L-ESCs (p30). (D) Immunostaining of <t>OCT4,</t> SOX2 and <t>NANOG</t> in L-ESCs. Scale bars, 50 μm. (E) Single cell clonogenicity efficiency in L-ESCs and 2i/L-ESCs. (F) L-ESCs were treated with JAK inhibitor I after day 3 p2 and day 10 p4. Scale bars, 100 μm. (G) 2i/L-ESCs were treated with JAK inhibitor I after day 3 p2, day 10 p6 and p10. Scale bars, 100 μm.
    Anti Nanog, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nanog monoclonal antibody
    Analysis of Rex1-Positive and -Negative Serum ES Cell Fractions, Related to Figure 1 (A) ES cell morphology in 2i or serum culture. (B) Oct4 (left) and Klf4 (right) immunostaining of ES cells in 2i or serum. Arrows point to Klf4-negative ES cells in serum. (C) GFP flow cytometry profile of the total population of Rex1-GFP serum ES cells (black) and ES cells without any GFP transgene (negative control, gray (largely overlapping red)). The GFP-negative and -positive sorted Rex1-GFP serum ES cells used in this study are shown in red and green, respectively. (D) Number of colonies after re-plating 2i ES cells and Rex1-positive and Rex1-negative ES cells at clonal density in serum (left) or 2i (right). After 5 days (serum) or 7 days (2i + LIF), alkaline phosphatase staining was performed to discriminate between colonies consisting of largely undifferentiated cells (undiff), mixed, or largely differentiated (diff) cells. (E) Klf4 and <t>Nanog</t> immunostaining of unsorted Rex1-GFP serum ES cells. The arrowheads in the DAPI stainings point to Rex1-, Klf4-, and Nanog-negative cells, the arrows in the GFP+Klf4+Nanog stainings to Rex1-positive, but Klf4- and Nanog-negative cells. (F) Comparison of expression of pluripotency and lineage-specific genes (as shown in Figures 1 B and 1D) of Rex1-positive (Serum-Rex-pos) and Rex1-negative (Serum-Rex-neg) ES cells. (G) Transcript levels of genes associated with the various germ layers in Rex1-positive and Rex1-negative serum ES cells. (H) Functional analysis of the differential genes between Rex1-positive and Rex1-negative serum ES cells. (I) Functional analysis of the differential genes between 2i ES cells and Rex1-positive serum ES cells.
    Nanog Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunocytochemistry of SOX2, <t>OCT4,</t> <t>NANOG,</t> EMD and SSEA1 in hESCs with and without BANF1 knockdown. CHB-4 hESCs were transduced with either Scrambled or human Banf1 shRNA lentiviral vectors, selected with puromycin to eliminate uninfected cells, and seeded
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    Image Search Results


    Mechanisms of aspartic acid effects against UVA-induced attenuation of stemness of stem cells. UVA irradiation induces production of PGE 2 and its downstream molecule, cAMP through activation of AP-1 and NF-κB. cAMP molecule sequentially reduces expression of HIF-1α gene through CREB activation, consequently downregulating expression of stemness genes such as NANOG, SOX2, and OCT4. In the UVA irradiation-induced signaling pathway, aspartic acid attenuates UVA-induced effects on expression of stemness genes by inhibiting AP-1, which is upstream of PGE 2 production. AA: aspartic acid

    Journal: PLoS ONE

    Article Title: Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0124417

    Figure Lengend Snippet: Mechanisms of aspartic acid effects against UVA-induced attenuation of stemness of stem cells. UVA irradiation induces production of PGE 2 and its downstream molecule, cAMP through activation of AP-1 and NF-κB. cAMP molecule sequentially reduces expression of HIF-1α gene through CREB activation, consequently downregulating expression of stemness genes such as NANOG, SOX2, and OCT4. In the UVA irradiation-induced signaling pathway, aspartic acid attenuates UVA-induced effects on expression of stemness genes by inhibiting AP-1, which is upstream of PGE 2 production. AA: aspartic acid

    Article Snippet: The following assays were used: HIF-1α (ID: Hs00936371_m1), HIF-2α (ID: Hs01026149_m1), OCT4 (ID: Hs03005111_g1), NANOG (ID: Hs02387400_g1), SOX2 (ID: Hs01053049_s1), REX1 (ID: Hs01938187_s1), GAPDH (ID: Hs00266705_g1).

    Techniques: Irradiation, Activation Assay, Expressing

    Antagonizing effects of aspartic acid against UVA-induced downregulation of stemness genes are mediated by downregulating PGE 2 -cAMP-HIF-1α signaling through inhibition of AP-1. hAMSCs were irradiated with 5 J/cm 2 UVA or transfected with the siRNA for HIF-1α and then incubated for three days with aspartic acid (100 μM) in the presence of the indicated concentration of PGE 2 or cAMP under serum-free conditions. After three days of incubation, total RNA was isolated and the mRNA levels of the HIF-1α gene (A) and OCT4, NANOG, SOX2 genes (B) were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against GAPDH. Data are expressed as the means ± S.D. *, p

    Journal: PLoS ONE

    Article Title: Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0124417

    Figure Lengend Snippet: Antagonizing effects of aspartic acid against UVA-induced downregulation of stemness genes are mediated by downregulating PGE 2 -cAMP-HIF-1α signaling through inhibition of AP-1. hAMSCs were irradiated with 5 J/cm 2 UVA or transfected with the siRNA for HIF-1α and then incubated for three days with aspartic acid (100 μM) in the presence of the indicated concentration of PGE 2 or cAMP under serum-free conditions. After three days of incubation, total RNA was isolated and the mRNA levels of the HIF-1α gene (A) and OCT4, NANOG, SOX2 genes (B) were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against GAPDH. Data are expressed as the means ± S.D. *, p

    Article Snippet: The following assays were used: HIF-1α (ID: Hs00936371_m1), HIF-2α (ID: Hs01026149_m1), OCT4 (ID: Hs03005111_g1), NANOG (ID: Hs02387400_g1), SOX2 (ID: Hs01053049_s1), REX1 (ID: Hs01938187_s1), GAPDH (ID: Hs00266705_g1).

    Techniques: Inhibition, Irradiation, Transfection, Incubation, Concentration Assay, Isolation, Quantitative RT-PCR

    Aspartic acid attenuates the effects of UVA irradiation on self-renewal of hAMSCs. hAMSCs were irradiated with 5 J/cm 2 UVA and then incubated for three days in the presence of the indicated concentrations of aspartic acid under serum-free conditions. (A) After three days of incubation under serum-free conditions, total RNA was isolated and the mRNA levels of the OCT4, NANOG, SOX2, and REX1 genes were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against the GAPDH. Data are expressed as the means ± S.D. *, p

    Journal: PLoS ONE

    Article Title: Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0124417

    Figure Lengend Snippet: Aspartic acid attenuates the effects of UVA irradiation on self-renewal of hAMSCs. hAMSCs were irradiated with 5 J/cm 2 UVA and then incubated for three days in the presence of the indicated concentrations of aspartic acid under serum-free conditions. (A) After three days of incubation under serum-free conditions, total RNA was isolated and the mRNA levels of the OCT4, NANOG, SOX2, and REX1 genes were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against the GAPDH. Data are expressed as the means ± S.D. *, p

    Article Snippet: The following assays were used: HIF-1α (ID: Hs00936371_m1), HIF-2α (ID: Hs01026149_m1), OCT4 (ID: Hs03005111_g1), NANOG (ID: Hs02387400_g1), SOX2 (ID: Hs01053049_s1), REX1 (ID: Hs01938187_s1), GAPDH (ID: Hs00266705_g1).

    Techniques: Irradiation, Incubation, Isolation, Quantitative RT-PCR

    Old passage E14 mESCs show differentiation defects and loss of methylation at ICRs. ( a ) Schematic representation of embryoid body (EB) differentiation protocol of YP- and OP- mESCs. ( b ) Representative bright field images showing EBs at day 4 (D4) and 9 (D9) obtained from both YP- and OP- E14 mESCs. Scale bar is 400 μm. ( c ) Quantitative real-time PCR showing the expression profiles of differentiation genes ( Nkx2.5, Gata6, Otx2 ) and pluripotency genes ( Rex1, Oct4, Nanog ) in YP- and OP- E14 mESCs (ESC) and during EB differentiation at day 6 (D6) and day 12 (D12). ( d ) Schematic representation of neural differentiation protocol of YP- and OP- mESCs. ( e ) Quantitative real-time PCR showing the expression profiles of Sox1 at day 3 (D3) of N2B27 + retinoic acid (RA) treatment in YP- and OP-E14 mESCs (ESC). ( f ) Representative immunofluorescence images showing Nestin (left panels) and III β-tubulin (TUJ1, right panels) protein expression in YP- and OP- mESCs at day 8 (D8) of neural differentiation. ( g ) Quantitative real-time PCR experiment showing the expression profiles of Pax6 and Fgf5 at day 8 (D8) of N2B27 + retinoic acid (RA) treatment in YP- and OP- E14 mESCs (ESC). (c , e , g ) The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and the results are means of n = 3 independent experiments ± SE ( c , e ) and means of n = 3 technical replicated for SD ( g ). ( c , e ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p

    Journal: Scientific Reports

    Article Title: Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells

    doi: 10.1038/s41598-018-37442-5

    Figure Lengend Snippet: Old passage E14 mESCs show differentiation defects and loss of methylation at ICRs. ( a ) Schematic representation of embryoid body (EB) differentiation protocol of YP- and OP- mESCs. ( b ) Representative bright field images showing EBs at day 4 (D4) and 9 (D9) obtained from both YP- and OP- E14 mESCs. Scale bar is 400 μm. ( c ) Quantitative real-time PCR showing the expression profiles of differentiation genes ( Nkx2.5, Gata6, Otx2 ) and pluripotency genes ( Rex1, Oct4, Nanog ) in YP- and OP- E14 mESCs (ESC) and during EB differentiation at day 6 (D6) and day 12 (D12). ( d ) Schematic representation of neural differentiation protocol of YP- and OP- mESCs. ( e ) Quantitative real-time PCR showing the expression profiles of Sox1 at day 3 (D3) of N2B27 + retinoic acid (RA) treatment in YP- and OP-E14 mESCs (ESC). ( f ) Representative immunofluorescence images showing Nestin (left panels) and III β-tubulin (TUJ1, right panels) protein expression in YP- and OP- mESCs at day 8 (D8) of neural differentiation. ( g ) Quantitative real-time PCR experiment showing the expression profiles of Pax6 and Fgf5 at day 8 (D8) of N2B27 + retinoic acid (RA) treatment in YP- and OP- E14 mESCs (ESC). (c , e , g ) The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and the results are means of n = 3 independent experiments ± SE ( c , e ) and means of n = 3 technical replicated for SD ( g ). ( c , e ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p

    Article Snippet: The membranes were blocked with 5% non-fat dry milk (SIGMA 70166) in TBS-Tween 20 (0,1%) (SIGMA P1379) for 60 min, incubated with primary antibodies (β-catenin (BD, 610153), NANOG (Calbiochem, #SC1000), OCT-4 (Santa Cruz, sc-5279), β-tubulin (SIGMA, T0198), DNMT1 (Abcam, ab87656), KAP1 (Abcam, ab10483)) overnight at 4 °C.

