anti-oct4 Search Results


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  • 94
    Millipore anti pou5f1 oct4 antibody
    Schematics of developmental events in the ventral and dorsal ovarian regions during the embryonic period and first wave of follicle activation. Early onset of meiosis in the ventral region is represented in the insert <t>(OCT4</t> and STRA8). First primordial
    Anti Pou5f1 Oct4 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend pe anti oct4 oct3
    Metformin decreases K7M2 xenograft tumor growth and inhibits lung metastasis in vivo . (a) Metformin suppressed tumorigenicity of K7M2 OSCs in vivo by limited dilution assay. n = 7. (b) The tumor formation rate of K7M2 OSCs treated with metformin. (c) The tumor volumes and (d) tumor weights at the end of the experiments. (e) Images of resected tumor xenografts on day 21. n = 6. (f) The tumor volumes and (g) the tumor weights at the end of the experiments. (h) Numbers of mice with lung metastasis. (i) H E staining of lung tissues for metastatic nodules. The arrows represent smaller tumor nodules in the lung. Scale bars = 100 μ m. (j) Immunohistochemical staining of LC3, ATG5, Ki67, Sox2, and <t>Oct4</t> of three different tumors from each group. Scale bars = 100 μ m. ∗ P
    Pe Anti Oct4 Oct3, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology anti oct4 pou5f1
    GridMedian extracts features from fluorescent image data while reduce the size of data. ( A,B ) The median intensities of all 144 grids from the test image set ( Fig. 1A ) are plotted. <t>Anti-Oct4</t> intensity does not correlate with EGFP intensity ( A ), while well correlate with H33342 intensity ( B ). The 144 grids are classified to 4 sub-classes by Mclust according to the 3-channel intensities (H33342, <t>anti-Oct4</t> and EGFP) without drawing threshold lines arbitrarily. The 4 sub-classes are shown in 4 different colours and tentatively designated. Misclassified grids are encircled ( B ). ( C ) Kernel density estimations indicate very similar EGFP expression profiles between EGFP(−) and void sub-classes. High-peak of sub-class designated “other” (blue) corresponds cytoplasmic region of EGFP(+) mESCs. ( D ) Oct4 expression profiles between EGFP(−) and EGFP(+) sub-classes suggests higher expression of Oct4 in EGFP(+) mESCs. Smoothing kernel = Gaussian, bandwidth = 0.05. ( E ) After successful application of “Median_IQR filter” (see Supplementary Fig. S2 ) on the 144 grids, 67 of class 3 grids are again classified by Mclust leading EGFP(+) and EGFP(−) sub-classes. Suspected misclassified grids are encircled. ( F ) The Oct4 expression profiles between 51 of EGFP(+) and 16 of EGFP(−) sub-classes indicate higher expression of Oct4 in mESCs. Sample script (GBIQ_ESC.R) to reproduce the result and figures is available from https://github.com/yo-ninomy/DemoScripts .
    Anti Oct4 Pou5f1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ABclonal anti oct4
    1α,25(OH)2D3 inhibited the stemness and impaired the mitochondrial respiration and ATP production in acidic condition. a Immunoblotting of the expression of stemness markers NESTIN, CD133, <t>OCT4,</t> and SOX2 in GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM for 4, 8, 12, 24, and 48 h. b and c Self-renewal ability of SLCs with 1α,25(OH) 2 D 3 treatment under pH 7.4 or pH 6.8 culture conditions. Neurosphere formation assay showed the number of neurospheres (diameters larger than 50 µm) formed from GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM ( b ), * P
    Anti Oct4, supplied by ABclonal, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend alexa fluor 647 anti oct4 oct3
    Low efficiency and slow kinetics during reprogramming of SIRT2-knockout cells. a Delayed morphological changes in SIRT2-KO-MEFs. Scale bar = 400 μm. b Number of alkaline phosphatase-positive colonies. c Proportion of the SSEA-1(+)/CD44(−) fraction. d Proportion of the <t>OCT4(+)/CD44(−)</t> fraction. Non-reprogrammed MEFs are shown as negative control and mouse ESCs are shown as positive control. Samples treated with FITC- and <t>Alexa</t> Fluor 647-conjugated mouse immunoglobulin Gs (IgGs) are presented in the upper panel as negative controls for staining. * P
    Alexa Fluor 647 Anti Oct4 Oct3, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend alexa fluor 488 anti oct4 oct3
    Low efficiency and slow kinetics during reprogramming of SIRT2-knockout cells. a Delayed morphological changes in SIRT2-KO-MEFs. Scale bar = 400 μm. b Number of alkaline phosphatase-positive colonies. c Proportion of the SSEA-1(+)/CD44(−) fraction. d Proportion of the <t>OCT4(+)/CD44(−)</t> fraction. Non-reprogrammed MEFs are shown as negative control and mouse ESCs are shown as positive control. Samples treated with FITC- and <t>Alexa</t> Fluor 647-conjugated mouse immunoglobulin Gs (IgGs) are presented in the upper panel as negative controls for staining. * P
    Alexa Fluor 488 Anti Oct4 Oct3, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti oct 4
    <t>Oct-4</t> immunocytochemical staining of sheep spermatogonial stem cells (×40).
    Anti Oct 4, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti oct 4 antibody
    The effect of B7-H3 on mouse SSC proliferation A. The expression of C-kit and <t>Oct-4</t> on mouse SSCs was analyzed by flow cytometry. B. Mouse SSCs were incubated with different concentrations of B7-H3 (0, 5, 10, 25 ng/mL) for various time points (1, 6, 12, 24, 48, 72 h) in vitro . Then, cells were collected for proliferation assessment using CCK8 assays. The results are expressed as the mean±standard deviation ( n = 3). Differences were analyzed by two-way ANOVA followed by Tukey's post hoc analysis. ###P
    Anti Oct 4 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    oct4  (Abcam)
    94
    Abcam oct4
    Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of <t>Oct4,</t> Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p
    Oct4, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 2436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti oct4
    Characterization and differentiation of patient-derived NLSDM-iPSC. (A) Immunostaining with primary antibodies <t>anti-OCT4,</t> anti-SSEA-4 and anti-TRA-1-81 revealed that NLSDM-iPSCs express markers of pluripotency, including OCT4 (red), SSEA4 (green), and TRA-1-81 (green). Nuclei were stained with DAPI. Magnification: 10 ×. (B) Real-time quantitative RT-PCR assays for eight pluripotency-associate genes in two patient-derived iPSCs compared with fibroblast cell lines (basal level reported as 1). Data represent the mean of three independent experiments, performed in triplicate. PCR reactions were normalized against an internal control (ACTB). (C) Quantification of TG content in iPSCs derived from 2 controls and 2 NLSDM fibroblast cell lines. The graph represents TG content obtained from three independent experiments. The analysis was performed using Student t -test. Thick bar: mean value; error bar: SD. P-values of
    Anti Oct4, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stemgent oct4
    Characterization of human iPS A. Human iPS cells observed in phase contrast. B. Human iPS labeled with pluripotency markers: anti-SOX2, anti-NANOG and <t>anti-OCT4</t> with DAPI counterstaining (right). C. iPS cells labeled for alkaline phosphatase (AP) and observed in phase contrast. D. An example of typical DNA content of iPS cells after propidium iodide labeling measured by flow cytometry.
    Oct4, supplied by Stemgent, used in various techniques. Bioz Stars score: 93/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Marque oct4
    Distinct expression patterns of CSC markers in lung adenocarcinoma and squamous cell carcinoma A. CD133, B. CD44, C. ALDH1, D. SOX2, E. <t>OCT4,</t> and F. Nanog (10x magnification).
    Oct4, supplied by Cell Marque, used in various techniques. Bioz Stars score: 90/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    STEMCELL Technologies Inc oct4
    Experimental design and response of hiPSCs to RA treatment over time a hiPSCs were treated with 0.5 µM RA every second passage for a total of 8 steps over time during a culture period of 14 passages; cells that have undergone to 8 passages RA treatment are named hiPSCs-RA 8 . b Representation of AP activity in hiPSCs-RA 8 and hiPSCs. Maintenance of pluripotency in hiPSCs-RA 8 was confirmed by immunofluorescence for <t>Oct4</t> (Red) and Nanog (Green) c and with depth-gene expression analysis by PluriTest algorithm d . e Quantitative real-time PCR (qPCR) of relative telomere length (RTL) assessed in hiPSCs-F and hiPSCs-TL, in three experimental conditions (regular medium, P0; regular medium P14; RA 8 exposure P14) showing maintenance of length of telomeres during RA exposure. A statistical comparison was made between hiPSCs P14 RA 8 and hiPSCs P0 or hiPSCs P14 RA 8 and hiPSCs P14 by paired Student’s t -test (* p
    Oct4, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 93/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti oct4
    Msl1, Nsl1 and Mof regulate mESC-unspecific genes. ( A ) GO analysis ( Tassy and Pourquie, 2013 ) of downregulated genes in sh Msl1, sh Nsl1 and Mof KO mESCs. ( B ) <t>Oct4</t> expression in cell extracts prepared from sh control, sh Msl1, sh Nsl1 and sh Msl1/Nsl1 mESCs was analysed by Western Blot. Extracts from mouse embryonic fibroblasts (mEFs) were used as negative control. 20 μg of proteins were loaded per lane and normalized by ponceau staining, and the blots were developed with an anti-Oct4 antibody. DOI: http://dx.doi.org/10.7554/eLife.02104.019
    Anti Oct4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc oct4
    Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, <t>OCT4,</t> SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and
    Oct4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend oct4
    Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, <t>OCT4,</t> SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and
    Oct4, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti oct4 antibody epr17980
    Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, <t>OCT4,</t> SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and
    Anti Oct4 Antibody Epr17980, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Schematics of developmental events in the ventral and dorsal ovarian regions during the embryonic period and first wave of follicle activation. Early onset of meiosis in the ventral region is represented in the insert (OCT4 and STRA8). First primordial

    Journal: Biology of Reproduction

    Article Title: Geography of Follicle Formation in the Embryonic Mouse Ovary Impacts Activation Pattern During the First Wave of Folliculogenesis 1

    doi: 10.1095/biolreprod.115.131227

    Figure Lengend Snippet: Schematics of developmental events in the ventral and dorsal ovarian regions during the embryonic period and first wave of follicle activation. Early onset of meiosis in the ventral region is represented in the insert (OCT4 and STRA8). First primordial

    Article Snippet: Sections were incubated overnight at 4°C with primary antibodies: HA tag (1:200; H6908; Sigma-Aldrich), MVH (Mouse Vasa Homolog; 1:50; DDX4/MVH antibody; ab138440; Abcam), OCT4 (octamer-binding protein 4, POU5F1, 1:50; P0056; Sigma-Aldrich); STRA8 (stimulated by retinoic acid 8, 1:50; ab49602; Abcam), FOXL2 (forkhead box L2, 1:50; ab5096; Abcam), laminin (1:100; L9393; Sigma-Aldrich), and p63 (1:100; sc-8431; Santa Cruz Biotechnology).