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Two Tailed Test

    Prolonged in vitro cell culture of E14 mouse embryonic stem cells (mESCs) correlates with low Wnt/β-catenin activity. ( a ) Schematic representation of Young (YP) and Old passage (OP) E14 mESCs. ( b ) Representative bright field images of YP- and OP-mESCs. Round-shaped and flat colonies are indicated by white and yellow arrow, respectively. ( c ) Quantitative real-time PCR showing the expression profiles of Axin2 , Lef1 , Tcf1 , Sp5 in YP- and OP- mESCs. The transcriptional levels are normalized to Gapdh as reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and means of n = 3 independent experiments ± SE. ( d , e ) Representative immunofluorescence ( d ) and confocal microphotographs ( e ) of β-catenin. Nuclear demarcation is indicated by white circles (right panel). ( f ) Western blot analysis showing total and nuclear β-catenin protein in YP- and OP-mESCs and its quantification (n = 1) relative to total β-catenin in YP-mESCs. For quantification, densitometric analysis was performed with ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. ( g ) Representative immunofluorescence images showing OCT4 (green), NANOG (red) and their merge in YP- and OP-E14 mESCs. ( h , i ) Representative western blot analysis of OCT4 and NANOG in YP- and OP-mESCs ( h ) and its quantification represented as fold change over the protein amount in YP-mESCs and means of n = 3 independent experiments ± SE ( i ). ( f , h , i . ( j – m ) FACS-plot showing the percentage of E-cadherin +( j ) and SSEA1 +cells ( l ) in YP- and OP- mESCs and its quantification ( k , m ) as means of 3 technical replicates ± SE (NS: non stained). ( n , o ) Representative cell cycle FACS profile analyzed with Flowjo software ( n ) and its quantification ( o ) represented as percentage of total cells and means of n = 3 independent experiments ± SE. Scale bar is 200 ( b , d , g ) and 10 μm ( e ). ( d , e left panel, and g ) Nuclei were stained with DAPI. ( f , h ) β-tubulin and H3 were used as loading controls. ( c , i , k , m , o ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p-value

    Journal: Scientific Reports

    Article Title: Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells

    doi: 10.1038/s41598-018-37442-5

    Figure Lengend Snippet: Prolonged in vitro cell culture of E14 mouse embryonic stem cells (mESCs) correlates with low Wnt/β-catenin activity. ( a ) Schematic representation of Young (YP) and Old passage (OP) E14 mESCs. ( b ) Representative bright field images of YP- and OP-mESCs. Round-shaped and flat colonies are indicated by white and yellow arrow, respectively. ( c ) Quantitative real-time PCR showing the expression profiles of Axin2 , Lef1 , Tcf1 , Sp5 in YP- and OP- mESCs. The transcriptional levels are normalized to Gapdh as reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and means of n = 3 independent experiments ± SE. ( d , e ) Representative immunofluorescence ( d ) and confocal microphotographs ( e ) of β-catenin. Nuclear demarcation is indicated by white circles (right panel). ( f ) Western blot analysis showing total and nuclear β-catenin protein in YP- and OP-mESCs and its quantification (n = 1) relative to total β-catenin in YP-mESCs. For quantification, densitometric analysis was performed with ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. ( g ) Representative immunofluorescence images showing OCT4 (green), NANOG (red) and their merge in YP- and OP-E14 mESCs. ( h , i ) Representative western blot analysis of OCT4 and NANOG in YP- and OP-mESCs ( h ) and its quantification represented as fold change over the protein amount in YP-mESCs and means of n = 3 independent experiments ± SE ( i ). ( f , h , i . ( j – m ) FACS-plot showing the percentage of E-cadherin +( j ) and SSEA1 +cells ( l ) in YP- and OP- mESCs and its quantification ( k , m ) as means of 3 technical replicates ± SE (NS: non stained). ( n , o ) Representative cell cycle FACS profile analyzed with Flowjo software ( n ) and its quantification ( o ) represented as percentage of total cells and means of n = 3 independent experiments ± SE. Scale bar is 200 ( b , d , g ) and 10 μm ( e ). ( d , e left panel, and g ) Nuclei were stained with DAPI. ( f , h ) β-tubulin and H3 were used as loading controls. ( c , i , k , m , o ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p-value

    Article Snippet: The membranes were blocked with 5% non-fat dry milk (SIGMA 70166) in TBS-Tween 20 (0,1%) (SIGMA P1379) for 60 min, incubated with primary antibodies (β-catenin (BD, 610153), NANOG (Calbiochem, #SC1000), OCT-4 (Santa Cruz, sc-5279), β-tubulin (SIGMA, T0198), DNMT1 (Abcam, ab87656), KAP1 (Abcam, ab10483)) overnight at 4 °C.

    Techniques: In Vitro, Cell Culture, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Western Blot, Software, FACS, Staining, Two Tailed Test

    β-catenin silencing impairs mESC differentiation. ( a ) Quantitative real-time PCR analysis showing β-catenin silencing efficiency, Axin2 and pluripotency marker ( Nanog , Oct4 ) levels. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the shCtrl-infected mESCs and the results are means of n = 3 technical replicated ± SD. ( b ) Western blot analysis showing protein levels of total and nuclear β-catenin in shCtrl-, shβcat#1-, shβcat#2- and shβcat#3- transduced E14 mESCs (n = 1). Quantification of total and nuclear β-catenin is represented as relative to total β-catenin in shCtrl-transduced mESCs. β-tubulin and H3 were used as loading controls. For western-blot quantification densitometric analysis was carried out by using ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. ( c ) Representative bright field images of mESCs and embryoid bodies (EBs) at day 3 (D3), 8 (D8) after β-catenin silencing (shβcat #1, #2, #3) versus the control condition (shCtrl). Scale bar is 400 μm. ( d ) Quantitative real-time PCR experiments showing the expression profiles of ERVs ( IAP , MusD , MERVL ) in shCtrl-, shβcat#1-, shβcat#2- and shβcat#3- transduced E14 mESCs. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to shCtrl-infected mESCs and are means of n = 3 independent experiments ± SE. Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p

    Journal: Scientific Reports

    Article Title: Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells

    doi: 10.1038/s41598-018-37442-5

    Figure Lengend Snippet: β-catenin silencing impairs mESC differentiation. ( a ) Quantitative real-time PCR analysis showing β-catenin silencing efficiency, Axin2 and pluripotency marker ( Nanog , Oct4 ) levels. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the shCtrl-infected mESCs and the results are means of n = 3 technical replicated ± SD. ( b ) Western blot analysis showing protein levels of total and nuclear β-catenin in shCtrl-, shβcat#1-, shβcat#2- and shβcat#3- transduced E14 mESCs (n = 1). Quantification of total and nuclear β-catenin is represented as relative to total β-catenin in shCtrl-transduced mESCs. β-tubulin and H3 were used as loading controls. For western-blot quantification densitometric analysis was carried out by using ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. ( c ) Representative bright field images of mESCs and embryoid bodies (EBs) at day 3 (D3), 8 (D8) after β-catenin silencing (shβcat #1, #2, #3) versus the control condition (shCtrl). Scale bar is 400 μm. ( d ) Quantitative real-time PCR experiments showing the expression profiles of ERVs ( IAP , MusD , MERVL ) in shCtrl-, shβcat#1-, shβcat#2- and shβcat#3- transduced E14 mESCs. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to shCtrl-infected mESCs and are means of n = 3 independent experiments ± SE. Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p

    Article Snippet: The membranes were blocked with 5% non-fat dry milk (SIGMA 70166) in TBS-Tween 20 (0,1%) (SIGMA P1379) for 60 min, incubated with primary antibodies (β-catenin (BD, 610153), NANOG (Calbiochem, #SC1000), OCT-4 (Santa Cruz, sc-5279), β-tubulin (SIGMA, T0198), DNMT1 (Abcam, ab87656), KAP1 (Abcam, ab10483)) overnight at 4 °C.

    Techniques: Real-time Polymerase Chain Reaction, Marker, Infection, Western Blot, Software, Expressing, Two Tailed Test

    Morphological, protein and gene expression changes in HFFs induced by hESC extract treatment. (A) Morphology of HFFs treated with its own extract (a) or hESC extract on day 3 (b), day 10 (c) and passage 2 (d). (B) OCT4 expression was induced in hESC extract-treated HFFs on day 7. (C) LAMIN A/C was demolished in hESC extract-treated HFFs on day 7. (D) Imunoblotting analysis of NANOG, OCT4 and LAMIN A/C expression on day 7. Lanes from 1 to 6 were loaded with proteins (2 µg) from HFFs, hESCs, hESC extract and HFF extract-treated HFFs, water and hESC extracts. (E) Expression of OCT4 , NANOG , SOX2 , KLF4 and C-MYC were upregulated in hESC extract-treated HFFs after 7 days of hESC extract treatment. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., *** P

    Journal: PLoS ONE

    Article Title: A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

    doi: 10.1371/journal.pone.0012297

    Figure Lengend Snippet: Morphological, protein and gene expression changes in HFFs induced by hESC extract treatment. (A) Morphology of HFFs treated with its own extract (a) or hESC extract on day 3 (b), day 10 (c) and passage 2 (d). (B) OCT4 expression was induced in hESC extract-treated HFFs on day 7. (C) LAMIN A/C was demolished in hESC extract-treated HFFs on day 7. (D) Imunoblotting analysis of NANOG, OCT4 and LAMIN A/C expression on day 7. Lanes from 1 to 6 were loaded with proteins (2 µg) from HFFs, hESCs, hESC extract and HFF extract-treated HFFs, water and hESC extracts. (E) Expression of OCT4 , NANOG , SOX2 , KLF4 and C-MYC were upregulated in hESC extract-treated HFFs after 7 days of hESC extract treatment. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., *** P

    Article Snippet: Antibodies used included: Rabbit polyclonal to OCT4 (Abcam), Rabbit polyclonal to NANOG (Abcam), Goat polyclonal to LAMIN A/C (Santa Cruz Biotechnology), Mouse monoclonal to 5-Methyl Cytidine (Abcam), Rabbit polyclonal to Acetyl-Histone H3 (Lys9) (Cell signaling), Rabbit polyclonal to PAX6 (Abcam), Rabbit polyclonal to SOX1 (Abcam), Mouse monoclonal to NESTIN (Millipore), Rabbit polyclonal to TUJ (Covance), Mouse monoclonal to MAP2 (Covance), Mouse monoclonal to TH (R & D systems), Goat anti-Mouse IgG (FITC) (Millipore), Goat anti- Rabbit IgG (FITC), Abcam, Rabbit anti-Goat IgG (FITC) (Millipore), Goat anti-Rabbit IgG-H+L (Alexa Fluor 594) (Invitrogen).

    Techniques: Expressing

    Protein and gene expression changes of HFFs after reprogramming treatment. (A) Pluripotency marker NANOG and OCT4 were induced in HFFs after reprogramming. (B), (C) and (D) qPCR analysis of pluripotency-related gene and apoptotic gene expression in controls and reprogrammed HFFs. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., * P

    Journal: PLoS ONE

    Article Title: A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

    doi: 10.1371/journal.pone.0012297

    Figure Lengend Snippet: Protein and gene expression changes of HFFs after reprogramming treatment. (A) Pluripotency marker NANOG and OCT4 were induced in HFFs after reprogramming. (B), (C) and (D) qPCR analysis of pluripotency-related gene and apoptotic gene expression in controls and reprogrammed HFFs. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., * P

    Article Snippet: Antibodies used included: Rabbit polyclonal to OCT4 (Abcam), Rabbit polyclonal to NANOG (Abcam), Goat polyclonal to LAMIN A/C (Santa Cruz Biotechnology), Mouse monoclonal to 5-Methyl Cytidine (Abcam), Rabbit polyclonal to Acetyl-Histone H3 (Lys9) (Cell signaling), Rabbit polyclonal to PAX6 (Abcam), Rabbit polyclonal to SOX1 (Abcam), Mouse monoclonal to NESTIN (Millipore), Rabbit polyclonal to TUJ (Covance), Mouse monoclonal to MAP2 (Covance), Mouse monoclonal to TH (R & D systems), Goat anti-Mouse IgG (FITC) (Millipore), Goat anti- Rabbit IgG (FITC), Abcam, Rabbit anti-Goat IgG (FITC) (Millipore), Goat anti-Rabbit IgG-H+L (Alexa Fluor 594) (Invitrogen).