    Techniques: Activation Assay

    Timing of meiosis entry differed between ventral and dorsal regions of the mouse ovary. Meiosis onset was visualized using immunofluorescence assay in serial sections with OCT4, STRA8, and MVH antibodies, allowing detection of germ cells before, during,

    Journal: Biology of Reproduction

    Article Title: Geography of Follicle Formation in the Embryonic Mouse Ovary Impacts Activation Pattern During the First Wave of Folliculogenesis 1

    doi: 10.1095/biolreprod.115.131227

    Figure Lengend Snippet: Timing of meiosis entry differed between ventral and dorsal regions of the mouse ovary. Meiosis onset was visualized using immunofluorescence assay in serial sections with OCT4, STRA8, and MVH antibodies, allowing detection of germ cells before, during,

    Article Snippet: Sections were incubated overnight at 4°C with primary antibodies: HA tag (1:200; H6908; Sigma-Aldrich), MVH (Mouse Vasa Homolog; 1:50; DDX4/MVH antibody; ab138440; Abcam), OCT4 (octamer-binding protein 4, POU5F1, 1:50; P0056; Sigma-Aldrich); STRA8 (stimulated by retinoic acid 8, 1:50; ab49602; Abcam), FOXL2 (forkhead box L2, 1:50; ab5096; Abcam), laminin (1:100; L9393; Sigma-Aldrich), and p63 (1:100; sc-8431; Santa Cruz Biotechnology).

    Techniques: Immunofluorescence

    MiR-219 induces neural development in mouse embryos. MiR-219 agomirs, Foxj3/Zbtb18 mRNAs, and their corresponding negative controls were microinjected into the cytoplasm of zygotes as indicated. The expression patterns of Oct4 and Nestin in the serially sectioned E6.5 and E7.5 embryos were examined by immunohistochemistry analysis. ( A ) Immunohistochemistry analysis of the E6.5 embryos. Representative images are shown. Black dashed frames in E6.5 embryos indicate the embryonic region. ( B ) Immunohistochemistry (a–d) and H E staining (e, f) analysis of E7.5 embryos. a and b show the representative control embryo and indicate how the embryonic region is organized and how the ectodermal and endodermal tissues can be distinguished. c and d show the representative abnormal miR-219-injected embryo, which exhibits a severe overdevelopment in its anterior region. (e, f) show representative abnormal Foxj3/Zbtb18 mRNA-injected embryo with disorganized embryonic region, apparent arrested development, or resorption. Full images of serially sectioned embryos are shown in Supplementary Figure S4 . a, anterior; ac, amniotic cavity; al, allantois; d, distal; exc, exocoelomic cavity; p, posterior; px, proximal. Scale bars, 200 μ m

    Journal: Cell Death & Disease

    Article Title: Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells

    doi: 10.1038/cddis.2017.336

    Figure Lengend Snippet: MiR-219 induces neural development in mouse embryos. MiR-219 agomirs, Foxj3/Zbtb18 mRNAs, and their corresponding negative controls were microinjected into the cytoplasm of zygotes as indicated. The expression patterns of Oct4 and Nestin in the serially sectioned E6.5 and E7.5 embryos were examined by immunohistochemistry analysis. ( A ) Immunohistochemistry analysis of the E6.5 embryos. Representative images are shown. Black dashed frames in E6.5 embryos indicate the embryonic region. ( B ) Immunohistochemistry (a–d) and H E staining (e, f) analysis of E7.5 embryos. a and b show the representative control embryo and indicate how the embryonic region is organized and how the ectodermal and endodermal tissues can be distinguished. c and d show the representative abnormal miR-219-injected embryo, which exhibits a severe overdevelopment in its anterior region. (e, f) show representative abnormal Foxj3/Zbtb18 mRNA-injected embryo with disorganized embryonic region, apparent arrested development, or resorption. Full images of serially sectioned embryos are shown in Supplementary Figure S4 . a, anterior; ac, amniotic cavity; al, allantois; d, distal; exc, exocoelomic cavity; p, posterior; px, proximal. Scale bars, 200 μ m

    Article Snippet: The sections were incubated with mouse anti-Nestin (1 : 100, #MAB5326; Sigma-Aldrich) or rabbit anti-Oct4 antibody (1 : 100, #SAB2701972; Sigma-Aldrich) at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Photographs were obtained with fluorescent microscope (Carl Zeiss).

    Techniques: Expressing, Immunohistochemistry, Staining, Injection

    MiR-219 mediates ESCs to differentiate into neural cells. ( a ) Confirmation of the effects of miR-219 mimics and inhibitors. ( b , c ) ESCs were transfected with miR-219 mimics for 48 h; the relative levels of Oct4 , Nestin , and Map2 were detected through qRT-PCR ( b ) and western blot ( c ). ( d ) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. ( e, f ) ESCs were pretreated with RA for 48 h and transfected with miR-219 inhibitors. The relative levels of Oct4 , Nestin , and Map2 were detected using qRT-PCR ( e ) and western blot ( f ). ( g ) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. ** P

    Journal: Cell Death & Disease

    Article Title: Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells

    doi: 10.1038/cddis.2017.336

    Figure Lengend Snippet: MiR-219 mediates ESCs to differentiate into neural cells. ( a ) Confirmation of the effects of miR-219 mimics and inhibitors. ( b , c ) ESCs were transfected with miR-219 mimics for 48 h; the relative levels of Oct4 , Nestin , and Map2 were detected through qRT-PCR ( b ) and western blot ( c ). ( d ) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. ( e, f ) ESCs were pretreated with RA for 48 h and transfected with miR-219 inhibitors. The relative levels of Oct4 , Nestin , and Map2 were detected using qRT-PCR ( e ) and western blot ( f ). ( g ) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. ** P

    Article Snippet: The sections were incubated with mouse anti-Nestin (1 : 100, #MAB5326; Sigma-Aldrich) or rabbit anti-Oct4 antibody (1 : 100, #SAB2701972; Sigma-Aldrich) at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Photographs were obtained with fluorescent microscope (Carl Zeiss).

    Techniques: Transfection, Quantitative RT-PCR, Western Blot, Immunofluorescence

    Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. ( a, b ) Relative levels of Nestin , Map2, and Tuj1 were detected through qRT-PCR ( a ) and western blot ( b ) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. ( c, d ) ESCs were transfected with siRNAs that were targeted to Olig1 / Olig2 for 12 h and treated with RA or miR-219 mimics for another 36 h. Relative levels of Nestin , Map2 , and Tuj1 were detected through qRT-PCR ( c ) and western blot. ( e ) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. ** P

    Journal: Cell Death & Disease

    Article Title: Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells

    doi: 10.1038/cddis.2017.336

    Figure Lengend Snippet: Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. ( a, b ) Relative levels of Nestin , Map2, and Tuj1 were detected through qRT-PCR ( a ) and western blot ( b ) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. ( c, d ) ESCs were transfected with siRNAs that were targeted to Olig1 / Olig2 for 12 h and treated with RA or miR-219 mimics for another 36 h. Relative levels of Nestin , Map2 , and Tuj1 were detected through qRT-PCR ( c ) and western blot. ( e ) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. ** P

    Article Snippet: The sections were incubated with mouse anti-Nestin (1 : 100, #MAB5326; Sigma-Aldrich) or rabbit anti-Oct4 antibody (1 : 100, #SAB2701972; Sigma-Aldrich) at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Photographs were obtained with fluorescent microscope (Carl Zeiss).

    Techniques: Quantitative RT-PCR, Western Blot, Transfection, Immunofluorescence

    Metformin decreases K7M2 xenograft tumor growth and inhibits lung metastasis in vivo . (a) Metformin suppressed tumorigenicity of K7M2 OSCs in vivo by limited dilution assay. n = 7. (b) The tumor formation rate of K7M2 OSCs treated with metformin. (c) The tumor volumes and (d) tumor weights at the end of the experiments. (e) Images of resected tumor xenografts on day 21. n = 6. (f) The tumor volumes and (g) the tumor weights at the end of the experiments. (h) Numbers of mice with lung metastasis. (i) H E staining of lung tissues for metastatic nodules. The arrows represent smaller tumor nodules in the lung. Scale bars = 100 μ m. (j) Immunohistochemical staining of LC3, ATG5, Ki67, Sox2, and Oct4 of three different tumors from each group. Scale bars = 100 μ m. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Metformin decreases K7M2 xenograft tumor growth and inhibits lung metastasis in vivo . (a) Metformin suppressed tumorigenicity of K7M2 OSCs in vivo by limited dilution assay. n = 7. (b) The tumor formation rate of K7M2 OSCs treated with metformin. (c) The tumor volumes and (d) tumor weights at the end of the experiments. (e) Images of resected tumor xenografts on day 21. n = 6. (f) The tumor volumes and (g) the tumor weights at the end of the experiments. (h) Numbers of mice with lung metastasis. (i) H E staining of lung tissues for metastatic nodules. The arrows represent smaller tumor nodules in the lung. Scale bars = 100 μ m. (j) Immunohistochemical staining of LC3, ATG5, Ki67, Sox2, and Oct4 of three different tumors from each group. Scale bars = 100 μ m. ∗ P

    Article Snippet: Alexa Fluor® 488 mouse/human anti-Sox2 antibody (656109), PE anti-mouse/human Oct4 antibody (653703), APC anti-mouse/human CD44 antibody (103011), APC anti-mouse CD133 antibody (141207), and PE/Cy7 anti-human CD133 antibody (372809) were obtained from Biolegend (San Diego, USA).

    Techniques: In Vivo, Dilution Assay, Mouse Assay, Staining, Immunohistochemistry

    Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Article Snippet: Alexa Fluor® 488 mouse/human anti-Sox2 antibody (656109), PE anti-mouse/human Oct4 antibody (653703), APC anti-mouse/human CD44 antibody (103011), APC anti-mouse CD133 antibody (141207), and PE/Cy7 anti-human CD133 antibody (372809) were obtained from Biolegend (San Diego, USA).

    Techniques: Transmission Electron Microscopy, Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    Autophagy regulates the homeostasis of pluripotency in OSCs. (a) TEM images of autophagosomes in metformin-treated K7M2 and MG63 OSCs. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (b) Immunofluorescence analysis of autophagy in K7M2 and MG63 OSCs. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 50 μ m. n = 3. (c) Flow cytometry analysis of the effect of autophagy on ALDH1 activity. The upper panel shows that OSCs were incubated with DEAB, a specific ALDH inhibitor. The lower panel shows ALDH1-positive OSCs. n = 3. (d) The percentage of ALDH1-positive OSCs. (e) Clone formation of K7M2 and MG63 OSCs. Scale bars = 50 μ m. n = 5. (f) SEM images of sphere forming of K7M2 and MG63 OSCs. Scale bars = 20 μ m. n = 3. (g) Western blot analysis of the autophagy markers ATG5 and ATG7 and the pluripotency-associated proteins Sox2 and Oct4 in K7M2 and MG63 OSCs. n = 3. (h) Densitometric analyses of autophagy markers and pluripotency-associated proteins. Each experiment was conducted at least three occasions. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Autophagy regulates the homeostasis of pluripotency in OSCs. (a) TEM images of autophagosomes in metformin-treated K7M2 and MG63 OSCs. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (b) Immunofluorescence analysis of autophagy in K7M2 and MG63 OSCs. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 50 μ m. n = 3. (c) Flow cytometry analysis of the effect of autophagy on ALDH1 activity. The upper panel shows that OSCs were incubated with DEAB, a specific ALDH inhibitor. The lower panel shows ALDH1-positive OSCs. n = 3. (d) The percentage of ALDH1-positive OSCs. (e) Clone formation of K7M2 and MG63 OSCs. Scale bars = 50 μ m. n = 5. (f) SEM images of sphere forming of K7M2 and MG63 OSCs. Scale bars = 20 μ m. n = 3. (g) Western blot analysis of the autophagy markers ATG5 and ATG7 and the pluripotency-associated proteins Sox2 and Oct4 in K7M2 and MG63 OSCs. n = 3. (h) Densitometric analyses of autophagy markers and pluripotency-associated proteins. Each experiment was conducted at least three occasions. ∗ P

    Article Snippet: Alexa Fluor® 488 mouse/human anti-Sox2 antibody (656109), PE anti-mouse/human Oct4 antibody (653703), APC anti-mouse/human CD44 antibody (103011), APC anti-mouse CD133 antibody (141207), and PE/Cy7 anti-human CD133 antibody (372809) were obtained from Biolegend (San Diego, USA).

    Techniques: Transmission Electron Microscopy, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Activity Assay, Incubation, Western Blot

    GridMedian extracts features from fluorescent image data while reduce the size of data. ( A,B ) The median intensities of all 144 grids from the test image set ( Fig. 1A ) are plotted. Anti-Oct4 intensity does not correlate with EGFP intensity ( A ), while well correlate with H33342 intensity ( B ). The 144 grids are classified to 4 sub-classes by Mclust according to the 3-channel intensities (H33342, anti-Oct4 and EGFP) without drawing threshold lines arbitrarily. The 4 sub-classes are shown in 4 different colours and tentatively designated. Misclassified grids are encircled ( B ). ( C ) Kernel density estimations indicate very similar EGFP expression profiles between EGFP(−) and void sub-classes. High-peak of sub-class designated “other” (blue) corresponds cytoplasmic region of EGFP(+) mESCs. ( D ) Oct4 expression profiles between EGFP(−) and EGFP(+) sub-classes suggests higher expression of Oct4 in EGFP(+) mESCs. Smoothing kernel = Gaussian, bandwidth = 0.05. ( E ) After successful application of “Median_IQR filter” (see Supplementary Fig. S2 ) on the 144 grids, 67 of class 3 grids are again classified by Mclust leading EGFP(+) and EGFP(−) sub-classes. Suspected misclassified grids are encircled. ( F ) The Oct4 expression profiles between 51 of EGFP(+) and 16 of EGFP(−) sub-classes indicate higher expression of Oct4 in mESCs. Sample script (GBIQ_ESC.R) to reproduce the result and figures is available from https://github.com/yo-ninomy/DemoScripts .

    Journal: Scientific Reports

    Article Title: GBIQ: a non-arbitrary, non-biased method for quantification of fluorescent images

    doi: 10.1038/srep26454

    Figure Lengend Snippet: GridMedian extracts features from fluorescent image data while reduce the size of data. ( A,B ) The median intensities of all 144 grids from the test image set ( Fig. 1A ) are plotted. Anti-Oct4 intensity does not correlate with EGFP intensity ( A ), while well correlate with H33342 intensity ( B ). The 144 grids are classified to 4 sub-classes by Mclust according to the 3-channel intensities (H33342, anti-Oct4 and EGFP) without drawing threshold lines arbitrarily. The 4 sub-classes are shown in 4 different colours and tentatively designated. Misclassified grids are encircled ( B ). ( C ) Kernel density estimations indicate very similar EGFP expression profiles between EGFP(−) and void sub-classes. High-peak of sub-class designated “other” (blue) corresponds cytoplasmic region of EGFP(+) mESCs. ( D ) Oct4 expression profiles between EGFP(−) and EGFP(+) sub-classes suggests higher expression of Oct4 in EGFP(+) mESCs. Smoothing kernel = Gaussian, bandwidth = 0.05. ( E ) After successful application of “Median_IQR filter” (see Supplementary Fig. S2 ) on the 144 grids, 67 of class 3 grids are again classified by Mclust leading EGFP(+) and EGFP(−) sub-classes. Suspected misclassified grids are encircled. ( F ) The Oct4 expression profiles between 51 of EGFP(+) and 16 of EGFP(−) sub-classes indicate higher expression of Oct4 in mESCs. Sample script (GBIQ_ESC.R) to reproduce the result and figures is available from https://github.com/yo-ninomy/DemoScripts .

    Article Snippet: Anti-Oct4 (Pou5f1) (Santa Cruz, sc-5279, 1:100) with indicated dilution ratio in the blocking solution was applied for overnight at 4 °C.

    Techniques: Expressing

    GBIQ reproduces a comparative result to a standard cell-segmentation method. ( A,B ) The 903 observations after applying the “Median_IQR filter” and Mclust on the filtered dataset of 3 full resolution image sets are plotted. ( C,D ) Kernel density estimation of EGFP and Oct4 expression profiles show distinctive properties among the 3 sub-classes (red, green and blue). ( E,F ) Expression level of Oct4 is measured by TQ on the same image sets that consist 3 sets of Dox-treated fluorescent micrographs. From the 3 sets of images, TQ identifies 530 cells that are classified into 3 sub-classes (red, green and blue) by Mclust ( E ). Kernel density estimation shows TQ also reports higher expression of Oct4 in EGFP(+) mESCs ( F ). Smoothing kernel = Gaussian. ( C,D ) GBIQ, bandwidth = 0.05. ( F ) TQ, bandwidth = 10.

    Journal: Scientific Reports

    Article Title: GBIQ: a non-arbitrary, non-biased method for quantification of fluorescent images

    doi: 10.1038/srep26454

    Figure Lengend Snippet: GBIQ reproduces a comparative result to a standard cell-segmentation method. ( A,B ) The 903 observations after applying the “Median_IQR filter” and Mclust on the filtered dataset of 3 full resolution image sets are plotted. ( C,D ) Kernel density estimation of EGFP and Oct4 expression profiles show distinctive properties among the 3 sub-classes (red, green and blue). ( E,F ) Expression level of Oct4 is measured by TQ on the same image sets that consist 3 sets of Dox-treated fluorescent micrographs. From the 3 sets of images, TQ identifies 530 cells that are classified into 3 sub-classes (red, green and blue) by Mclust ( E ). Kernel density estimation shows TQ also reports higher expression of Oct4 in EGFP(+) mESCs ( F ). Smoothing kernel = Gaussian. ( C,D ) GBIQ, bandwidth = 0.05. ( F ) TQ, bandwidth = 10.

    Article Snippet: Anti-Oct4 (Pou5f1) (Santa Cruz, sc-5279, 1:100) with indicated dilution ratio in the blocking solution was applied for overnight at 4 °C.

    Techniques: Expressing

    Acquisition of mESC-like signaling pathways in LIF-3i-reverted hPSCs. (A) Stabilization of nuclear P-STAT3 and cytoplasmic activated (activ) β-catenin in LIF-3-reverted hPSCs. Nuclear (Nu), cytoplasmic (Cy) and total (Tot) fractions are shown for H9 hESC, E5C3 sa-MP-iPSC and C1.2 fibro-iPSC lines. TBP and actin are protein loading controls. +, LIF-3i; −, primed culture. (B) Inhibition/proliferation assays of sa-MP-iPSC line E5C3. Shown are mESC signaling pathways [e.g. LIF/JAK/STAT, LIF-receptor/gp130, CREB, BMP4 (dorsomorphin) and PI3K]. Cumulative proliferations of SSEA4 + TRA-1-81 + E5C3 cells were measured after 12 days of culture in the presence of indicated inhibitors, normalized to LIF-3i-alone conditions (at 100%). (C) Western blots of key WNT components AXIN1 and activated β-catenin in both nuclear and cytoplasmic fractions in primed (−) versus LIF-3i reverted (+) hPSC cultures. (D,E) Confocal microscopy of WNT proteins in indicated primed (bFGF) versus LIF-3i-reverted hPSC lines. Activated β-catenin is shown to be distinctly sequestered outside of the nuclear compartment, whereas uniform expression of OCT4 was localized strictly within nuclei. Scale bars: 20 µm.

    Journal: Development (Cambridge, England)

    Article Title: Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality

    doi: 10.1242/dev.138982

    Figure Lengend Snippet: Acquisition of mESC-like signaling pathways in LIF-3i-reverted hPSCs. (A) Stabilization of nuclear P-STAT3 and cytoplasmic activated (activ) β-catenin in LIF-3-reverted hPSCs. Nuclear (Nu), cytoplasmic (Cy) and total (Tot) fractions are shown for H9 hESC, E5C3 sa-MP-iPSC and C1.2 fibro-iPSC lines. TBP and actin are protein loading controls. +, LIF-3i; −, primed culture. (B) Inhibition/proliferation assays of sa-MP-iPSC line E5C3. Shown are mESC signaling pathways [e.g. LIF/JAK/STAT, LIF-receptor/gp130, CREB, BMP4 (dorsomorphin) and PI3K]. Cumulative proliferations of SSEA4 + TRA-1-81 + E5C3 cells were measured after 12 days of culture in the presence of indicated inhibitors, normalized to LIF-3i-alone conditions (at 100%). (C) Western blots of key WNT components AXIN1 and activated β-catenin in both nuclear and cytoplasmic fractions in primed (−) versus LIF-3i reverted (+) hPSC cultures. (D,E) Confocal microscopy of WNT proteins in indicated primed (bFGF) versus LIF-3i-reverted hPSC lines. Activated β-catenin is shown to be distinctly sequestered outside of the nuclear compartment, whereas uniform expression of OCT4 was localized strictly within nuclei. Scale bars: 20 µm.