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

    Effects of epigenetic modifications on HFFs. (A) Bisulfite sequencing of NANOG and (B) OCT4 promoter region. Black circles represent methylated sites, white circles represent unmethylated sites. Global methylated cytosines are shown as %M. (C) qPCR analysis of NANOG , OCT4 and SOX2 and (D) KLF4 and c-MYC expression in controls and HFFs with chemical treatment in different culture medium. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., * P

    Journal: PLoS ONE

    Article Title: A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

    doi: 10.1371/journal.pone.0012297

    Figure Lengend Snippet: Effects of epigenetic modifications on HFFs. (A) Bisulfite sequencing of NANOG and (B) OCT4 promoter region. Black circles represent methylated sites, white circles represent unmethylated sites. Global methylated cytosines are shown as %M. (C) qPCR analysis of NANOG , OCT4 and SOX2 and (D) KLF4 and c-MYC expression in controls and HFFs with chemical treatment in different culture medium. The gene expression levels were normalized to the GAPDH and compared relative to gene expression in control HFFs. Error bar, S.D., * P

    Article Snippet: Antibodies used included: Rabbit polyclonal to OCT4 (Abcam), Rabbit polyclonal to NANOG (Abcam), Goat polyclonal to LAMIN A/C (Santa Cruz Biotechnology), Mouse monoclonal to 5-Methyl Cytidine (Abcam), Rabbit polyclonal to Acetyl-Histone H3 (Lys9) (Cell signaling), Rabbit polyclonal to PAX6 (Abcam), Rabbit polyclonal to SOX1 (Abcam), Mouse monoclonal to NESTIN (Millipore), Rabbit polyclonal to TUJ (Covance), Mouse monoclonal to MAP2 (Covance), Mouse monoclonal to TH (R & D systems), Goat anti-Mouse IgG (FITC) (Millipore), Goat anti- Rabbit IgG (FITC), Abcam, Rabbit anti-Goat IgG (FITC) (Millipore), Goat anti-Rabbit IgG-H+L (Alexa Fluor 594) (Invitrogen).

    Techniques: Methylation Sequencing, Methylation, Real-time Polymerase Chain Reaction, Expressing

    Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) NANOG and OCT-4 were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.

    Journal: Neurochemistry International

    Article Title: Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

    doi: 10.1016/j.neuint.2018.10.014

    Figure Lengend Snippet: Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) NANOG and OCT-4 were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.

    Article Snippet: Primary antibodies, OCT-4 (1:400; Cell Signaling, D73G4), NANOG (1:800, Cell Signaling, C30A3), TUJ-1 (1:2000; Abcam ab14545), SV-2 (1:500; DSHB), PSD-95 (1:100; NeuroMab, P78352), KI-67 (1:600; BD Pharmingen™, 550609), PAX-6 (1:600; BioLegend, 901301), nestin (1:50; R & D Systems, MAB1259), CTIP-2 (1:300; Abcam, ab18465), TBR-1 (1:300; Abcam, ab31940), BRN-2 (1:400; Santa Cruz, sc-6029), CUX-1 (1:300; Santa Cruz, sc-13024), GAP-43 (1:1000; Abcam, ab75810), tau (1:1000; Biorbyt, orb175815), NRGN (1:100; Upstate Biotechnologies, 07–425), SNAP-25 (1:400; Sigma Aldrich, S9684-100UL) and SYT-1 antibody (1:200; Synaptic systems, 105,011) were diluted in block buffer and incubated at 4 °C overnight.

    Techniques: Immunocytochemistry, Staining, Expressing

    FOXD2-AS1 conferred cancer stemness and chemotherapy resistance in laryngeal cancer through STAT3 activation. a Representative micrographs and quantification of tumor spheres formed by FOXD2-AS1-overexpressing laryngeal cancer cells treated with vehicle or STAT3 inhibitor stattic. Histograms showed the mean number of spheres formed by the indicated cells. b Real-time PCR analysis of OCT4, SOX2, and NANOG expression in indicated cells that under stattic treatment and control cells. Expression levels were normalized to GAPDH. c Viability of indicated cells after 24 h cisplatin treatment or treating with cisplatin plus stattic, implicating that stattic-abolished FOXD2-AS1 induced cisplatin resistance of laryngeal cancer cells. d Colony formation of FOXD2-AS1-overexpressing cells with indicated treatment. e , f The effect of STAT3 inhibition by stattic on tumor formation of FOXD2-AS1-overexpressing laryngeal cancer cells during cisplatin treatment ( n = 6/group). Stattic treatment significantly rescued the sensitivity to cisplatin in FOXD2-AS1-overexpressing cells. g IHC staining of Ki67 and TUNEL staining were performed to verified the proliferation and anti-apoptosis potential of FOXD2-AS1-overexpressing cells accompanied with indicated treatment. * P

    Journal: Cell Death & Disease

    Article Title: Long noncoding RNA FOXD2-AS1 enhances chemotherapeutic resistance of laryngeal squamous cell carcinoma via STAT3 activation

    doi: 10.1038/s41419-020-2232-7

    Figure Lengend Snippet: FOXD2-AS1 conferred cancer stemness and chemotherapy resistance in laryngeal cancer through STAT3 activation. a Representative micrographs and quantification of tumor spheres formed by FOXD2-AS1-overexpressing laryngeal cancer cells treated with vehicle or STAT3 inhibitor stattic. Histograms showed the mean number of spheres formed by the indicated cells. b Real-time PCR analysis of OCT4, SOX2, and NANOG expression in indicated cells that under stattic treatment and control cells. Expression levels were normalized to GAPDH. c Viability of indicated cells after 24 h cisplatin treatment or treating with cisplatin plus stattic, implicating that stattic-abolished FOXD2-AS1 induced cisplatin resistance of laryngeal cancer cells. d Colony formation of FOXD2-AS1-overexpressing cells with indicated treatment. e , f The effect of STAT3 inhibition by stattic on tumor formation of FOXD2-AS1-overexpressing laryngeal cancer cells during cisplatin treatment ( n = 6/group). Stattic treatment significantly rescued the sensitivity to cisplatin in FOXD2-AS1-overexpressing cells. g IHC staining of Ki67 and TUNEL staining were performed to verified the proliferation and anti-apoptosis potential of FOXD2-AS1-overexpressing cells accompanied with indicated treatment. * P

    Article Snippet: The PVDF membrane was hybridized with the following primary antibodies overnight at 4 °C: anti‐OCT4 (1:1000, #75463, CST), anti‐NANOG (1:2000, #4903, CST), anti‐SOX2 (1:1000, #4900, CST), anti‐GAPDH (1:1000, #5174, CST), anti‐STAT3 (1:1000, #9139, CST), anti‐phosphorylated-STAT3 (1:1000, #9145, CST), and anti‐PRMT5 (1:1000, #79998, CST).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Inhibition, Immunohistochemistry, Staining, TUNEL Assay

    Upregulation of FOXD2-AS1 increased CSC-like potential in laryngeal cancer. a Representative micrographs and quantification of tumor spheres formed by indicated Hep-2 and Tu-212 cells. Histograms showed the mean number of spheres formed by the indicated cells. b , c Flow cytometry analyses showed proportion of side population b and CD133 + population d in laryngeal cancer cells. d Real-time PCR analysis of OCT4, SOX2, and NANOG expression in FOXD2-AS1-overexpressing and -silencing cells. Expression levels were normalized to GAPDH. e Western blot analysis of OCT4, SOX2, and NANOG expression in indicated cells. GAPDH was used as a loading control. * P

    Journal: Cell Death & Disease

    Article Title: Long noncoding RNA FOXD2-AS1 enhances chemotherapeutic resistance of laryngeal squamous cell carcinoma via STAT3 activation

    doi: 10.1038/s41419-020-2232-7

    Figure Lengend Snippet: Upregulation of FOXD2-AS1 increased CSC-like potential in laryngeal cancer. a Representative micrographs and quantification of tumor spheres formed by indicated Hep-2 and Tu-212 cells. Histograms showed the mean number of spheres formed by the indicated cells. b , c Flow cytometry analyses showed proportion of side population b and CD133 + population d in laryngeal cancer cells. d Real-time PCR analysis of OCT4, SOX2, and NANOG expression in FOXD2-AS1-overexpressing and -silencing cells. Expression levels were normalized to GAPDH. e Western blot analysis of OCT4, SOX2, and NANOG expression in indicated cells. GAPDH was used as a loading control. * P

    Article Snippet: The PVDF membrane was hybridized with the following primary antibodies overnight at 4 °C: anti‐OCT4 (1:1000, #75463, CST), anti‐NANOG (1:2000, #4903, CST), anti‐SOX2 (1:1000, #4900, CST), anti‐GAPDH (1:1000, #5174, CST), anti‐STAT3 (1:1000, #9139, CST), anti‐phosphorylated-STAT3 (1:1000, #9145, CST), and anti‐PRMT5 (1:1000, #79998, CST).

    Techniques: Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    Assessment of pluripotent marker expression and characterization of young and old BM-MSCs: (A) Passage 3 BM-MSCs from young and old rats were harvested and labeled with a PE-coupled antibody against CD45 and CD31, and FITC-coupled antibodies against CD105, CD90 and CD73 or immunoglobin isotype control IgGs and analyzed with FACS. Histograms show counts for specific antibody staining profiles (blue). (B) Immunocytochemical analysis demonstrates that old BM-MSCs express the pluripotent maker Oct-4 but fail to express Sox-2 and NANOG. (C) qPCR analysis for the relative expression of Oct-4, Sox-2 and NANOG in young and old BM-MSCs. We failed to detect Sox-2 and NANOG in old BM-MSCs. Columns show the combined mean ΔΔC q values of young (n = 4) and old rats (n = 4) for each marker. Data represent relative expression of transcripts normalized relative to GAPDH and expressed as the Mean ± SEM for three biologically independent experiments (n = 3). The '‡' sign indicates a significant difference between the indicated group and any passage 3 groups from younger donors ( p

    Journal: BMC Cell Biology

    Article Title: Age-related changes in rat bone-marrow mesenchymal stem cell plasticity

    doi: 10.1186/1471-2121-12-44

    Figure Lengend Snippet: Assessment of pluripotent marker expression and characterization of young and old BM-MSCs: (A) Passage 3 BM-MSCs from young and old rats were harvested and labeled with a PE-coupled antibody against CD45 and CD31, and FITC-coupled antibodies against CD105, CD90 and CD73 or immunoglobin isotype control IgGs and analyzed with FACS. Histograms show counts for specific antibody staining profiles (blue). (B) Immunocytochemical analysis demonstrates that old BM-MSCs express the pluripotent maker Oct-4 but fail to express Sox-2 and NANOG. (C) qPCR analysis for the relative expression of Oct-4, Sox-2 and NANOG in young and old BM-MSCs. We failed to detect Sox-2 and NANOG in old BM-MSCs. Columns show the combined mean ΔΔC q values of young (n = 4) and old rats (n = 4) for each marker. Data represent relative expression of transcripts normalized relative to GAPDH and expressed as the Mean ± SEM for three biologically independent experiments (n = 3). The '‡' sign indicates a significant difference between the indicated group and any passage 3 groups from younger donors ( p

    Article Snippet: ; 1:200), SOX-2 (sc-17320; 1:100), Oct-3/4 (sc-9081; 1:100) and NANOG (sc-33760; 1:100) were from (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    Techniques: Marker, Expressing, Labeling, FACS, Staining, Real-time Polymerase Chain Reaction

    Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of OCT4 and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , NANOG , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.

    Journal: Scientific Reports

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    doi: 10.1038/s41598-018-26238-2

    Figure Lengend Snippet: Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of OCT4 and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , NANOG , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.

    Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.

    Techniques: Derivative Assay, Cell Culture, Immunofluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    Optimizing procedure of methanol fixation. The J1 mES cells were cultured on methanol fixed MEF cells (MT-MEF) under different conditions. ( A ) Schematic diagram of procedure and application of MT-MEF. ( B ) Methanol in different temperatures was used to fix MEFs. ( C ) MEFs were fixed by methanol for 5 to 20 min. ( D ) MEFs in the density from 5 × 10 3 /cm 2 to 3 × 10 4 /cm 2 were fixed by methanol. ( E ) MT-MEFs stored at room temperature (RT) for 0 to 21 days. ( F ) qRT-PCR analysis of Oct4 and Nanog expressions in J1 cells cultured on MT-MEF that was stored at RT for different dates (0 to 21 days). Phase 1, morphology of MT-MEF; Phase 2, morphology of J1 cells cultured on MT-MEF. Scale bar, 400 μm for B–D, 200 μm for E.