    Article Snippet: Cells were incubated overnight at 4°C in humid chambers with: primary rabbit anti-human unconjugated antibodies for AXIN1 (1:200; 06-1049, Millipore), KLF2 (1:50; HPA055964, Sigma), KLF4 (1:50; HPA002926, Sigma), KLF5 (1:50; HPA040398, Sigma), KLF17 (1:200; HPA024629, Sigma), Nanog (1:100; 3369-1, Epitomics), NR5A2 (1:50; HPA005455, Sigma), OCT4 (POU5F1) (1:50; sc9081, Santa Cruz), STELLA (DPPA3) (1:200; HPA045695, Sigma) and TFCP2L1 (1:50; HPA029708, Sigma) or alternatively with mouse anti-human STELLA (1:50; MAB4388, Millipore) or AXIN1 (clone C76H11, 1:500; 2087, Cell Signaling).

    Techniques: Inhibition, Western Blot, Confocal Microscopy, Expressing

    Generation of PEPT1-KO Human iPSCs (A) The schematic overview shows the targeting strategy for PEPT1/SLC15A1 . The PCR primers that can distinguish WT and mutant alleles are shown with red arrows. Donor plasmid: EF1α, elongation factor 1 alpha promoter; PuroR, puromycin resistant protein; pA, polyadenylation sequence; HDR, homology-directed repair. (B) The genotyping was performed in the PEPT1/SLC15A1 locus. (C) Sequencing analyses were performed to examine whether the PEPT1/SLC15A1-KO iPSC clone was correctly targeted. To confirm the DNA sequence, the PCR products were purified and subjected to sequencing analyses. The single-guide RNA (sgRNA)-targeting sequences are shown in red. (D) The gene expression levels of POU5F1 , NANOG , and SOX2 in WT-iPSCs and PEPT1-KO iPSCs were examined by real-time RT-PCR analysis. The gene expression levels in the WT-iPSCs (WT) were taken as 1.0. Data represent the means ± SD (n = 8, technical replicate). (E) Immunostaining analysis of POU5F1 (red) was performed in the WT-iPSCs and PEPT1/SLC15A1-KO cells. Nuclei were stained with DAPI (blue). Scale bars represent 50 μm.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Establishment of SLC15A1/PEPT1-Knockout Human-Induced Pluripotent Stem Cell Line for Intestinal Drug Absorption Studies

    doi: 10.1016/j.omtm.2019.11.008

    Figure Lengend Snippet: Generation of PEPT1-KO Human iPSCs (A) The schematic overview shows the targeting strategy for PEPT1/SLC15A1 . The PCR primers that can distinguish WT and mutant alleles are shown with red arrows. Donor plasmid: EF1α, elongation factor 1 alpha promoter; PuroR, puromycin resistant protein; pA, polyadenylation sequence; HDR, homology-directed repair. (B) The genotyping was performed in the PEPT1/SLC15A1 locus. (C) Sequencing analyses were performed to examine whether the PEPT1/SLC15A1-KO iPSC clone was correctly targeted. To confirm the DNA sequence, the PCR products were purified and subjected to sequencing analyses. The single-guide RNA (sgRNA)-targeting sequences are shown in red. (D) The gene expression levels of POU5F1 , NANOG , and SOX2 in WT-iPSCs and PEPT1-KO iPSCs were examined by real-time RT-PCR analysis. The gene expression levels in the WT-iPSCs (WT) were taken as 1.0. Data represent the means ± SD (n = 8, technical replicate). (E) Immunostaining analysis of POU5F1 (red) was performed in the WT-iPSCs and PEPT1/SLC15A1-KO cells. Nuclei were stained with DAPI (blue). Scale bars represent 50 μm.

    Article Snippet: Immunocytochemistry To perform the immunocytochemistry, the human iPSCs and their derivatives were fixed with 4% paraformaldehyde (FUJIFILM Wako) in PBS for 10 min. After blocking the cells with PBS containing 2% BSA and 0.2% Triton X-100 for 20 min, the cells were incubated with the anti-OCT4 (POU5F1) antibody (sc5279, Santa Cruz Biotechnology) or anti-villin 1 antibody (ab130751, Abcam) at 4°C overnight, and, finally, with an immunoglobulin (Ig)G secondary antibody, Alexa Fluor 594 conjugate (Thermo Fisher Scientific), at room temperature for 1 h.

    Techniques: Polymerase Chain Reaction, Mutagenesis, Plasmid Preparation, Sequencing, Purification, Expressing, Quantitative RT-PCR, Immunostaining, Staining

    Multiple iPSC lines derived from a PD patient with SNCA triplication and an unaffected relative exhibit hallmarks of pluripotency. ( a ) Western blot analysis of AST and NAS fibroblasts confirms a lack of α-synuclein protein. Lysate of SH-SY5Y neuroblastoma cells transfected with an α-synuclein expressing plasmid was used as a positive control, and β-actin as a loading control. ( b ) Phase contrast images of AST and NAS fibroblasts and de novo iPSC colonies on SNL feeder cells at day 20 post-viral transduction. Scale bar, 100 μm. ( c ) Quantitative RT–PCR for retroviral transgene expression of the four reprogramming factors ( Oct4 , Sox2 , c-Myc , Klf4 ). Expression in iPSC lines was normalized to transgene expression of NAS fibroblasts 3 days post-viral transduction (NAS VT). Also shown are negative controls: untransduced fibroblasts (NAS fibs) and human ESCs (Shef4 hESC). ( d, e ) Representative iPSC colonies derived from NAS fibroblasts ( d ) and AST fibroblasts ( e ) stained positive for the pluripotency markers OCT4, SSEA4, TRA-1-81 and NANOG. Scale bar, 100 μm. ( f ) Endogenous expression of OCT4 , SOX2 , NANOG and REX1 in two AST and two NAS iPSC lines, normalized to expression in human ESCs (Shef4), found a similar expression profile in all lines examined. Expression of these genes was absent in human fibroblasts (not shown). Error bars represent standard deviation of technical replicates, n =3.

    Journal: Nature Communications

    Article Title: Parkinson's disease induced pluripotent stem cells with triplication of the ?-synuclein locus

    doi: 10.1038/ncomms1453

    Figure Lengend Snippet: Multiple iPSC lines derived from a PD patient with SNCA triplication and an unaffected relative exhibit hallmarks of pluripotency. ( a ) Western blot analysis of AST and NAS fibroblasts confirms a lack of α-synuclein protein. Lysate of SH-SY5Y neuroblastoma cells transfected with an α-synuclein expressing plasmid was used as a positive control, and β-actin as a loading control. ( b ) Phase contrast images of AST and NAS fibroblasts and de novo iPSC colonies on SNL feeder cells at day 20 post-viral transduction. Scale bar, 100 μm. ( c ) Quantitative RT–PCR for retroviral transgene expression of the four reprogramming factors ( Oct4 , Sox2 , c-Myc , Klf4 ). Expression in iPSC lines was normalized to transgene expression of NAS fibroblasts 3 days post-viral transduction (NAS VT). Also shown are negative controls: untransduced fibroblasts (NAS fibs) and human ESCs (Shef4 hESC). ( d, e ) Representative iPSC colonies derived from NAS fibroblasts ( d ) and AST fibroblasts ( e ) stained positive for the pluripotency markers OCT4, SSEA4, TRA-1-81 and NANOG. Scale bar, 100 μm. ( f ) Endogenous expression of OCT4 , SOX2 , NANOG and REX1 in two AST and two NAS iPSC lines, normalized to expression in human ESCs (Shef4), found a similar expression profile in all lines examined. Expression of these genes was absent in human fibroblasts (not shown). Error bars represent standard deviation of technical replicates, n =3.

    Article Snippet: Immunocytochemistry Cells were fixed in 4% paraformaldehyde at room temperature, or 100% methanol at −20°C for 15 minutes, then blocked and permeabilized with blocking buffer (2% goat serum, 0.1% Triton-X in phosphate buffered saline) overnight at 4°C before probing with the following primary antibodies (overnight in goat blocking buffer at 4°C): OCT4 (Santa Cruz #5279, mouse IgG2b, 1:200), TRA 1-81 (Biolegend #330701, mouse IgMκ, 1:600), SSEA4 (DSHB, mouse IgG3κ, neat), NANOG (Abcam #ab21624, rabbit IgG, 1:1000), TuJ-1 (R & D systems #MAB1195, mouse IgG2A), TH (R & D systems #MAB1423 clone TH-2, mouse IgG1), LMX1B (Rabbit polyclonal from Dai et al ., 1:2000) and α-synuclein (BD Transduction Laboratories #610787, mouse IgG1).

    Techniques: Derivative Assay, Western Blot, AST Assay, Transfection, Expressing, Plasmid Preparation, Positive Control, Transduction, Quantitative RT-PCR, Staining, Standard Deviation

    Exogenous OCT4 expression inhibits apoptosis by transactivating miR-125b-1. ( a ) Apoptosis-related miRNAs were screened by real-time PCR in control and OCT4-expressing HeLa and SiHa cells. Real-time PCR analysis of pri-miR-125b-1 ( b ) and pri-miR-125b-2 ( c ) in OCT4-expressing and control cells. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( d ) Schematic structure of miR-125b-1 promoter. The pink and red ovals indicate the location of two potential OCT4-binding sites (RR1 and RR2). The bidirectional arrows (P1, P2 and P3) represent the primers used in ChIP analysis. Dual luciferase reporter assays in HeLa-GFP/OCT4 cells with a series of miR-125b-1 promoter 5′ deletion and rearrangement mutants. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( e ) Quantitative ChIP analysis of OCT4 binding to miR-125b-1 promoter sequences is shown for HeLa-GFP/OCT4 and SiHa-GFP/OCT4 cells. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( f ) Apoptosis assays were performed on OCT4-expressing or control cells following transfection with the miR-125b-expressing or -sponge vector. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t -test)

    Journal: Cell Death & Disease

    Article Title: OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    doi: 10.1038/cddis.2013.272

    Figure Lengend Snippet: Exogenous OCT4 expression inhibits apoptosis by transactivating miR-125b-1. ( a ) Apoptosis-related miRNAs were screened by real-time PCR in control and OCT4-expressing HeLa and SiHa cells. Real-time PCR analysis of pri-miR-125b-1 ( b ) and pri-miR-125b-2 ( c ) in OCT4-expressing and control cells. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( d ) Schematic structure of miR-125b-1 promoter. The pink and red ovals indicate the location of two potential OCT4-binding sites (RR1 and RR2). The bidirectional arrows (P1, P2 and P3) represent the primers used in ChIP analysis. Dual luciferase reporter assays in HeLa-GFP/OCT4 cells with a series of miR-125b-1 promoter 5′ deletion and rearrangement mutants. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( e ) Quantitative ChIP analysis of OCT4 binding to miR-125b-1 promoter sequences is shown for HeLa-GFP/OCT4 and SiHa-GFP/OCT4 cells. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( f ) Apoptosis assays were performed on OCT4-expressing or control cells following transfection with the miR-125b-expressing or -sponge vector. Data are presented as the mean±S.E.M. of triplicate analyses (Student's t -test)

    Article Snippet: After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies as follows: anti-OCT4 (1:500, H-134, sc-9081, Santa Cruz Biotechnology), anti-BAK1 (1:500, N-20, sc-1035, Santa Cruz Biotechnology) and anti-β -actin (1:500, C4, sc-47778, Santa Cruz Biotechnology).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Transfection, Plasmid Preparation

    Exogenous OCT4 expression downregulates BAK1 through the activation of miR-125b. ( a ) BAK1 expression was detected in the indicated cell lines by western blot analysis. ( b ) Highly conserved, predicted binding sites for the seed sequences of miR-125b among human (629-636), mouse (607-614) and rat (87-94) in the 3′-UTR (untranslated region) of BAK1 . ( c ) BAK1 protein levels were determined by western blot analysis in HeLa-GFP or SiHa-GFP cells following transfection with the miR-125b-1-expressing or control vector and in HeLa-OCT4 or SiHa-OCT4 cells transfected with the miR-125b-sponge or control vector. ( d ) Schematic representation of the constructs used in the luciferase assay. HeLa-GFP/OCT4 cells were transfected with plasmid of BAK1wt, BAK1mut or BAK1del. Dual luciferase reporter activity was expressed relative to the pmiR-Report vector and data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( e ) Expression of OCT4 and BAK1 was examined by IHC staining in 20 cervical cancers specimens. The expression levels of two representative samples are shown. Scale bars=200 μ m for upper panel, 50 μ m for lower panel. ( f ) Nuclear OCT4 and cytoplasmic BAK1 staining were scored from 1 to 15. The correlation was significant, as determined by the Pearson's correlation test ( r =−0.565; P =0.0094). ( g ) Expression of miR-125b was detected in 13 clinical samples from Figure 1 by real-time PCR and the correlation between OCT4 and miR-125b was significant, as determined by the Pearson's correlation test ( r =0.849; P =0.0002)

    Journal: Cell Death & Disease

    Article Title: OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    doi: 10.1038/cddis.2013.272

    Figure Lengend Snippet: Exogenous OCT4 expression downregulates BAK1 through the activation of miR-125b. ( a ) BAK1 expression was detected in the indicated cell lines by western blot analysis. ( b ) Highly conserved, predicted binding sites for the seed sequences of miR-125b among human (629-636), mouse (607-614) and rat (87-94) in the 3′-UTR (untranslated region) of BAK1 . ( c ) BAK1 protein levels were determined by western blot analysis in HeLa-GFP or SiHa-GFP cells following transfection with the miR-125b-1-expressing or control vector and in HeLa-OCT4 or SiHa-OCT4 cells transfected with the miR-125b-sponge or control vector. ( d ) Schematic representation of the constructs used in the luciferase assay. HeLa-GFP/OCT4 cells were transfected with plasmid of BAK1wt, BAK1mut or BAK1del. Dual luciferase reporter activity was expressed relative to the pmiR-Report vector and data are presented as the mean±S.E.M. of triplicate analyses (Student's t- test). ( e ) Expression of OCT4 and BAK1 was examined by IHC staining in 20 cervical cancers specimens. The expression levels of two representative samples are shown. Scale bars=200 μ m for upper panel, 50 μ m for lower panel. ( f ) Nuclear OCT4 and cytoplasmic BAK1 staining were scored from 1 to 15. The correlation was significant, as determined by the Pearson's correlation test ( r =−0.565; P =0.0094). ( g ) Expression of miR-125b was detected in 13 clinical samples from Figure 1 by real-time PCR and the correlation between OCT4 and miR-125b was significant, as determined by the Pearson's correlation test ( r =0.849; P =0.0002)

    Article Snippet: After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies as follows: anti-OCT4 (1:500, H-134, sc-9081, Santa Cruz Biotechnology), anti-BAK1 (1:500, N-20, sc-1035, Santa Cruz Biotechnology) and anti-β -actin (1:500, C4, sc-47778, Santa Cruz Biotechnology).

    Techniques: Expressing, Activation Assay, Western Blot, Binding Assay, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    OCT4 promotes tumor formation by cervical cancer cells in vivo . ( a ) OCT4 expression in cervical cancer cell lines was detected by immunocytochemistry and the positive rates were summarized. The arrow indicates the OCT4 positive cell. ( b ) OCT4 expression in cervical cancer cell lines was detected by western blotting. ( c ) Western blot analysis of OCT4 protein and in HeLa and SiHa parental, control- and OCT4-stably transfected cells. For western blot analyses, Tera-1 cells were used as a positive control, and β -actin served as the loading control. A tumor formation assay was performed with three mice per group. Tumor growth curves were calculated based on monitoring performed every 4 days post-transplant. At 36 days post-transplant for HeLa cells ( d ) and 32 days post-transplant for SiHa cells ( e ), the xenograft tumors were dissociated and weighed ( f and g ). The data were analyzed and shown as mean±S.E.M. ( d and e were determined by two-way ANOVA test, * P

    Journal: Cell Death & Disease

    Article Title: OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    doi: 10.1038/cddis.2013.272

    Figure Lengend Snippet: OCT4 promotes tumor formation by cervical cancer cells in vivo . ( a ) OCT4 expression in cervical cancer cell lines was detected by immunocytochemistry and the positive rates were summarized. The arrow indicates the OCT4 positive cell. ( b ) OCT4 expression in cervical cancer cell lines was detected by western blotting. ( c ) Western blot analysis of OCT4 protein and in HeLa and SiHa parental, control- and OCT4-stably transfected cells. For western blot analyses, Tera-1 cells were used as a positive control, and β -actin served as the loading control. A tumor formation assay was performed with three mice per group. Tumor growth curves were calculated based on monitoring performed every 4 days post-transplant. At 36 days post-transplant for HeLa cells ( d ) and 32 days post-transplant for SiHa cells ( e ), the xenograft tumors were dissociated and weighed ( f and g ). The data were analyzed and shown as mean±S.E.M. ( d and e were determined by two-way ANOVA test, * P

    Article Snippet: After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies as follows: anti-OCT4 (1:500, H-134, sc-9081, Santa Cruz Biotechnology), anti-BAK1 (1:500, N-20, sc-1035, Santa Cruz Biotechnology) and anti-β -actin (1:500, C4, sc-47778, Santa Cruz Biotechnology).

    Techniques: In Vivo, Expressing, Immunocytochemistry, Western Blot, Stable Transfection, Transfection, Positive Control, Tube Formation Assay, Mouse Assay

    Exogenous OCT4 expression inhibits apoptosis of human cervical cancer cells in vitro and in vivo . ( a ) Apoptosis analysis based on flow cytometry was performed triplicate. All of the data were summarized and presented as the mean±S.E.M. (Student's t- test), * P

    Journal: Cell Death & Disease

    Article Title: OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    doi: 10.1038/cddis.2013.272

    Figure Lengend Snippet: Exogenous OCT4 expression inhibits apoptosis of human cervical cancer cells in vitro and in vivo . ( a ) Apoptosis analysis based on flow cytometry was performed triplicate. All of the data were summarized and presented as the mean±S.E.M. (Student's t- test), * P

    Article Snippet: After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies as follows: anti-OCT4 (1:500, H-134, sc-9081, Santa Cruz Biotechnology), anti-BAK1 (1:500, N-20, sc-1035, Santa Cruz Biotechnology) and anti-β -actin (1:500, C4, sc-47778, Santa Cruz Biotechnology).

    Techniques: Expressing, In Vitro, In Vivo, Flow Cytometry, Cytometry

    OCT4 protein expression in normal cervical (NC) tissue and cervical cancer lesions. ( a ) Representative immunostained specimens showing OCT4 expression in NC ( n =42), cervical carcinoma in situ (CIS; n =20) and invasive cervical carcinoma (ICC; n =44). Scale bars=20 μ m. ( b ) Bar chart showing the frequency of OCT4-positive samples among different cervical lesions. ( c ) The scatter plot shows the immunoreactivity scores (IRS) for OCT4 staining in tissue samples (NC versus CIS, P =0.027; CIS versus ICC, P =0.0030; NC versus ICC, P =1.61E-09, one-way ANOVA test). ( d ) Western blot analysis of OCT4 protein levels in NC ( n =8) and ICC ( n =8) specimens. ( e ) Ratio of OCT4 to β -actin staining density in NC and ICC specimens determined with the AlphaView SA software ( P =0.0055; Student's t- test)

    Journal: Cell Death & Disease

    Article Title: OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    doi: 10.1038/cddis.2013.272

    Figure Lengend Snippet: OCT4 protein expression in normal cervical (NC) tissue and cervical cancer lesions. ( a ) Representative immunostained specimens showing OCT4 expression in NC ( n =42), cervical carcinoma in situ (CIS; n =20) and invasive cervical carcinoma (ICC; n =44). Scale bars=20 μ m. ( b ) Bar chart showing the frequency of OCT4-positive samples among different cervical lesions. ( c ) The scatter plot shows the immunoreactivity scores (IRS) for OCT4 staining in tissue samples (NC versus CIS, P =0.027; CIS versus ICC, P =0.0030; NC versus ICC, P =1.61E-09, one-way ANOVA test). ( d ) Western blot analysis of OCT4 protein levels in NC ( n =8) and ICC ( n =8) specimens. ( e ) Ratio of OCT4 to β -actin staining density in NC and ICC specimens determined with the AlphaView SA software ( P =0.0055; Student's t- test)

    Article Snippet: After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies as follows: anti-OCT4 (1:500, H-134, sc-9081, Santa Cruz Biotechnology), anti-BAK1 (1:500, N-20, sc-1035, Santa Cruz Biotechnology) and anti-β -actin (1:500, C4, sc-47778, Santa Cruz Biotechnology).

    Techniques: Expressing, In Situ, Immunocytochemistry, Staining, Western Blot, Software

    A model of the OCT4/miR-125b/BAK1 pathway in human cervical carcinogenesis. OCT4 expression activates miR-125b-1. In turn, miR-125b-1 inhibits BAK1 translation, which inhibits apoptosis and promotes tumor progression

    Journal: Cell Death & Disease

    Article Title: OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    doi: 10.1038/cddis.2013.272

    Figure Lengend Snippet: A model of the OCT4/miR-125b/BAK1 pathway in human cervical carcinogenesis. OCT4 expression activates miR-125b-1. In turn, miR-125b-1 inhibits BAK1 translation, which inhibits apoptosis and promotes tumor progression

    Article Snippet: After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies as follows: anti-OCT4 (1:500, H-134, sc-9081, Santa Cruz Biotechnology), anti-BAK1 (1:500, N-20, sc-1035, Santa Cruz Biotechnology) and anti-β -actin (1:500, C4, sc-47778, Santa Cruz Biotechnology).