    Journal: Scientific Reports

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    doi: 10.1038/s41598-018-26238-2

    Figure Lengend Snippet: Optimizing procedure of methanol fixation. The J1 mES cells were cultured on methanol fixed MEF cells (MT-MEF) under different conditions. ( A ) Schematic diagram of procedure and application of MT-MEF. ( B ) Methanol in different temperatures was used to fix MEFs. ( C ) MEFs were fixed by methanol for 5 to 20 min. ( D ) MEFs in the density from 5 × 10 3 /cm 2 to 3 × 10 4 /cm 2 were fixed by methanol. ( E ) MT-MEFs stored at room temperature (RT) for 0 to 21 days. ( F ) qRT-PCR analysis of Oct4 and Nanog expressions in J1 cells cultured on MT-MEF that was stored at RT for different dates (0 to 21 days). Phase 1, morphology of MT-MEF; Phase 2, morphology of J1 cells cultured on MT-MEF. Scale bar, 400 μm for B–D, 200 μm for E.

    Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.

    Techniques: Cell Culture, Quantitative RT-PCR

    Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P

    Journal: Scientific Reports

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    doi: 10.1038/s41598-018-26238-2

    Figure Lengend Snippet: Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P

    Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.

    Techniques: Cell Culture, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Microscopy

    Effect of EV co‐culture on stemness maintenance in LoVo and HT29 cells. LoVo‐CSCs and HT29‐CSCs were first transfected with miR‐200c mimics (miR‐200c‐mim) or inhibitors (miR‐200c‐inh). LoVo cells were co‐cultured for 48 h with EVs isolated from nontransfected (Control) or transfected (A) LoVo‐CSCs or (B) HT29‐CSCs, with or without ATL‐1 administration at 200 μM. Similarly, HT29 cells were co‐cultured for 48 h with EVs isolated from nontransfected (Control) or transfected (C) LoVo‐CSCs or (D) HT29‐CSCs, with or without ATL‐1 administration at 200 μM. Western blot and quantification of stemness maintenance markers OCT‐4, SOX‐9, and Nanog relative to GAPDH were carried out. Overexpression of miR‐200c in EVs enhanced the stem‐like properties of LoVo and HT29 cells via horizontal transfer, whereas miR‐200c interference suppressed these traits. The data represent the mean ± SD of three independent technical replicates (ANOVA); * P

    Journal: Clinical and Translational Medicine

    Article Title: Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I. Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I

    doi: 10.1002/ctm2.139

    Figure Lengend Snippet: Effect of EV co‐culture on stemness maintenance in LoVo and HT29 cells. LoVo‐CSCs and HT29‐CSCs were first transfected with miR‐200c mimics (miR‐200c‐mim) or inhibitors (miR‐200c‐inh). LoVo cells were co‐cultured for 48 h with EVs isolated from nontransfected (Control) or transfected (A) LoVo‐CSCs or (B) HT29‐CSCs, with or without ATL‐1 administration at 200 μM. Similarly, HT29 cells were co‐cultured for 48 h with EVs isolated from nontransfected (Control) or transfected (C) LoVo‐CSCs or (D) HT29‐CSCs, with or without ATL‐1 administration at 200 μM. Western blot and quantification of stemness maintenance markers OCT‐4, SOX‐9, and Nanog relative to GAPDH were carried out. Overexpression of miR‐200c in EVs enhanced the stem‐like properties of LoVo and HT29 cells via horizontal transfer, whereas miR‐200c interference suppressed these traits. The data represent the mean ± SD of three independent technical replicates (ANOVA); * P

    Article Snippet: Thereafter, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following proteins: octamer‐binding transcription factor 4 (OCT‐4, 1:1000, AB18976, Abcam, Cambridge, UK), sex‐determining region Y‐box (SOX‐9, 1:1000, AB185966, Abcam), Nanog (1:1000, AB106465, Abcam), CD63 (1:1000, PAB33929, Bioswamp), CD81 (1:1000, PAB33928, Bioswamp), TSG101 (1:1000, PAB32949, Bioswamp), PI3K (1:1000, AB191606, Abcam), phospho‐PI3K (p‐PI3K, 1:1000, AB182651, Abcam), Akt (1:1000, AB8805, Abcam), p‐Akt (1:1000, AB38449, Abcam), mTOR (1:2000, AB32028, Abcam), p‐mTOR (1:1000, AB84400, Abcam), and GAPDH (1:5000, PAB36264, Bioswamp).

    Techniques: Co-Culture Assay, Transfection, Cell Culture, Isolation, Western Blot, Over Expression

    Characterization of colorectal CSCs isolated from LoVo and HT29 cells. A, Flow cytometric sorting of CSCs using CD44 and EpCAM as markers. The percentage of parental LoVo and HT29 cells expressing both CD44 and EpCAM was approximately 15%, representing the proportion of CSCs. CSCs isolated from parental LoVo and HT29 cells (denoted as LoVo‐CSCs and HT29‐CSCs, respectively) exhibited high expression of both markers ( > 90%). Relative proliferation of (B) LoVo‐CSCs and (C) HT29‐CSCs was inhibited by ATL‐1 at 200 μM for up to 72 h, compared to that of control (+PBS) CSCs. The protein expression of stemness markers OCT‐4, SOX‐9, and Nanog, relative to GAPDH, in (D) LoVo‐CSCs and (E) HT29‐CSCs was downregulated by ATL‐1 at 200 μM after 48 h of culture, compared to that of control (+PBS) CSCs. Transwell assay of the (F) migration and (G) invasion of LoVo‐CSCs demonstrated decreased cell counts in both cases when ATL‐1 was administered at 200 μM, compared to those of control (+PBS) CSCs. H, The percentage of apoptotic LoVo‐CSCs and HT29‐CSCs was elevated in the presence of ATL‐1 at 200 μM, compared to that of control (+PBS) CSCs. The data represent the mean ± SD of three independent technical replicates ( t ‐test); * P

    Journal: Clinical and Translational Medicine

    Article Title: Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I. Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I

    doi: 10.1002/ctm2.139

    Figure Lengend Snippet: Characterization of colorectal CSCs isolated from LoVo and HT29 cells. A, Flow cytometric sorting of CSCs using CD44 and EpCAM as markers. The percentage of parental LoVo and HT29 cells expressing both CD44 and EpCAM was approximately 15%, representing the proportion of CSCs. CSCs isolated from parental LoVo and HT29 cells (denoted as LoVo‐CSCs and HT29‐CSCs, respectively) exhibited high expression of both markers ( > 90%). Relative proliferation of (B) LoVo‐CSCs and (C) HT29‐CSCs was inhibited by ATL‐1 at 200 μM for up to 72 h, compared to that of control (+PBS) CSCs. The protein expression of stemness markers OCT‐4, SOX‐9, and Nanog, relative to GAPDH, in (D) LoVo‐CSCs and (E) HT29‐CSCs was downregulated by ATL‐1 at 200 μM after 48 h of culture, compared to that of control (+PBS) CSCs. Transwell assay of the (F) migration and (G) invasion of LoVo‐CSCs demonstrated decreased cell counts in both cases when ATL‐1 was administered at 200 μM, compared to those of control (+PBS) CSCs. H, The percentage of apoptotic LoVo‐CSCs and HT29‐CSCs was elevated in the presence of ATL‐1 at 200 μM, compared to that of control (+PBS) CSCs. The data represent the mean ± SD of three independent technical replicates ( t ‐test); * P

    Article Snippet: Thereafter, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following proteins: octamer‐binding transcription factor 4 (OCT‐4, 1:1000, AB18976, Abcam, Cambridge, UK), sex‐determining region Y‐box (SOX‐9, 1:1000, AB185966, Abcam), Nanog (1:1000, AB106465, Abcam), CD63 (1:1000, PAB33929, Bioswamp), CD81 (1:1000, PAB33928, Bioswamp), TSG101 (1:1000, PAB32949, Bioswamp), PI3K (1:1000, AB191606, Abcam), phospho‐PI3K (p‐PI3K, 1:1000, AB182651, Abcam), Akt (1:1000, AB8805, Abcam), p‐Akt (1:1000, AB38449, Abcam), mTOR (1:2000, AB32028, Abcam), p‐mTOR (1:1000, AB84400, Abcam), and GAPDH (1:5000, PAB36264, Bioswamp).

    Techniques: Isolation, Expressing, Transwell Assay, Migration

    Human iPSCs expanded in a bioreactor have typical hiPSC morphology and express hPSC-associated markers post-harvest. ( A ) Phase contrast images of iPSCs expanded in a bioreactor plated either as single cells after cell release from MCs (left panel), or as clusters of cells on MCs (right panel). Magnification, 100×; Scale bar: 100 µm; ( B ) Immunofluorescence staining of OCT3/4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81 of iPSCs expanded in a bioreactor and plated onto 2D cell culture plates. Representative images from two different cell lines are shown. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers OCT3/4, SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of RTiPSC3B and LiPSC18R cells expanded in a bioreactor. Data obtained from either three independent runs (RTiPSC3B) or two independent runs (LiPSC18R). Data plotted are mean ± standard deviation (SD); ( D ) Pluripotency of RTiPSC3B cells expanded in a bioreactor demonstrated by embryoid body (EB) formation followed by immunofluorescence staining for germ layer-specific markers. Magnification, 100× (ectoderm, endoderm), 200× (mesoderm). ( E ) Representative immunofluorescence staining for lineage-specific markers RTiPSC3B and LiPSC18R cell lines. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)

    Journal: International Journal of Molecular Sciences

    Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

    doi: 10.3390/ijms21010089

    Figure Lengend Snippet: Human iPSCs expanded in a bioreactor have typical hiPSC morphology and express hPSC-associated markers post-harvest. ( A ) Phase contrast images of iPSCs expanded in a bioreactor plated either as single cells after cell release from MCs (left panel), or as clusters of cells on MCs (right panel). Magnification, 100×; Scale bar: 100 µm; ( B ) Immunofluorescence staining of OCT3/4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81 of iPSCs expanded in a bioreactor and plated onto 2D cell culture plates. Representative images from two different cell lines are shown. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers OCT3/4, SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of RTiPSC3B and LiPSC18R cells expanded in a bioreactor. Data obtained from either three independent runs (RTiPSC3B) or two independent runs (LiPSC18R). Data plotted are mean ± standard deviation (SD); ( D ) Pluripotency of RTiPSC3B cells expanded in a bioreactor demonstrated by embryoid body (EB) formation followed by immunofluorescence staining for germ layer-specific markers. Magnification, 100× (ectoderm, endoderm), 200× (mesoderm). ( E ) Representative immunofluorescence staining for lineage-specific markers RTiPSC3B and LiPSC18R cell lines. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)

    Article Snippet: The following primary antibodies were used to detect hPSC-associated markers: OCT4/POU5F1 (Abcam, ab19857, Cambridge, UK), NANOG (R & D systems, AF1997, Minneapolis, MN, USA), TRA-1-81 (ReproCELL USA, Inc., 09-001, Beltsville, MD, USA), TRA-1-60 (Millipore, MAB4360, Burlington, MA, USA) and SSEA-4 (Millipore, MAB4304, Burlington, MA, USA).