    Techniques: Expressing

    1α,25(OH)2D3 inhibited the stemness and impaired the mitochondrial respiration and ATP production in acidic condition. a Immunoblotting of the expression of stemness markers NESTIN, CD133, OCT4, and SOX2 in GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM for 4, 8, 12, 24, and 48 h. b and c Self-renewal ability of SLCs with 1α,25(OH) 2 D 3 treatment under pH 7.4 or pH 6.8 culture conditions. Neurosphere formation assay showed the number of neurospheres (diameters larger than 50 µm) formed from GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM ( b ), * P

    Journal: Cell Death & Disease

    Article Title: Acidosis enhances the self-renewal and mitochondrial respiration of stem cell-like glioma cells through CYP24A1-mediated reduction of vitamin D

    doi: 10.1038/s41419-018-1242-1

    Figure Lengend Snippet: 1α,25(OH)2D3 inhibited the stemness and impaired the mitochondrial respiration and ATP production in acidic condition. a Immunoblotting of the expression of stemness markers NESTIN, CD133, OCT4, and SOX2 in GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM for 4, 8, 12, 24, and 48 h. b and c Self-renewal ability of SLCs with 1α,25(OH) 2 D 3 treatment under pH 7.4 or pH 6.8 culture conditions. Neurosphere formation assay showed the number of neurospheres (diameters larger than 50 µm) formed from GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM ( b ), * P

    Article Snippet: The antibodies used included anti-CYP24A1 (Abcam, UK), anti-GFAP (Abcam, UK), anti-Tuj1 (Sigma, USA), anti-Sox2 (Abcam, UK; Abclonal, Beijing, China), anti-CD133 (Miltenyi Biotec, Germany; Abclonal, Beijing, China), anti-Nestin (Abclonal, Beijing, China), anti-Oct4 (Abclonal, Beijing, China), anti-CNPase (Abclonal, Beijing, China), and anti-β-actin (Sigma, USA).

    Techniques: Expressing, Tube Formation Assay

    Low efficiency and slow kinetics during reprogramming of SIRT2-knockout cells. a Delayed morphological changes in SIRT2-KO-MEFs. Scale bar = 400 μm. b Number of alkaline phosphatase-positive colonies. c Proportion of the SSEA-1(+)/CD44(−) fraction. d Proportion of the OCT4(+)/CD44(−) fraction. Non-reprogrammed MEFs are shown as negative control and mouse ESCs are shown as positive control. Samples treated with FITC- and Alexa Fluor 647-conjugated mouse immunoglobulin Gs (IgGs) are presented in the upper panel as negative controls for staining. * P

    Journal: Cell Death & Disease

    Article Title: SIRT2 is required for efficient reprogramming of mouse embryonic fibroblasts toward pluripotency

    doi: 10.1038/s41419-018-0920-3

    Figure Lengend Snippet: Low efficiency and slow kinetics during reprogramming of SIRT2-knockout cells. a Delayed morphological changes in SIRT2-KO-MEFs. Scale bar = 400 μm. b Number of alkaline phosphatase-positive colonies. c Proportion of the SSEA-1(+)/CD44(−) fraction. d Proportion of the OCT4(+)/CD44(−) fraction. Non-reprogrammed MEFs are shown as negative control and mouse ESCs are shown as positive control. Samples treated with FITC- and Alexa Fluor 647-conjugated mouse immunoglobulin Gs (IgGs) are presented in the upper panel as negative controls for staining. * P

    Article Snippet: Fluorescein isothiocyanate (FITC) anti-mouse CD44 antibodies (#553133, BD Biosciences, NJ, USA) as a MEF marker and Alexa Fluor 647 anti-mouse SSEA-1 antibodies (#125607, BioLegend, CA, USA) or OCT3/4− Alexa Fluor 647 (#653710, BioLegend, CA, USA) as pluripotent stem cell markers were simultaneously used for staining.

    Techniques: Knock-Out, Negative Control, Positive Control, Staining

    Oct-4 immunocytochemical staining of sheep spermatogonial stem cells (×40).

    Journal: Avicenna Journal of Medical Biotechnology

    Article Title: Isolation and Proliferation of Spermatogonial Cells from Ghezel Sheep

    doi:

    Figure Lengend Snippet: Oct-4 immunocytochemical staining of sheep spermatogonial stem cells (×40).

    Article Snippet: Briefly, anti Oct-4 (Abcam) diluted in TBS/BSA was applied over slides.

    Techniques: Staining

    Revertant iPSCs from RDEB skin (a) Persistent mRNA expression of OCT4, SOX2 , NANOG , LIN28 , and DNMT3b , and transient mRNA expression of c-MYC and KLF4 , both consistent with fully reprogrammed mutant and revertant iPSCs. (b) Protein expression in mutant iPSCs of embryonic stem cell surface markers TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase (AF), and transcription factors Nanog and OCT3/4. (c) Same embryonic stem cell protein expression panel in revertant iPSCs. Nuclei are stained with DAPI (blue). Lower panels show corresponding isotype controls. Scale bar = 50 μm.

    Journal: The Journal of investigative dermatology

    Article Title: Patient-specific naturally gene-reverted induced pluripotent stem cells in recessive dystrophic epidermolysis bullosa

    doi: 10.1038/jid.2013.523

    Figure Lengend Snippet: Revertant iPSCs from RDEB skin (a) Persistent mRNA expression of OCT4, SOX2 , NANOG , LIN28 , and DNMT3b , and transient mRNA expression of c-MYC and KLF4 , both consistent with fully reprogrammed mutant and revertant iPSCs. (b) Protein expression in mutant iPSCs of embryonic stem cell surface markers TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase (AF), and transcription factors Nanog and OCT3/4. (c) Same embryonic stem cell protein expression panel in revertant iPSCs. Nuclei are stained with DAPI (blue). Lower panels show corresponding isotype controls. Scale bar = 50 μm.

    Article Snippet: The following antibodies were used: TRA1-60 (clone MAB4360, 1:400), TRA1-81 (clone MAB4381, 1:400), SSEA4 (clone MAB4304, 1:100) and SSEA3 (clone MAB-4303, 1:100) from Millipore; NANOG (clone EB06860, 1:100) from Everest Biotech, Upper Heyford, Oxfordshire, UK; OCT3/4 (clone AB27985, 1:200) from ABCAM, Cambridge, MA; and SOX2 (clone 630802, 1:500) from Biolegend, San Diego, CA.

    Techniques: Expressing, Mutagenesis, Staining

    The effect of B7-H3 on mouse SSC proliferation A. The expression of C-kit and Oct-4 on mouse SSCs was analyzed by flow cytometry. B. Mouse SSCs were incubated with different concentrations of B7-H3 (0, 5, 10, 25 ng/mL) for various time points (1, 6, 12, 24, 48, 72 h) in vitro . Then, cells were collected for proliferation assessment using CCK8 assays. The results are expressed as the mean±standard deviation ( n = 3). Differences were analyzed by two-way ANOVA followed by Tukey's post hoc analysis. ###P

    Journal: Oncotarget

    Article Title: B7-H3 promoted proliferation of mouse spermatogonial stem cells via the PI3K signaling pathway

    doi: 10.18632/oncotarget.23457

    Figure Lengend Snippet: The effect of B7-H3 on mouse SSC proliferation A. The expression of C-kit and Oct-4 on mouse SSCs was analyzed by flow cytometry. B. Mouse SSCs were incubated with different concentrations of B7-H3 (0, 5, 10, 25 ng/mL) for various time points (1, 6, 12, 24, 48, 72 h) in vitro . Then, cells were collected for proliferation assessment using CCK8 assays. The results are expressed as the mean±standard deviation ( n = 3). Differences were analyzed by two-way ANOVA followed by Tukey's post hoc analysis. ###P

    Article Snippet: Anti-phospho-PI3K antibody, anti-phospho-ERK1/2 antibody, anti-phospho-JNK1/2/3 antibody, anti-C-kit antibody, anti-Oct-4 antibody, FITC-conjugated anti-B7-H3 antibody, PE-conjugated goat anti-rat IgG, BRDU, anti-BrdU antibody and Alexa Fluor 488- conjugated goat anti-rat IgG were obtained from Abcam (Cambridge, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Incubation, In Vitro, Standard Deviation

    Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of Oct4, Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p

    Journal: PLoS ONE

    Article Title: Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells

    doi: 10.1371/journal.pone.0074968

    Figure Lengend Snippet: Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of Oct4, Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p

    Article Snippet: The membrane with transferred proteins was incubated in buffer containing specific rabbit polyclonal antibodies for Sox2/Nanog or goat polyclonal antibodies for Oct4 (Abcam, Kendall square, MA, USA, 1∶1000 dilution), followed by incubating with goat anti-rabbit or donkey anti-goat secondary antibody conjugated with horseradish peroxidase at 1∶1000.

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    Gain of H3.3 at pluripotency genes is required. a Line plot demonstrating the dynamic H3.3 enrichment on the pluripotency genes-set during cellular reprogramming. Y -axis represents the normalized number of reads mapping to the genebodies of these genes at the indicated time-points ( x -axis). b Line plot demonstrating the dynamic H3.3 enrichment on the epithelial genes-set during cellular reprogramming. Y -axis represents the normalized number of reads mapping to the genebodies of these genes at the indicated time-points ( x -axis). c , d Average enrichment profile of H3.3 reads, of the indicated time-points, around Nanog-bound ( c ) and Eset-bound ( d ) genomic loci. The y -axis represents average normalized number of fragments mapping to the corresponding regions indicated in the x -axis. e Venn diagram demonstrating uniquely and commonly bound regions among D0 H3.3 (left), D16S+ H3.3 (middle), iPSC H3.3 (right) with mESCs Oct4, Sox2 or Nanog (OSN). f Differential GO analysis revealing the enriched biological processes by genes commonly bound by OSN and H3.3 at the indicated time-points. The colour ranges from white (no enrichment) to dark red (high enrichment). g Heatmap revealing the enrichment of the motifs of the indicated transcription factors in the intergenic regions bound by H3.3 in the reprogramming cells (both successful and unsuccessful). Enrichment ranges from not enriched (white) to highly enriched (dark red). h Schematics of the knockdown experiment (top). The bar chart below represents the number of AP-stained colonies ( y -axis) observed in wells in which the knockdown of the H3.3 had been performed. Non-targeting siNT constructs were used as controls. Values are mean ± s.e.m from independent replicate experiments ( n = 3). Two-tailed t -test was used for statistical analysis. Error bars represent standard deviation

    Journal: Nature Communications

    Article Title: Global H3.3 dynamic deposition defines its bimodal role in cell fate transition

    doi: 10.1038/s41467-018-03904-7

    Figure Lengend Snippet: Gain of H3.3 at pluripotency genes is required. a Line plot demonstrating the dynamic H3.3 enrichment on the pluripotency genes-set during cellular reprogramming. Y -axis represents the normalized number of reads mapping to the genebodies of these genes at the indicated time-points ( x -axis). b Line plot demonstrating the dynamic H3.3 enrichment on the epithelial genes-set during cellular reprogramming. Y -axis represents the normalized number of reads mapping to the genebodies of these genes at the indicated time-points ( x -axis). c , d Average enrichment profile of H3.3 reads, of the indicated time-points, around Nanog-bound ( c ) and Eset-bound ( d ) genomic loci. The y -axis represents average normalized number of fragments mapping to the corresponding regions indicated in the x -axis. e Venn diagram demonstrating uniquely and commonly bound regions among D0 H3.3 (left), D16S+ H3.3 (middle), iPSC H3.3 (right) with mESCs Oct4, Sox2 or Nanog (OSN). f Differential GO analysis revealing the enriched biological processes by genes commonly bound by OSN and H3.3 at the indicated time-points. The colour ranges from white (no enrichment) to dark red (high enrichment). g Heatmap revealing the enrichment of the motifs of the indicated transcription factors in the intergenic regions bound by H3.3 in the reprogramming cells (both successful and unsuccessful). Enrichment ranges from not enriched (white) to highly enriched (dark red). h Schematics of the knockdown experiment (top). The bar chart below represents the number of AP-stained colonies ( y -axis) observed in wells in which the knockdown of the H3.3 had been performed. Non-targeting siNT constructs were used as controls. Values are mean ± s.e.m from independent replicate experiments ( n = 3). Two-tailed t -test was used for statistical analysis. Error bars represent standard deviation

    Article Snippet: After blocking, cells were stained with anti-HA antibodies (Santa Cruz), anti-Oct4 antibodies (ab19857, Abcam), or anti-SSEA-1 antibodies (Miltenyi Biotec), or anti-Nanog antibodies (ReproCELL Inc) followed by Alexa 488 or Alexa 555 conjugated secondary antibodies (Life Technologies) and counterstained with Hoechst 33342 (Life Technologies).

    Techniques: Staining, Construct, Two Tailed Test, Standard Deviation

    Characterization and differentiation of patient-derived NLSDM-iPSC. (A) Immunostaining with primary antibodies anti-OCT4, anti-SSEA-4 and anti-TRA-1-81 revealed that NLSDM-iPSCs express markers of pluripotency, including OCT4 (red), SSEA4 (green), and TRA-1-81 (green). Nuclei were stained with DAPI. Magnification: 10 ×. (B) Real-time quantitative RT-PCR assays for eight pluripotency-associate genes in two patient-derived iPSCs compared with fibroblast cell lines (basal level reported as 1). Data represent the mean of three independent experiments, performed in triplicate. PCR reactions were normalized against an internal control (ACTB). (C) Quantification of TG content in iPSCs derived from 2 controls and 2 NLSDM fibroblast cell lines. The graph represents TG content obtained from three independent experiments. The analysis was performed using Student t -test. Thick bar: mean value; error bar: SD. P-values of

    Journal: Molecular Genetics and Metabolism

    Article Title: Generation of induced Pluripotent Stem Cells as disease modelling of NLSDM

    doi: 10.1016/j.ymgme.2017.03.009

    Figure Lengend Snippet: Characterization and differentiation of patient-derived NLSDM-iPSC. (A) Immunostaining with primary antibodies anti-OCT4, anti-SSEA-4 and anti-TRA-1-81 revealed that NLSDM-iPSCs express markers of pluripotency, including OCT4 (red), SSEA4 (green), and TRA-1-81 (green). Nuclei were stained with DAPI. Magnification: 10 ×. (B) Real-time quantitative RT-PCR assays for eight pluripotency-associate genes in two patient-derived iPSCs compared with fibroblast cell lines (basal level reported as 1). Data represent the mean of three independent experiments, performed in triplicate. PCR reactions were normalized against an internal control (ACTB). (C) Quantification of TG content in iPSCs derived from 2 controls and 2 NLSDM fibroblast cell lines. The graph represents TG content obtained from three independent experiments. The analysis was performed using Student t -test. Thick bar: mean value; error bar: SD. P-values of

    Article Snippet: The cells (patient 1: 14th passage; patient 2: 16th passage) were characterized for the expression of the following pluripotent markers: anti-OCT4 (Mouse mAb; Sigma Aldrich, Milan, Italy), anti-SSEA-4 (Mouse mAb; Life Technology, Carlsbad, USA) and anti-TRA-1-81 (Mouse mAb; Millipore, Darmstadt, Germany); the nuclei were counter-stained with DAPI (working DAPI solution was 200 ng/ml; Sigma Aldrich, Milan, Italy) for 2 min.

    Techniques: Derivative Assay, Immunostaining, Staining, Quantitative RT-PCR, Polymerase Chain Reaction

    Characterization of human iPS A. Human iPS cells observed in phase contrast. B. Human iPS labeled with pluripotency markers: anti-SOX2, anti-NANOG and anti-OCT4 with DAPI counterstaining (right). C. iPS cells labeled for alkaline phosphatase (AP) and observed in phase contrast. D. An example of typical DNA content of iPS cells after propidium iodide labeling measured by flow cytometry.

    Journal: Oncotarget

    Article Title: The expression pattern of PFKFB3 enzyme distinguishes between induced-pluripotent stem cells and cancer stem cells

    doi:

    Figure Lengend Snippet: Characterization of human iPS A. Human iPS cells observed in phase contrast. B. Human iPS labeled with pluripotency markers: anti-SOX2, anti-NANOG and anti-OCT4 with DAPI counterstaining (right). C. iPS cells labeled for alkaline phosphatase (AP) and observed in phase contrast. D. An example of typical DNA content of iPS cells after propidium iodide labeling measured by flow cytometry.

    Article Snippet: Cells were incubated overnight at 4°C with primary antibodies directed against Nanog (Stemgent), Sox2 (R & D systems) or Oct4 (Stemgent).

    Techniques: Labeling, Flow Cytometry, Cytometry

    Distinct expression patterns of CSC markers in lung adenocarcinoma and squamous cell carcinoma A. CD133, B. CD44, C. ALDH1, D. SOX2, E. OCT4, and F. Nanog (10x magnification).

    Journal: Oncotarget

    Article Title: Prognostic significance of stem cell-related marker expression and its correlation with histologic subtypes in lung adenocarcinoma

    doi: 10.18632/oncotarget.9894

    Figure Lengend Snippet: Distinct expression patterns of CSC markers in lung adenocarcinoma and squamous cell carcinoma A. CD133, B. CD44, C. ALDH1, D. SOX2, E. OCT4, and F. Nanog (10x magnification).

    Article Snippet: The primary antibodies used against the follows: CD133 (1:200; Spring Bioscience, Pleasanton, CA), CD44 (1:200; Thermo Scientific, Fremont, CA), ALDH1 (1:100; BD Biosciences, San Diego, CA), SOX2 (1:100; Cell Signalling, Beverly, MA), OCT4 (1:100; Cell Marque, Rocklin, CA), Nanog (1:100; Epitomics, CA), E-cadherin (1:100; BD Biosciences, San Jose, CA) and Snail-1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing

    Experimental design and response of hiPSCs to RA treatment over time a hiPSCs were treated with 0.5 µM RA every second passage for a total of 8 steps over time during a culture period of 14 passages; cells that have undergone to 8 passages RA treatment are named hiPSCs-RA 8 . b Representation of AP activity in hiPSCs-RA 8 and hiPSCs. Maintenance of pluripotency in hiPSCs-RA 8 was confirmed by immunofluorescence for Oct4 (Red) and Nanog (Green) c and with depth-gene expression analysis by PluriTest algorithm d . e Quantitative real-time PCR (qPCR) of relative telomere length (RTL) assessed in hiPSCs-F and hiPSCs-TL, in three experimental conditions (regular medium, P0; regular medium P14; RA 8 exposure P14) showing maintenance of length of telomeres during RA exposure. A statistical comparison was made between hiPSCs P14 RA 8 and hiPSCs P0 or hiPSCs P14 RA 8 and hiPSCs P14 by paired Student’s t -test (* p

    Journal: Cell Death & Disease

    Article Title: Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells

    doi: 10.1038/s41419-017-0028-1

    Figure Lengend Snippet: Experimental design and response of hiPSCs to RA treatment over time a hiPSCs were treated with 0.5 µM RA every second passage for a total of 8 steps over time during a culture period of 14 passages; cells that have undergone to 8 passages RA treatment are named hiPSCs-RA 8 . b Representation of AP activity in hiPSCs-RA 8 and hiPSCs. Maintenance of pluripotency in hiPSCs-RA 8 was confirmed by immunofluorescence for Oct4 (Red) and Nanog (Green) c and with depth-gene expression analysis by PluriTest algorithm d . e Quantitative real-time PCR (qPCR) of relative telomere length (RTL) assessed in hiPSCs-F and hiPSCs-TL, in three experimental conditions (regular medium, P0; regular medium P14; RA 8 exposure P14) showing maintenance of length of telomeres during RA exposure. A statistical comparison was made between hiPSCs P14 RA 8 and hiPSCs P0 or hiPSCs P14 RA 8 and hiPSCs P14 by paired Student’s t -test (* p

    Article Snippet: After blocking in TBS-T (0.05% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibodies against Nanog (PA1-097, Thermo Fisher Scientific), Oct4 (60093, STEMCELL Technologies), β-catenin (sc-7963, Santacruz), phospho-Akt Ser473 (9271, Cell Signalling), phospho-Akt Thr308 (4056, Cell Signalling), Akt1 (2967, Cell Signalling), mTOR (2983, Cell Signalling), phospho-mTOR Ser2448 (5536, Cell Signalling), S6K1 (2708, Cell Signalling), phospho-S6K1 Thr389 (9206, Cell Signalling), and Actin (sc-1616, Santa Cruz).