    Techniques: Immunofluorescence, Staining, Cell Culture, Flow Cytometry, Standard Deviation

    Cells expanded in a bioreactor and concentrated by kSep show hPSC characteristics of morphology and marker expression. ( A ) Phase contrast images of single cells (RTiPSC3B and LiPSC18R) post concentration by kSep400, 24 h and 72 h post plating onto 2D cell culture plates. Magnification, 40×; ( B ) Immunofluorescence staining RTiPSC3B cells expanded in a bioreactor and concentrated via kSep show expression of OCT3/4, NANOG, SSEA-4 and TRA-1-60. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of iPSCs expanded in a bioreactor and concentrated via kSep. Results are shown for RTiPSC3B and LiPSC18R lines; ( D ) Pluripotency of RTiPSC3B and LiPSC18R cells expanded in a bioreactor and concentrated by kSep were demonstrated by directed differentiation into definitive endoderm, neural stem cells and cardiomyocytes. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)

    Journal: International Journal of Molecular Sciences

    Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

    doi: 10.3390/ijms21010089

    Figure Lengend Snippet: Cells expanded in a bioreactor and concentrated by kSep show hPSC characteristics of morphology and marker expression. ( A ) Phase contrast images of single cells (RTiPSC3B and LiPSC18R) post concentration by kSep400, 24 h and 72 h post plating onto 2D cell culture plates. Magnification, 40×; ( B ) Immunofluorescence staining RTiPSC3B cells expanded in a bioreactor and concentrated via kSep show expression of OCT3/4, NANOG, SSEA-4 and TRA-1-60. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of iPSCs expanded in a bioreactor and concentrated via kSep. Results are shown for RTiPSC3B and LiPSC18R lines; ( D ) Pluripotency of RTiPSC3B and LiPSC18R cells expanded in a bioreactor and concentrated by kSep were demonstrated by directed differentiation into definitive endoderm, neural stem cells and cardiomyocytes. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)

    Article Snippet: The following primary antibodies were used to detect hPSC-associated markers: OCT4/POU5F1 (Abcam, ab19857, Cambridge, UK), NANOG (R & D systems, AF1997, Minneapolis, MN, USA), TRA-1-81 (ReproCELL USA, Inc., 09-001, Beltsville, MD, USA), TRA-1-60 (Millipore, MAB4360, Burlington, MA, USA) and SSEA-4 (Millipore, MAB4304, Burlington, MA, USA).

    Techniques: Marker, Expressing, Concentration Assay, Cell Culture, Immunofluorescence, Staining, Flow Cytometry

    Isolation and characterization of pES-269 cells with commonly used markers. ( A ) pES-269 cells isolation, Alkaline phosphatase (AKP) staining and immunofluorescence staining using anti-OCT4, -NANOG and -TRA-1-60 antibodies; ( B ) RT-PCR assays for the expression of pluripotency-associated markers in pES-269 cells; ( C ) Immunostaining of differentiated cells from embryoid bodies (EBs) formed by pES-269 cells, using antibodies against β-tubulin (ectoderm), SMA (mesoderm), and AFP (endoderm); ( D ) Karyotype analysis of pES-269 cells; ( E ) Genome-wide SNP analysis of pES-269 cells using an Affymetrix 250 K SNP Array. A homozygous AA allele maps to approximately +1, and a homozygous BB allele maps to approximately −1, with the heterozygote mapping to approximately 0.

    Journal: Scientific Reports

    Article Title: Self-diploidization of human haploid parthenogenetic embryos through the Rho pathway regulates endomitosis and failed cytokinesis

    doi: 10.1038/s41598-017-04602-y

    Figure Lengend Snippet: Isolation and characterization of pES-269 cells with commonly used markers. ( A ) pES-269 cells isolation, Alkaline phosphatase (AKP) staining and immunofluorescence staining using anti-OCT4, -NANOG and -TRA-1-60 antibodies; ( B ) RT-PCR assays for the expression of pluripotency-associated markers in pES-269 cells; ( C ) Immunostaining of differentiated cells from embryoid bodies (EBs) formed by pES-269 cells, using antibodies against β-tubulin (ectoderm), SMA (mesoderm), and AFP (endoderm); ( D ) Karyotype analysis of pES-269 cells; ( E ) Genome-wide SNP analysis of pES-269 cells using an Affymetrix 250 K SNP Array. A homozygous AA allele maps to approximately +1, and a homozygous BB allele maps to approximately −1, with the heterozygote mapping to approximately 0.

    Article Snippet: Zona-free embryos or ESCs were fixed in microtubule stabilizing buffer (to stain α-Tubulin, F-actin and p-MRLC: 0.1 M PIPES, PH 6.9, 2 mM MgCl2 .6H2 O, 2.5 mM EGTA, 2% formaldehyde, 0.5% Triton X-100 and 10μM taxol), ice-cold 10% trichloroacetic acid (TCA, to stain RhoA), or 4% paraformaldehyde (PFA, to stain β-tubulin, SMA, AFP, OCT-4, NANOG and TRA-1-60) for 30 min. Immunostaining was performed according to standard procedures and the following primary antibodies were used: mouse anti-RhoA (1:100; Santa Cruz Biotechnology, California, USA), mouse anti-α-Tubulin (1:200, Sigma), mouse antibodies recognizing the serine 19-phosphorylated form of myosin regulatory light chain (p-MRLC, 1:200; Cell Signaling Technology, Boston, USA), BODIPY-FL phallacidin (F-actin, 1:800, Sigma), mouse anti-OCT4 (1:50; Santa Cruz Biotechnology), rabbit anti-NANOG (1:50; Abcam, Cambridge, UK), mouse anti-TRA-1-60 (1:50; Millipore, Boston, MA, USA), mouse anti-β tubulin (1:800; Sigma), mouse anti-AFP (1:500; sigma), and mouse anti-SMA (1:100; Chemicon, California, USA).

    Techniques: Isolation, ALP Assay, Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Genome Wide

    Characterization of iPS cells for their pluripotency. Number 1 represents the cell line UTA.04602.WT and 2 the line UTA.04607.WT. A : iPS cells formed typical colonies for pluripotent stem cells that are rather compact and round in shape. B : The iPS cell colonies typically had well defined edges and distinct cell borders, and the iPS cells had a high nucleus to cytoplasm -ratio and a large nucleoli characteristic for stem cells. C : Endogenous pluripotency gene expression was studied using RT-PCR. Nanog, Oct4, Sox2 and Rex1 were all expressed at mRNA level in the iPS cells. β-actin and GAPDH were used as housekeeping control genes for both endogenous and exogenous markers. D : The expression of pluripotency genes was also studied at the protein level by immunocytochemical staining. The iPS cell expressed several markers for the pluripotent state: Nanog, Oct4, Sox2, SSEA4, TRA-1-60, and TRA-1-81 (all in red). Pictures in the left panel are from the line UTA.04602.WT and the ones on the right side are from UTA.04607.WT. Blue in all pictures indicates the DAPI staining of nuclei. E : Using RT-PCR, it was shown that all the transgenes were silenced in the iPS cells. Negative control is marked with “-” and positive control with “+”. F : Embryoid body (EB) -assay was used to define the pluripotency of the iPS cells in vitro . Markers for all three germ layers were detected from the EBs formed from both cell lines. Alpha - fetoprotein (AFP) was used as a marker for endoderm, kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR-2) was used as a marker for mesoderm and nestin was used as an ectoderm marker. GAPDH was used as an endogenous control gene.

    Journal: BioMedical Engineering OnLine

    Article Title: Video image-based analysis of single human induced pluripotent stem cell derived cardiomyocyte beating dynamics using digital image correlation

    doi: 10.1186/1475-925X-13-39

    Figure Lengend Snippet: Characterization of iPS cells for their pluripotency. Number 1 represents the cell line UTA.04602.WT and 2 the line UTA.04607.WT. A : iPS cells formed typical colonies for pluripotent stem cells that are rather compact and round in shape. B : The iPS cell colonies typically had well defined edges and distinct cell borders, and the iPS cells had a high nucleus to cytoplasm -ratio and a large nucleoli characteristic for stem cells. C : Endogenous pluripotency gene expression was studied using RT-PCR. Nanog, Oct4, Sox2 and Rex1 were all expressed at mRNA level in the iPS cells. β-actin and GAPDH were used as housekeeping control genes for both endogenous and exogenous markers. D : The expression of pluripotency genes was also studied at the protein level by immunocytochemical staining. The iPS cell expressed several markers for the pluripotent state: Nanog, Oct4, Sox2, SSEA4, TRA-1-60, and TRA-1-81 (all in red). Pictures in the left panel are from the line UTA.04602.WT and the ones on the right side are from UTA.04607.WT. Blue in all pictures indicates the DAPI staining of nuclei. E : Using RT-PCR, it was shown that all the transgenes were silenced in the iPS cells. Negative control is marked with “-” and positive control with “+”. F : Embryoid body (EB) -assay was used to define the pluripotency of the iPS cells in vitro . Markers for all three germ layers were detected from the EBs formed from both cell lines. Alpha - fetoprotein (AFP) was used as a marker for endoderm, kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR-2) was used as a marker for mesoderm and nestin was used as an ectoderm marker. GAPDH was used as an endogenous control gene.

    Article Snippet: Immunocytochemistry for pluripotency. iPS cells at passage 8 were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) and stained with anti-Oct3/4 (1:400, R & D Systems), anti-TRA1-60 (1:200, Millipore), anti-Sox2, anti-Nanog, anti-SSEA4, and anti-TRA1-81 (all 1:200, Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Negative Control, Positive Control, In Vitro, Marker

    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.

    Journal: International Journal of Biological Sciences

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

    doi: 10.7150/ijbs.3723

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: International Journal of Biological Sciences

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

    doi: 10.7150/ijbs.3723

    Figure Lengend 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.

    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.

    Techniques: Expressing, Marker, Immunofluorescence, Staining

    Embryonic stem cell markers. Immunohistochemistry: A . NANOG; B . OCT4. The majority of nuclei in the NANOG IHC are brown indicating the presence of NANOG. Some, but not all, nuclei are positive for OCT4. Negative (secondary antibody only) and positive (seminoma) controls are presented in the thumbnails below the photomicrographs. C . Immunofluorescence against nucleostemin. Left: anti-nucleostemin antibody, center: DAPI, right: merge. Bar = 10 microns.

    Journal: BMC Cell Biology

    Article Title: Phenotypic plasticity in normal breast derived epithelial cells

    doi: 10.1186/1471-2121-15-20

    Figure Lengend Snippet: Embryonic stem cell markers. Immunohistochemistry: A . NANOG; B . OCT4. The majority of nuclei in the NANOG IHC are brown indicating the presence of NANOG. Some, but not all, nuclei are positive for OCT4. Negative (secondary antibody only) and positive (seminoma) controls are presented in the thumbnails below the photomicrographs. C . Immunofluorescence against nucleostemin. Left: anti-nucleostemin antibody, center: DAPI, right: merge. Bar = 10 microns.

    Article Snippet: 100 ng of cDNA mixed with 5 μL 2× SensiMix SYBR N0-Rox Kit (Bioline) and 200 nM of each primer (Sigma, St. Louis, MO, USA; Applied Biosystems, Carlsbad, CA, USA: OCT4 (POU5F1 ) primer/probe set, Hs04260367_gH, cat# 4351372, NANOG primer/probe set Hs04260366_g1, cat# 4351372; and GAPDH primer/probe set, Hs99999905_m1, cat# 4331182) in a final volume of 10 μL.

    Techniques: Immunohistochemistry, Immunofluorescence

    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.

    Journal: Bioscience Reports

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

    doi: 10.1042/BSR20160247

    Figure Lengend 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.

    Article Snippet: Silencer Select siRNAs targeting Renilla luciferase or NANOG were obtained from Life Technologies.

    Techniques: 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.

    Journal: Bioscience Reports

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

    doi: 10.1042/BSR20160247

    Figure Lengend 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.

    Article Snippet: Silencer Select siRNAs targeting Renilla luciferase or NANOG were obtained from Life Technologies.

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

    PAF1c function during early lineage decisions. Our data suggests that PAF1c is required for molecular identity of ICM and TE cells after the first cell fate decision. Loss of function of CTR9 or RTF1 results in increased OCT4 and NANOG positive TE cells,

    Journal: Developmental biology

    Article Title: CTR9/PAF1c regulates molecular lineage identity, histone H3K36 trimethylation and genomic imprinting during preimplantation development

    doi: 10.1016/j.ydbio.2013.09.005

    Figure Lengend Snippet: PAF1c function during early lineage decisions. Our data suggests that PAF1c is required for molecular identity of ICM and TE cells after the first cell fate decision. Loss of function of CTR9 or RTF1 results in increased OCT4 and NANOG positive TE cells,

    Article Snippet: CDH1 (Abcam, Ab53033, 1:200, Rabbit); CDX2 (Biogenex, AM392-5 M, 1:200, Mouse); CTR9 (Abcam, Ab84487, 1:200, Rabbit); ELF5 (Santa Cruz, sc9645, 1:100, Goat); EOMES (Abcam, Ab23345, 1:200, Rabbit); GATA6 (R & D Systems, AF1700, 1:200, Goat); H3K36me3 (Abcam, Ab9050, 1:200, Rabbit); H3K4me3 (Abcam, Ab8580, 1:200, Rabbit); H3K9me3 (Abcam, Ab8988, 1:200 Rabbit); NANOG (CosmoBio, RCAB0002P-F, 1:200, Rabbit); OCT4 (Santa Cruz, sc5279, 1:200, Mouse); SOX17 (R & D Systems, NL1924R, 1:300, Goat); and SOX2 (Santa Cruz, sc17320, 1:200, Goat).