    Techniques: Activity Assay, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction

    Msl1, Nsl1 and Mof regulate mESC-unspecific genes. ( A ) GO analysis ( Tassy and Pourquie, 2013 ) of downregulated genes in sh Msl1, sh Nsl1 and Mof KO mESCs. ( B ) Oct4 expression in cell extracts prepared from sh control, sh Msl1, sh Nsl1 and sh Msl1/Nsl1 mESCs was analysed by Western Blot. Extracts from mouse embryonic fibroblasts (mEFs) were used as negative control. 20 μg of proteins were loaded per lane and normalized by ponceau staining, and the blots were developed with an anti-Oct4 antibody. DOI: http://dx.doi.org/10.7554/eLife.02104.019

    Journal: eLife

    Article Title: Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation

    doi: 10.7554/eLife.02104

    Figure Lengend Snippet: Msl1, Nsl1 and Mof regulate mESC-unspecific genes. ( A ) GO analysis ( Tassy and Pourquie, 2013 ) of downregulated genes in sh Msl1, sh Nsl1 and Mof KO mESCs. ( B ) Oct4 expression in cell extracts prepared from sh control, sh Msl1, sh Nsl1 and sh Msl1/Nsl1 mESCs was analysed by Western Blot. Extracts from mouse embryonic fibroblasts (mEFs) were used as negative control. 20 μg of proteins were loaded per lane and normalized by ponceau staining, and the blots were developed with an anti-Oct4 antibody. DOI: http://dx.doi.org/10.7554/eLife.02104.019

    Article Snippet: To analyse the pluripotency state of KD mESCs the anti-Oct4 (611202; BD Labs) antibody was used.

    Techniques: Expressing, Western Blot, Negative Control, Staining

    Msl1 and Mof binding at pluripotency genes. ( A and B ) Msl1, Nsl1 and Mof binding together with Pol II and H4K16ac profiles at the ( A ) Pou5f1 (Oct4) and ( B ) Sox2 locus at the UCSC genome browser. The Input serves as control. DOI: http://dx.doi.org/10.7554/eLife.02104.020

    Journal: eLife

    Article Title: Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation

    doi: 10.7554/eLife.02104

    Figure Lengend Snippet: Msl1 and Mof binding at pluripotency genes. ( A and B ) Msl1, Nsl1 and Mof binding together with Pol II and H4K16ac profiles at the ( A ) Pou5f1 (Oct4) and ( B ) Sox2 locus at the UCSC genome browser. The Input serves as control. DOI: http://dx.doi.org/10.7554/eLife.02104.020

    Article Snippet: To analyse the pluripotency state of KD mESCs the anti-Oct4 (611202; BD Labs) antibody was used.

    Techniques: Binding Assay

    Contribution of rat EG cells to embryonic chimaeras including germ cells. ( A ) Phase contrast and fluorescence images of Red1.2 EG cell clone. ( B ) Graph showing chromosome number per cell in 50 metaphase spreads from line Red1.2. Inset is a karyotype of Red1.2, showing 42 chromosomes. ( C ) Bright-field and fluorescence images of E13.5 chimaeric embryos. ( D ) Confocal section of genital ridge and surrounding mesonephric tissue dissected from a chimaeric embryo and immunostained for DsRed and Oct4. ( E ) Higher magnification confocal section of D shows cells co-stained for DsRed and Oct4, demonstrating the contribution of EG cells to germ cells. Scale bars: 100 μm in A,D; 25 μm in E.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Contribution of rat EG cells to embryonic chimaeras including germ cells. ( A ) Phase contrast and fluorescence images of Red1.2 EG cell clone. ( B ) Graph showing chromosome number per cell in 50 metaphase spreads from line Red1.2. Inset is a karyotype of Red1.2, showing 42 chromosomes. ( C ) Bright-field and fluorescence images of E13.5 chimaeric embryos. ( D ) Confocal section of genital ridge and surrounding mesonephric tissue dissected from a chimaeric embryo and immunostained for DsRed and Oct4. ( E ) Higher magnification confocal section of D shows cells co-stained for DsRed and Oct4, demonstrating the contribution of EG cells to germ cells. Scale bars: 100 μm in A,D; 25 μm in E.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Fluorescence, Staining

    Mouse EG cells can be maintained and derived in 2i-LIF. ( A ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells. ( B ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells cultured in: FCS-LIF on a MEF feeder layer; FCS-LIF after one passage without feeders; 2i-LIF without feeders. ( C ) Flow cytometry analysis of Oct4-ΔPE-GFP EG cells cultured in 2i-LIF compared with FCS-LIF on feeders. Gates were set to eliminate feeders and cell debris. ( D ) Colony-forming assay on Oct4-ΔPE-GFP EG cells. Colonies were scored for alkaline phosphatase (AP) and designated positive, negative or mixed. Data are means of two biological replicates. ( E ) Schematic of EG cell derivation protocol. On day 3, medium was changed to either FCS-LIF or 2i-LIF. ( F ) Comparison of GFP-positive EG cell colony formation following addition of 2i-LIF on day 3 compared with FCS-LIF. ( G ) High-contribution coat colour chimaeras generated with agouti EG cells derived in 2i-LIF injected into C57BL/6 blastocysts. ( H ) Chimaera, C57BL/6 mate, and mixed litter of agouti and black pups. Scale bars: 100 μm in A; 25 μm in B.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Mouse EG cells can be maintained and derived in 2i-LIF. ( A ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells. ( B ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells cultured in: FCS-LIF on a MEF feeder layer; FCS-LIF after one passage without feeders; 2i-LIF without feeders. ( C ) Flow cytometry analysis of Oct4-ΔPE-GFP EG cells cultured in 2i-LIF compared with FCS-LIF on feeders. Gates were set to eliminate feeders and cell debris. ( D ) Colony-forming assay on Oct4-ΔPE-GFP EG cells. Colonies were scored for alkaline phosphatase (AP) and designated positive, negative or mixed. Data are means of two biological replicates. ( E ) Schematic of EG cell derivation protocol. On day 3, medium was changed to either FCS-LIF or 2i-LIF. ( F ) Comparison of GFP-positive EG cell colony formation following addition of 2i-LIF on day 3 compared with FCS-LIF. ( G ) High-contribution coat colour chimaeras generated with agouti EG cells derived in 2i-LIF injected into C57BL/6 blastocysts. ( H ) Chimaera, C57BL/6 mate, and mixed litter of agouti and black pups. Scale bars: 100 μm in A; 25 μm in B.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Derivative Assay, Fluorescence, Cell Culture, Flow Cytometry, Cytometry, Generated, Injection

    Pre-migratory rat PGCs co-express Oct4 and Blimp1. ( A ) Bright-field image of an E10 rat embryo. Dashed line depicts line of incision to isolate the posterior fragment. ( B ) Bright-field image of an isolated posterior fragment of E10 rat embryo. ( C ) Confocal section through the posterior fragment of an E10 rat embryo immunostained in wholemount for Oct4 (green) and co-stained with DAPI (blue outline). ( D ) Higher magnification confocal section through a posterior embryo fragment showing nuclear staining of Oct4 in a cluster of cells at the base of the allantois. ( E ) Double immunostaining for Oct4 and Blimp1 on a cryosection through the posterior fragment. Scale bars: 100 μm in C, 10 μm in D,E.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Pre-migratory rat PGCs co-express Oct4 and Blimp1. ( A ) Bright-field image of an E10 rat embryo. Dashed line depicts line of incision to isolate the posterior fragment. ( B ) Bright-field image of an isolated posterior fragment of E10 rat embryo. ( C ) Confocal section through the posterior fragment of an E10 rat embryo immunostained in wholemount for Oct4 (green) and co-stained with DAPI (blue outline). ( D ) Higher magnification confocal section through a posterior embryo fragment showing nuclear staining of Oct4 in a cluster of cells at the base of the allantois. ( E ) Double immunostaining for Oct4 and Blimp1 on a cryosection through the posterior fragment. Scale bars: 100 μm in C, 10 μm in D,E.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Isolation, Staining, Double Immunostaining

    Rat EG cells can be derived and expanded in 2i-LIF and express markers of pluripotency. ( A ) Phase contrast image of a primary rat EG cell colony. ( B , C ) Phase contrast images of rat EG cells after 5 (B) and 35 (C) passages. ( D ) Oct4, Sox2 and Nanog immunostaining of rat EG cells. ( E ) Alkaline phosphatase (AP) staining of rat EG cells. ( F ) RT-PCR analysis of pluripotency markers in three early-passage rat EG cell lines (WBY1 P7, WBY2 P7, WBY3 P6) and two late-passage rat EG cell lines (WBY2 P35, WBY7 P33). Controls are rat ES cells (DAX11), rat neural stem (rNS) cells (Ras2-GFP), posterior fragments of E10 embryos and whole E13.5 genital ridges. ( G ) Colony-forming assay on dissociated rat EG cells plated on laminin. Colonies were scored for AP and designated positive or negative. Data are means of two biological replicates. ( H ) Typical AP-positive colony. Scale bars: 100 μm in A-C,E; 25 μm in D.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Rat EG cells can be derived and expanded in 2i-LIF and express markers of pluripotency. ( A ) Phase contrast image of a primary rat EG cell colony. ( B , C ) Phase contrast images of rat EG cells after 5 (B) and 35 (C) passages. ( D ) Oct4, Sox2 and Nanog immunostaining of rat EG cells. ( E ) Alkaline phosphatase (AP) staining of rat EG cells. ( F ) RT-PCR analysis of pluripotency markers in three early-passage rat EG cell lines (WBY1 P7, WBY2 P7, WBY3 P6) and two late-passage rat EG cell lines (WBY2 P35, WBY7 P33). Controls are rat ES cells (DAX11), rat neural stem (rNS) cells (Ras2-GFP), posterior fragments of E10 embryos and whole E13.5 genital ridges. ( G ) Colony-forming assay on dissociated rat EG cells plated on laminin. Colonies were scored for AP and designated positive or negative. Data are means of two biological replicates. ( H ) Typical AP-positive colony. Scale bars: 100 μm in A-C,E; 25 μm in D.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Derivative Assay, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction

    Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, OCT4, SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and

    Journal: Stem Cells Translational Medicine

    Article Title: Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells

    doi: 10.5966/sctm.2013-0179

    Figure Lengend Snippet: Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, OCT4, SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and

    Article Snippet: The cells were incubated with antibodies against NANOG (Cell Signaling Technology, Beverly, MA, ; 1:200, rabbit polyclonal), OCT4 (Cell Signaling Technology; 1:200, rabbit polyclonal), SOX2 (Cell Signaling Technology, 1:200, rabbit polyclonal), SSEA4 (Cell Signaling Technology; 1:200, mouse IgG3 monoclonal), TRA-1-60 (Cell Signaling Technology; 1:200, mouse IgM monoclonal), TRA-1-81 (Cell Signaling Technology; 1:200, mouse IgM monoclonal), human keratin 5/14 (Covance, Princeton, NJ, ; 1:500, rabbit IgG polyclonal), and each negative control antibody (Dako) overnight at 4°C.

    Techniques: Immunofluorescence, Expressing, Quantitative RT-PCR