    Techniques:

    Generation of iPSCs and spinal MN differentiation. a iPSCs were generated from healthy control individuals and HMSN-P patients with TFG P285L mutation. Control and HMSN-P patient iPSCs were morphologically identical to human ESCs and expressed the pluripotent stem cell markers NANOG and SSEA4. Nucleus was stained with DAPI. Scale bars = 200 μm. Differentiated MNs were stained with neuronal marker βIII-tubulin, MAP2, and spinal MN marker SMI-32. Glial cells were stained with GFAP. Scale bars = 50 μm. b - e Proportions of control and HMSN-P patient neurons stained positive for βIII-tubulin ( b ), MAP2 ( c ), SMI-32 ( d ), and GFAP ( e ) ( n = 3, n.s. by Student t -test). There were no significant differences between control and HMSN-P groups. Error bars are ± s.e.m., n.s.: not significant

    Journal: Molecular Brain

    Article Title: Proteasome impairment in neural cells derived from HMSN-P patient iPSCs

    doi: 10.1186/s13041-017-0286-y

    Figure Lengend Snippet: Generation of iPSCs and spinal MN differentiation. a iPSCs were generated from healthy control individuals and HMSN-P patients with TFG P285L mutation. Control and HMSN-P patient iPSCs were morphologically identical to human ESCs and expressed the pluripotent stem cell markers NANOG and SSEA4. Nucleus was stained with DAPI. Scale bars = 200 μm. Differentiated MNs were stained with neuronal marker βIII-tubulin, MAP2, and spinal MN marker SMI-32. Glial cells were stained with GFAP. Scale bars = 50 μm. b - e Proportions of control and HMSN-P patient neurons stained positive for βIII-tubulin ( b ), MAP2 ( c ), SMI-32 ( d ), and GFAP ( e ) ( n = 3, n.s. by Student t -test). There were no significant differences between control and HMSN-P groups. Error bars are ± s.e.m., n.s.: not significant

    Article Snippet: The following primary antibodies were used in this assay: NANOG (1:500; ReproCELL, Yokohama, Japan), SSEA4 (1:1,000; Chemicon, Darmstadt, Germany), βIII-tubulin (1:2,000; Covance, Princeton, NJ), MAP2 (1:1,000; Millipore, Billerica, MA), SMI-32 (1:2,000; Covance), GFAP (1:1,000; DAKO, Glostrup, Denmark), TFG (1:1,000; Protein Tech, Rosemont, IL), Ubiquitin (1:1,000; DAKO), FK2 (1:1,000; MBL, Nagoya, Japan), SOX17 (1:1,000; R & D Systems, Minneapolis, MN), and αSMA (1:500; DAKO).

    Techniques: Generated, Mutagenesis, Staining, Marker

    Suppression of MEK/ERK signaling promotes self-renewal and colony morphology of mESCs. (a) PD promotes colony morphology of mESCs. J1 mESCs were treated with 1 μ M PD or equal volume of DMSO for 24 h. Morphological changes were observed and recorded under a phase contrast microscope. Scale bar = 50 μ m. (b) PD influences the expression pluripotent factors. ESCs were treated with or without 1 μ M PD for 24 h; then the expression levels of Nanog , Tfcp2l1 , Klf4 , c- Myc , and Egr1 were analyzed by RT-qPCR. Gapdh was used as a normalization control. Error bars indicate mean ± SD of three independent experiments, ∗ p

    Journal: Stem Cells International

    Article Title: Maintenance of Self-Renewal and Pluripotency in J1 Mouse Embryonic Stem Cells through Regulating Transcription Factor and MicroRNA Expression Induced by PD0325901

    doi: 10.1155/2016/1792573

    Figure Lengend Snippet: Suppression of MEK/ERK signaling promotes self-renewal and colony morphology of mESCs. (a) PD promotes colony morphology of mESCs. J1 mESCs were treated with 1 μ M PD or equal volume of DMSO for 24 h. Morphological changes were observed and recorded under a phase contrast microscope. Scale bar = 50 μ m. (b) PD influences the expression pluripotent factors. ESCs were treated with or without 1 μ M PD for 24 h; then the expression levels of Nanog , Tfcp2l1 , Klf4 , c- Myc , and Egr1 were analyzed by RT-qPCR. Gapdh was used as a normalization control. Error bars indicate mean ± SD of three independent experiments, ∗ p

    Article Snippet: The primary antibodies used were rabbit anti-Nanog (CST, Danvers, MA, USA), rabbit anti-Klf4 (Boster, Wuhan, China), mouse-anti-c-Myc (Santa Cruz, CA, USA), goat anti-Tet1 (Santa Cruz), rabbit anti-5hmC (Active Motif, Carlsbad, CA, USA), rabbit anti-Ezh2 (Abcam, Cambridge, UK), rabbit anti-H3K27me3 (Abcam), mouse anti-Oct3/4 (Santa Cruz), and mouse anti-Sox2 (Santa Cruz).

    Techniques: Microscopy, Expressing, Quantitative RT-PCR

    Generation, characterization, and differentiation efficiency of hESC-derived CEC. CEC were derived from H9 hESC, and three biological replicates were set up for each sample, and each experiment was repeated at least twice (the same hereafter unless stated otherwise). (A) A schematic showing the protocol for hESC differentiation into CEC in E6 with the cells split at day 40. (B) Morphology of the differentiated cells at days 10 and 40. Scale bar: 400 μm (a, b, c) and 100 μm (a', b', c'). (C) Flow cytometry analyses for the pluripotency marker NANOG, the corneal developmental markers PAX6, TP63, and KRT15, and the mature CEC markers KRT3 and KRT12 in hESC that differentiated in E6 for the designated times. (D) Immunostaining for TP63, PAX6, and KRT15 at day 45 of differentiation, and KRT3 and KRT12 at day 75. Scale bar: 50 μm for all images. (E) Real-time PCR analysis for expression of marker genes for pluripotency, CEC progenitors, and mature CEC, and transparency-associated genes during hESC differentiation to CEC for 8 weeks.

    Journal: Translational Vision Science & Technology

    Article Title: Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold

    doi: 10.1167/tvst.7.5.23

    Figure Lengend Snippet: Generation, characterization, and differentiation efficiency of hESC-derived CEC. CEC were derived from H9 hESC, and three biological replicates were set up for each sample, and each experiment was repeated at least twice (the same hereafter unless stated otherwise). (A) A schematic showing the protocol for hESC differentiation into CEC in E6 with the cells split at day 40. (B) Morphology of the differentiated cells at days 10 and 40. Scale bar: 400 μm (a, b, c) and 100 μm (a', b', c'). (C) Flow cytometry analyses for the pluripotency marker NANOG, the corneal developmental markers PAX6, TP63, and KRT15, and the mature CEC markers KRT3 and KRT12 in hESC that differentiated in E6 for the designated times. (D) Immunostaining for TP63, PAX6, and KRT15 at day 45 of differentiation, and KRT3 and KRT12 at day 75. Scale bar: 50 μm for all images. (E) Real-time PCR analysis for expression of marker genes for pluripotency, CEC progenitors, and mature CEC, and transparency-associated genes during hESC differentiation to CEC for 8 weeks.

    Article Snippet: The fixed cells were incubated in 0.1% Triton X-100 solution for 10 minutes, blocked in 5% bovine serum albumin (BSA) for 1 hour, and incubated with antibodies against NANOG (Cell Signaling Technologies), PAX6 (Invitrogen), TP63 (Boster, Pleasanton, CA) or KRT15 (Santa Cruz Biotechnology, Inc., Dallas, TX), HLA-A/B/C (ebioscience, San Diego, CA), B2M (Sigma-Aldrich Corp., St. Louis, MO) at the dilution ratio of 1:100 overnight in a cold room.

    Techniques: Derivative Assay, Capillary Electrochromatography, Flow Cytometry, Cytometry, Marker, Immunostaining, Real-time Polymerase Chain Reaction, Expressing

    Ythdf1, Ythdf2 and Ythdf3 are redundant in ESCs differentiation a) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in KH2 mESCs, showing a protein expression in the cytosolic compartment of the cell. b) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. c) Brightfield and immunostaining of Nanog (green), Esrrb (red) and DAPI (blue), in KO cells (single, triple and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb. d) Teratomas generated by the KO cell line and by WT control. Single-KO cell lines show all germ layers, while triple-KO teratomas as poorly differentiated. e) Immunostaining of triple-KO and WT control with Oct4 (red), Foxa (green), Tuj1 (purple) and DAPI (blue). Triple-KO contains patches of Oct4 staining, unlike WT teratomas. f) Alkaline phosphatase (AP) staining of disassociated teratomas from Triple-KO and WT control, showing a greater AP staining in the triple-KO cells. g) RT-PCR of pluripotent genes (left) and differentiation genes (right), measured in WT and KO EBs, and in WT mESCs as a control. In the triple-KO EBs, pluripotent markers are higher than the control and differentiation markers are lower than the WT control.

    Journal: bioRxiv

    Article Title: Context-dependent functional compensation between Ythdf m6A readers

    doi: 10.1101/2020.06.03.131441

    Figure Lengend Snippet: Ythdf1, Ythdf2 and Ythdf3 are redundant in ESCs differentiation a) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in KH2 mESCs, showing a protein expression in the cytosolic compartment of the cell. b) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. c) Brightfield and immunostaining of Nanog (green), Esrrb (red) and DAPI (blue), in KO cells (single, triple and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb. d) Teratomas generated by the KO cell line and by WT control. Single-KO cell lines show all germ layers, while triple-KO teratomas as poorly differentiated. e) Immunostaining of triple-KO and WT control with Oct4 (red), Foxa (green), Tuj1 (purple) and DAPI (blue). Triple-KO contains patches of Oct4 staining, unlike WT teratomas. f) Alkaline phosphatase (AP) staining of disassociated teratomas from Triple-KO and WT control, showing a greater AP staining in the triple-KO cells. g) RT-PCR of pluripotent genes (left) and differentiation genes (right), measured in WT and KO EBs, and in WT mESCs as a control. In the triple-KO EBs, pluripotent markers are higher than the control and differentiation markers are lower than the WT control.

    Article Snippet: The following primary antibodies were used: Ythdf1 (Proteintech, 17479-1-AP), Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Mettl3 (Proteintech Group 15073-1-AP), Nanog (Bethyl, A300-397A or eBioscience, 14-5761), Esrrb (R & D systems, PP-H6705-00), Oct4 (Santa Cruz, SC9081 or SC5279), Foxa2 (Santa Cruz, sc-6554), Tuj1 (BioLegend, 801202), Tubulin (Santa Cruz, sc-23950).

    Techniques: Immunostaining, Expressing, Generated, Staining, Reverse Transcription Polymerase Chain Reaction

    Depletion of MGP allows for multipotency and osteoinduction in human aortic endothelial cells (HAECs) (A) Expression of multipotent markers Sox2, Nanog and Oct3/4 in HAECs after transfection of scrambled control siRNA (SCR) or MGP siRNA only (lane 1, 2), siRNA transfection with BMP4 treatment (lane 3, 4), siRNA transfection with Noggin treatment (lane 5, 6), or siRNA transfection with BMP4 and Noggin treatment (lane 7, 8), as determined by immunoblotting. (B) Co-expression of CD31 and SSEA-3, or CD31 and SSEA-4 after transfection of scrambled control siRNA or MGP siRNA without additional treatment (left), and with BMP4 treatment (right), as determined by FACS analysis. (C) Expression of multipotent markers Sox2, Nanog and Oct3/4, osteogenic markers Cbfa1, Osterix (OSX), early SMC markers SM22α, α-SM actin (αSMA), and late SMC marker SM-myosin heavy chain (SM-MHC) (top) after transfection of scrambled control siRNA or MGP siRNA and treatment with control (C), BMP2 (B), osteogenic media (O) or both (B+O), as determined by immunoblotting. (D) Staining for alkaline phosphatase (ALP), and mineral (Alizarin Red and Von Kossa staining) in HAECS treated as described in (c). (E) Expression of multipotent markers Sox2, Nanog and Oct3/4, osteogenic markers Cbfa1, OSX, SM22α, αSMA, and SM-MHC (top) after transfection of scrambled control siRNA or MGP siRNA and treatment with control (C), BMP2 (B), high glucose medium (G) or both (B+G), as determined by immunoblotting. (F) Staining for ALP and mineral in HAECS treated as described in (E).

    Journal: Circulation research

    Article Title: A Role for the Endothelium in Vascular Calcification

    doi: 10.1161/CIRCRESAHA.113.301792

    Figure Lengend Snippet: Depletion of MGP allows for multipotency and osteoinduction in human aortic endothelial cells (HAECs) (A) Expression of multipotent markers Sox2, Nanog and Oct3/4 in HAECs after transfection of scrambled control siRNA (SCR) or MGP siRNA only (lane 1, 2), siRNA transfection with BMP4 treatment (lane 3, 4), siRNA transfection with Noggin treatment (lane 5, 6), or siRNA transfection with BMP4 and Noggin treatment (lane 7, 8), as determined by immunoblotting. (B) Co-expression of CD31 and SSEA-3, or CD31 and SSEA-4 after transfection of scrambled control siRNA or MGP siRNA without additional treatment (left), and with BMP4 treatment (right), as determined by FACS analysis. (C) Expression of multipotent markers Sox2, Nanog and Oct3/4, osteogenic markers Cbfa1, Osterix (OSX), early SMC markers SM22α, α-SM actin (αSMA), and late SMC marker SM-myosin heavy chain (SM-MHC) (top) after transfection of scrambled control siRNA or MGP siRNA and treatment with control (C), BMP2 (B), osteogenic media (O) or both (B+O), as determined by immunoblotting. (D) Staining for alkaline phosphatase (ALP), and mineral (Alizarin Red and Von Kossa staining) in HAECS treated as described in (c). (E) Expression of multipotent markers Sox2, Nanog and Oct3/4, osteogenic markers Cbfa1, OSX, SM22α, αSMA, and SM-MHC (top) after transfection of scrambled control siRNA or MGP siRNA and treatment with control (C), BMP2 (B), high glucose medium (G) or both (B+G), as determined by immunoblotting. (F) Staining for ALP and mineral in HAECS treated as described in (E).

    Article Snippet: Blots were incubated with specific antibodies to CD31 (300 ng/ml; Cell Signaling Technology); VE-cadherin (400 ng/ml; Santa Cruz Biotechnology); Flk-1 (200 ng/ml; Santa Cruz Biotechnology); Sox2 (200 ng/ml; Cell Signaling); Nanog (400 ng/ml; BD Pharmingen and eBioscience); Oct3/4 (200 ng/ml; R & D Systems); Cbfa1 (500 ng/ml; Oncogene Research Products); Osterix ); SM22α (200 ng/ml; Santa Cruz Biotechnology) and αSMA (200 ng/ml; R & D Systems). β-Actin (1:5,000 dilution; Sigma-Aldrich) was used as loading control.

    Techniques: Expressing, Transfection, FACS, Marker, Staining, ALP Assay

    Multipotent marker expression in Mgp −/− and Mgp −/− ;Tie2-Gfptg endothelium (A) Aortic expression of Sox2, Nanog and Oct3/4 in Mgp +/+ and Mgp − / − mice detected by immunoblotting (left) and immunostaining (right). β-actin was used as control. (B) Aortic expression of Sox2, Nanog and Oct3/4 in Tie2-Gfp tg and Mgp − / − ;Tie2-Gfp tgmice determined by immunoblotting (left) and immunostaining (right). β-actin was used as control. (C) Co-expression of CD31 and Sox2 in enzymatically dispersed CD45-negative aortic cells from Mgp +/+ and Mgp − / − mice, as determined by FACS. Scale bars, 50 μm. Non-specific IgG control showed no staining. Vessel lumen faces upwards in the photos.

    Journal: Circulation research

    Article Title: A Role for the Endothelium in Vascular Calcification

    doi: 10.1161/CIRCRESAHA.113.301792

    Figure Lengend Snippet: Multipotent marker expression in Mgp −/− and Mgp −/− ;Tie2-Gfptg endothelium (A) Aortic expression of Sox2, Nanog and Oct3/4 in Mgp +/+ and Mgp − / − mice detected by immunoblotting (left) and immunostaining (right). β-actin was used as control. (B) Aortic expression of Sox2, Nanog and Oct3/4 in Tie2-Gfp tg and Mgp − / − ;Tie2-Gfp tgmice determined by immunoblotting (left) and immunostaining (right). β-actin was used as control. (C) Co-expression of CD31 and Sox2 in enzymatically dispersed CD45-negative aortic cells from Mgp +/+ and Mgp − / − mice, as determined by FACS. Scale bars, 50 μm. Non-specific IgG control showed no staining. Vessel lumen faces upwards in the photos.

    Article Snippet: Blots were incubated with specific antibodies to CD31 (300 ng/ml; Cell Signaling Technology); VE-cadherin (400 ng/ml; Santa Cruz Biotechnology); Flk-1 (200 ng/ml; Santa Cruz Biotechnology); Sox2 (200 ng/ml; Cell Signaling); Nanog (400 ng/ml; BD Pharmingen and eBioscience); Oct3/4 (200 ng/ml; R & D Systems); Cbfa1 (500 ng/ml; Oncogene Research Products); Osterix ); SM22α (200 ng/ml; Santa Cruz Biotechnology) and αSMA (200 ng/ml; R & D Systems). β-Actin (1:5,000 dilution; Sigma-Aldrich) was used as loading control.

    Techniques: Marker, Expressing, Mouse Assay, Immunostaining, FACS, Staining

    Time course of aortic changes in Mgp −/− mouse aorta (A) Aortas were collected between postnatal day (P) 2–30 from Mgp +/+ and Mgp − / − mice as indicated, and stained with H E. (B) Time course of aortic expression in Mgp +/+ and Mgp − / − aorta (P2–30) of MGP, BMP4, EC markers VE-cadherin (VE-cad) and CD31, osteogenic markers Cbfa1 and Osterix (OSX), and multipotency markers Nanog, Sox2 and Oct3/4. The expression was compared to that on P2. Scale bars, 100 μm.

    Journal: Circulation research

    Article Title: A Role for the Endothelium in Vascular Calcification

    doi: 10.1161/CIRCRESAHA.113.301792

    Figure Lengend Snippet: Time course of aortic changes in Mgp −/− mouse aorta (A) Aortas were collected between postnatal day (P) 2–30 from Mgp +/+ and Mgp − / − mice as indicated, and stained with H E. (B) Time course of aortic expression in Mgp +/+ and Mgp − / − aorta (P2–30) of MGP, BMP4, EC markers VE-cadherin (VE-cad) and CD31, osteogenic markers Cbfa1 and Osterix (OSX), and multipotency markers Nanog, Sox2 and Oct3/4. The expression was compared to that on P2. Scale bars, 100 μm.

    Article Snippet: Blots were incubated with specific antibodies to CD31 (300 ng/ml; Cell Signaling Technology); VE-cadherin (400 ng/ml; Santa Cruz Biotechnology); Flk-1 (200 ng/ml; Santa Cruz Biotechnology); Sox2 (200 ng/ml; Cell Signaling); Nanog (400 ng/ml; BD Pharmingen and eBioscience); Oct3/4 (200 ng/ml; R & D Systems); Cbfa1 (500 ng/ml; Oncogene Research Products); Osterix ); SM22α (200 ng/ml; Santa Cruz Biotechnology) and αSMA (200 ng/ml; R & D Systems). β-Actin (1:5,000 dilution; Sigma-Aldrich) was used as loading control.

    Techniques: Mouse Assay, Staining, Expressing

    Endothelial origin of multipotent cells in aortas of diabetic Ins2 Akita/+ mice (A) Aortic expression of Sox2, Nanog and Oct3/4 in wild type (WT), Ins2 Akita/+ , Ins2 Akita/+ ;Tie2-Gfp tg and Ins2 Akita/+ ;Tie2-Gfp tg mice. β-actin was used as control. (B) Co-expression of CD31 with Sox2, Nanog and Oct3/4 in aortas of Ins2 Akita/+ mice detected by immunostaining. (C) Co-expression of GFP with Sox2, Nanog and Oct3/4 in aortas of Tie2-Gfp tg and Ins2 Akita/+ ;Tie2-Gfp tg mice detected by immunostaining. (D) Co-expression of CD31 and Sox2 in enzymatically dispersed CD45-negative aortic cells from WT and Ins2 Akita/+ mice , as determined by FACS. (E–G) Enhanced MGP expression limits aortic expression of Sox2, Nanog and Oct3/4 in Ins2 Akita/+ mice, as determined by (E) real-time PCR, (F) immunoblotting (β-actin was used as control), and (G) immunostaining in WT, Mgp tg/wt , Ins2 Akita/+ , and Ins2 Akita/+ ;Mgp tg/wt mice. Scale bars, 50 μm. DAPI (blue) was used to visualize nuclei. Vessel lumen faces upwards in the photos.

    Journal: Circulation research

    Article Title: A Role for the Endothelium in Vascular Calcification

    doi: 10.1161/CIRCRESAHA.113.301792

    Figure Lengend Snippet: Endothelial origin of multipotent cells in aortas of diabetic Ins2 Akita/+ mice (A) Aortic expression of Sox2, Nanog and Oct3/4 in wild type (WT), Ins2 Akita/+ , Ins2 Akita/+ ;Tie2-Gfp tg and Ins2 Akita/+ ;Tie2-Gfp tg mice. β-actin was used as control. (B) Co-expression of CD31 with Sox2, Nanog and Oct3/4 in aortas of Ins2 Akita/+ mice detected by immunostaining. (C) Co-expression of GFP with Sox2, Nanog and Oct3/4 in aortas of Tie2-Gfp tg and Ins2 Akita/+ ;Tie2-Gfp tg mice detected by immunostaining. (D) Co-expression of CD31 and Sox2 in enzymatically dispersed CD45-negative aortic cells from WT and Ins2 Akita/+ mice , as determined by FACS. (E–G) Enhanced MGP expression limits aortic expression of Sox2, Nanog and Oct3/4 in Ins2 Akita/+ mice, as determined by (E) real-time PCR, (F) immunoblotting (β-actin was used as control), and (G) immunostaining in WT, Mgp tg/wt , Ins2 Akita/+ , and Ins2 Akita/+ ;Mgp tg/wt mice. Scale bars, 50 μm. DAPI (blue) was used to visualize nuclei. Vessel lumen faces upwards in the photos.

    Article Snippet: Blots were incubated with specific antibodies to CD31 (300 ng/ml; Cell Signaling Technology); VE-cadherin (400 ng/ml; Santa Cruz Biotechnology); Flk-1 (200 ng/ml; Santa Cruz Biotechnology); Sox2 (200 ng/ml; Cell Signaling); Nanog (400 ng/ml; BD Pharmingen and eBioscience); Oct3/4 (200 ng/ml; R & D Systems); Cbfa1 (500 ng/ml; Oncogene Research Products); Osterix ); SM22α (200 ng/ml; Santa Cruz Biotechnology) and αSMA (200 ng/ml; R & D Systems). β-Actin (1:5,000 dilution; Sigma-Aldrich) was used as loading control.

    Techniques: Mouse Assay, Expressing, Immunostaining, FACS, Real-time Polymerase Chain Reaction

    The effects of miR-3622a-3p and SALL4 on stemness features of CRC cells. a – d Flow cytometric analysis was used to examine the percentage of CD133(+) CRC cells. e – h Sphere formation assay was performed to investigate the stem-cell like properties of stable transfected CRC cells. i , j Expression levels of Sox2, Nanog, Oct4, CD44, and CD133 were determined by qRT-PCR in stable transfected CRC cells. k , l Protein levels of CSC-related biomarkers and pluripotency-related genes were detected by western blot in stable transfected CRC cells. All data are from three independent experiments and are presented as the means ± SD (* p

    Journal: Cell Death & Disease

    Article Title: MiR-3622a-3p acts as a tumor suppressor in colorectal cancer by reducing stemness features and EMT through targeting spalt-like transcription factor 4

    doi: 10.1038/s41419-020-02789-z

    Figure Lengend Snippet: The effects of miR-3622a-3p and SALL4 on stemness features of CRC cells. a – d Flow cytometric analysis was used to examine the percentage of CD133(+) CRC cells. e – h Sphere formation assay was performed to investigate the stem-cell like properties of stable transfected CRC cells. i , j Expression levels of Sox2, Nanog, Oct4, CD44, and CD133 were determined by qRT-PCR in stable transfected CRC cells. k , l Protein levels of CSC-related biomarkers and pluripotency-related genes were detected by western blot in stable transfected CRC cells. All data are from three independent experiments and are presented as the means ± SD (* p

    Article Snippet: The primary antibodies included: anti-SALL4 (ab57577), anti-GAPDH (ab8245), anti-Sox2 (ab79351), anti-Nanog (ab21624), anti-Oct4 (ab19857), anti-CD44 (ab157107), anti-CD133 (ab216323), anti-E-cadherin (ab1416), anti-N-cadherin (ab76057), anti-Vimentin (ab92547), anti-Snail (ab53519), anti-TWIST (ab50581), anti-beta-catenin (ab32572) and anti-Cyclin D1 (ab16663) from Abcam (Cambridge, UK), anti-TCF1 (#2203) and anti-c-myc (#5605) from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Tube Formation Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    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

    LIF alone supports ESCs self-renew and pluripotency. (A) Experimental outline of the L-ESCs derivation procedures from ESCs. (B) 2i/L-ESCs were switched to L-medium and cultured to passages 3 (p3), p5, p25. Here we use 2i/L-ESCs with GOF/GFP reporter. Scale bars, 100 μm. (C) Karyotyping of L-ESCs (p30). (D) Immunostaining of OCT4, SOX2 and NANOG in L-ESCs. Scale bars, 50 μm. (E) Single cell clonogenicity efficiency in L-ESCs and 2i/L-ESCs. (F) L-ESCs were treated with JAK inhibitor I after day 3 p2 and day 10 p4. Scale bars, 100 μm. (G) 2i/L-ESCs were treated with JAK inhibitor I after day 3 p2, day 10 p6 and p10. Scale bars, 100 μm.

    Journal: bioRxiv

    Article Title: DNA methylation is indispensable for leukemia inhibitory factor dependent embryonic stem cells reprogramming

    doi: 10.1101/2020.03.17.994939

    Figure Lengend Snippet: LIF alone supports ESCs self-renew and pluripotency. (A) Experimental outline of the L-ESCs derivation procedures from ESCs. (B) 2i/L-ESCs were switched to L-medium and cultured to passages 3 (p3), p5, p25. Here we use 2i/L-ESCs with GOF/GFP reporter. Scale bars, 100 μm. (C) Karyotyping of L-ESCs (p30). (D) Immunostaining of OCT4, SOX2 and NANOG in L-ESCs. Scale bars, 50 μm. (E) Single cell clonogenicity efficiency in L-ESCs and 2i/L-ESCs. (F) L-ESCs were treated with JAK inhibitor I after day 3 p2 and day 10 p4. Scale bars, 100 μm. (G) 2i/L-ESCs were treated with JAK inhibitor I after day 3 p2, day 10 p6 and p10. Scale bars, 100 μm.

    Article Snippet: Primary antibodies used were: anti-OCT4 (BD Biosciences, Catalog Number: 611203, 1:200), anti-NANOG (eBioscience, Catalog Number: 14-5761, 1:500), anti-SOX2 (Santa cruz, Catalog Number: sc-17320, 1:200), anti-H3K27me3 (Upstate, Catalog Number: 07-449, 1:500), anti-ZSCAN4 (Abcam, Catalog Number: ab106646, 1:200), anti-MERVL (HuaAn Bio, Catalog Number: ER50102, 1:100), anti-DNMT3A (abcam, Catalog Number: ab79822, 1:500), anti-NESTIN (BOSTER Bio, Catalog Number: BM4494, 1:50), anti-BRACHYURY (R & D Systems, Catalog Number: AF2085, 1:100) and anti-SOX17 (R & D Systems, Catalog Number: AF1924, 1:100).

    Techniques: Cell Culture, Immunostaining

    Analysis of Rex1-Positive and -Negative Serum ES Cell Fractions, Related to Figure 1 (A) ES cell morphology in 2i or serum culture. (B) Oct4 (left) and Klf4 (right) immunostaining of ES cells in 2i or serum. Arrows point to Klf4-negative ES cells in serum. (C) GFP flow cytometry profile of the total population of Rex1-GFP serum ES cells (black) and ES cells without any GFP transgene (negative control, gray (largely overlapping red)). The GFP-negative and -positive sorted Rex1-GFP serum ES cells used in this study are shown in red and green, respectively. (D) Number of colonies after re-plating 2i ES cells and Rex1-positive and Rex1-negative ES cells at clonal density in serum (left) or 2i (right). After 5 days (serum) or 7 days (2i + LIF), alkaline phosphatase staining was performed to discriminate between colonies consisting of largely undifferentiated cells (undiff), mixed, or largely differentiated (diff) cells. (E) Klf4 and Nanog immunostaining of unsorted Rex1-GFP serum ES cells. The arrowheads in the DAPI stainings point to Rex1-, Klf4-, and Nanog-negative cells, the arrows in the GFP+Klf4+Nanog stainings to Rex1-positive, but Klf4- and Nanog-negative cells. (F) Comparison of expression of pluripotency and lineage-specific genes (as shown in Figures 1 B and 1D) of Rex1-positive (Serum-Rex-pos) and Rex1-negative (Serum-Rex-neg) ES cells. (G) Transcript levels of genes associated with the various germ layers in Rex1-positive and Rex1-negative serum ES cells. (H) Functional analysis of the differential genes between Rex1-positive and Rex1-negative serum ES cells. (I) Functional analysis of the differential genes between 2i ES cells and Rex1-positive serum ES cells.

    Journal: Cell

    Article Title: The Transcriptional and Epigenomic Foundations of Ground State Pluripotency

    doi: 10.1016/j.cell.2012.03.026

    Figure Lengend Snippet: Analysis of Rex1-Positive and -Negative Serum ES Cell Fractions, Related to Figure 1 (A) ES cell morphology in 2i or serum culture. (B) Oct4 (left) and Klf4 (right) immunostaining of ES cells in 2i or serum. Arrows point to Klf4-negative ES cells in serum. (C) GFP flow cytometry profile of the total population of Rex1-GFP serum ES cells (black) and ES cells without any GFP transgene (negative control, gray (largely overlapping red)). The GFP-negative and -positive sorted Rex1-GFP serum ES cells used in this study are shown in red and green, respectively. (D) Number of colonies after re-plating 2i ES cells and Rex1-positive and Rex1-negative ES cells at clonal density in serum (left) or 2i (right). After 5 days (serum) or 7 days (2i + LIF), alkaline phosphatase staining was performed to discriminate between colonies consisting of largely undifferentiated cells (undiff), mixed, or largely differentiated (diff) cells. (E) Klf4 and Nanog immunostaining of unsorted Rex1-GFP serum ES cells. The arrowheads in the DAPI stainings point to Rex1-, Klf4-, and Nanog-negative cells, the arrows in the GFP+Klf4+Nanog stainings to Rex1-positive, but Klf4- and Nanog-negative cells. (F) Comparison of expression of pluripotency and lineage-specific genes (as shown in Figures 1 B and 1D) of Rex1-positive (Serum-Rex-pos) and Rex1-negative (Serum-Rex-neg) ES cells. (G) Transcript levels of genes associated with the various germ layers in Rex1-positive and Rex1-negative serum ES cells. (H) Functional analysis of the differential genes between Rex1-positive and Rex1-negative serum ES cells. (I) Functional analysis of the differential genes between 2i ES cells and Rex1-positive serum ES cells.

    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.

    Techniques: Immunostaining, Flow Cytometry, Cytometry, Negative Control, Staining, Expressing, Functional Assay

    Immunocytochemistry of SOX2, OCT4, NANOG, EMD and SSEA1 in hESCs with and without BANF1 knockdown. CHB-4 hESCs were transduced with either Scrambled or human Banf1 shRNA lentiviral vectors, selected with puromycin to eliminate uninfected cells, and seeded

    Journal: Journal of Cell Science

    Article Title: Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells

    doi: 10.1242/jcs.083238

    Figure Lengend Snippet: Immunocytochemistry of SOX2, OCT4, NANOG, EMD and SSEA1 in hESCs with and without BANF1 knockdown. CHB-4 hESCs were transduced with either Scrambled or human Banf1 shRNA lentiviral vectors, selected with puromycin to eliminate uninfected cells, and seeded

    Article Snippet: Other primary antibodies used are against: OCT4 (1:500, sc-8628, Santa Cruz Biotechnology), SOX2 (1:1000, ab-75179, Abcam, Cambridge, MA), NANOG (1:500, AF2729, R & D Systems, Minneapolis, MN), HDAC1 (1:5000, ab-7028, Abcam), HDAC2 (1:5000, ab-7029, Abcam).

    Techniques: Immunocytochemistry, Transduction, shRNA

    Knockdown of Banf1 in mESCs causes changes in cell morphology, elevation of gene markers of differentiation and reduction in the protein levels of Oct4, Sox2 and Nanog. ( A ) Banf1 levels were examined by western blot analysis using nuclear proteins isolated

    Journal: Journal of Cell Science

    Article Title: Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells

    doi: 10.1242/jcs.083238

    Figure Lengend Snippet: Knockdown of Banf1 in mESCs causes changes in cell morphology, elevation of gene markers of differentiation and reduction in the protein levels of Oct4, Sox2 and Nanog. ( A ) Banf1 levels were examined by western blot analysis using nuclear proteins isolated

    Article Snippet: Other primary antibodies used are against: OCT4 (1:500, sc-8628, Santa Cruz Biotechnology), SOX2 (1:1000, ab-75179, Abcam, Cambridge, MA), NANOG (1:500, AF2729, R & D Systems, Minneapolis, MN), HDAC1 (1:5000, ab-7028, Abcam), HDAC2 (1:5000, ab-7029, Abcam).

    Techniques: Western Blot, Isolation

    Immunocytochemistry of Sox2, Oct4, Nanog, SSEA1 and Emd in mESCs with and without Banf1 knockdown. D3 mESCs were infected, selected with puromycin and seeded into a 12-well plate for immunocytochemistry as described in the Materials and Methods. Photomicrographs

    Journal: Journal of Cell Science

    Article Title: Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells

    doi: 10.1242/jcs.083238

    Figure Lengend Snippet: Immunocytochemistry of Sox2, Oct4, Nanog, SSEA1 and Emd in mESCs with and without Banf1 knockdown. D3 mESCs were infected, selected with puromycin and seeded into a 12-well plate for immunocytochemistry as described in the Materials and Methods. Photomicrographs

    Article Snippet: Other primary antibodies used are against: OCT4 (1:500, sc-8628, Santa Cruz Biotechnology), SOX2 (1:1000, ab-75179, Abcam, Cambridge, MA), NANOG (1:500, AF2729, R & D Systems, Minneapolis, MN), HDAC1 (1:5000, ab-7028, Abcam), HDAC2 (1:5000, ab-7029, Abcam).

    Techniques: Immunocytochemistry, Infection