anti nanog Search Results


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  • 93
    Thermo Fisher c terminus directed anti nanog ab
    Reactivity of rNanogP8 and rNanog proteins towards 8 <t>anti-Nanog</t> Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb <t>pAb.</t>
    C Terminus Directed Anti Nanog Ab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti nanog
    Continuous exposure to gemcitabine increases tumorigenicity but continuous exposure to sulforaphane or quercetin reduces it. ( a ) BxPC-3, Bx-GEM, Bx-Q and Bx-SF cells were seeded at a low density (2000 cells/well) in 6-well plates. After 2 weeks, cells were Coomassie-stained and colonies containing more than 50 cells were counted under a dissecting microscope. The survival fraction and representative photographs of colonies (first generation) are presented on the left. For second-generation colony formation, an equal amount of living cells from first-generation colonies were collected and 2000 cells per well were re-seeded. The colony formation was analyzed as described above and is presented on the right. ( b ) Cells were cultured to 90% confluence before the cell layer was scratched with the tip of a 10- μ l pipette. Closure of the wounded region was evaluated 24 h after scratching by microscopy at × 100 magnification. For quantification of the scratched area, the percentage of the gap area was evaluated and calculated by TScratch software (diagram below photographs). ( c ) Cells were seeded in 6-well plates, followed by exposure to NH Osteo-Diff medium to induce osteocytic differentiation. Fourteen days later, the cells were stained with BCIP/NBT substrate for alkaline phosphatases, expressed by cells differentiated into osteocytes, which appear dark. Representative images at × 200 magnification are shown. ( d ) Proteins were harvested and the expression of <t>EpCAM,</t> <t>Nanog,</t> Twist2 and E-cadherin was measured by western blot analysis. β -Actin was used as a loading control. Three independent experiments were performed at least in triplicates and the data are presented as means ±S.D. * P
    Anti Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ReproCELL anti nanog antibodies
    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 <t>Nanog-bound</t> ( 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 <t>Oct4,</t> 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
    Anti Nanog Antibodies, supplied by ReproCELL, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti nanog antibody
    SKM FK 2i-06 cell line expresses pluripotency markers SSEA1, <t>OCT4,</t> and <t>NANOG</t> in feeder-free conditions. SKM FK 2i-06 was transitioned to serum without small molecules at two passages postisolation. SKM FK 2i-06 is shown eight passages postisolation and
    Anti Nanog Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    BioLegend biolegend anti nanog ab
    Reactivity of rNanogP8 and rNanog proteins towards 8 <t>anti-Nanog</t> Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the <t>BioLegend</t> Rb pAb.
    Biolegend Anti Nanog Ab, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abcam anti nanog mouse monoclonal antibody ab
    Reactivity of rNanogP8 and rNanog proteins towards 8 <t>anti-Nanog</t> Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the <t>BioLegend</t> Rb pAb.
    Anti Nanog Mouse Monoclonal Antibody Ab, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Santa Cruz Biotechnology rrid ab 628051 rabbit anti nanog
    Reactivity of rNanogP8 and rNanog proteins towards 8 <t>anti-Nanog</t> Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the <t>BioLegend</t> Rb pAb.
    Rrid Ab 628051 Rabbit Anti Nanog, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti nanog
    The time window for induced Epi lineage conversion can be narrowed to a 4 hours time period prior E3.75. ( A ) Schematic of the time schedule of inhibitor treatment. Orange and grey lines indicate the culture periods in the presence of inhibitors or DMSO (vehicle), respectively. ( B ) Immunodetection of <t>NANOG</t> (green) and <t>GATA6</t> (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. Error bars indicate SEM. n , number of embryos analyzed.
    Anti Nanog, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Kamiya anti nanog abs
    Reactivity of rNanogP8 and <t>rNanog</t> proteins towards 8 <t>anti-Nanog</t> Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.
    Anti Nanog Abs, supplied by Kamiya, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    WuXi AppTec nanog
    The expression of reprogramming transcription factors in 2D- and 3D-grown A549 cells. ( A ) qRT-PCR for the expression of reprogramming transcription factors <t>OCT4,</t> SOX2, <t>NANOG,</t> c-MYC and LIN28 in 2D- and 3D-grown A549 cells. ( B ) Western blotting for expression of OCT4, SOX2 and NANOG in 2D- and 3D-grown A549 cells. ( C ) Gray analysis the result of Figure 3 B. ( D ) qRT-PCR for the expression of reprogramming factor miR-302a expression in 2D- and 3D-grown A549 cells. ( E ) Western blotting for the expression of β-catenin in 2D- and 3D-grown A549 cells. Data are presented as mean ± SE. Experiments were independently repeated at least three times.
    Nanog, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.

    Article Snippet: Similarly, when IP was done with another C-terminus directed anti-Nanog Ab, i.e., the R & D goat pAb, followed by WB using eBioscience mAb, we again detected the 42 kD Nanog protein in NTERA-2 NE ( , lane 3).

    Techniques: Western Blot, Clone Assay

    IP and ID studies with 5 anti-Nanog antibodies in NTERA-2 and cancer cells. ( A ) The NE of NTERA-2 and various cancer cells was used in IP with the Kamiya anti-Nanog Rb pAb followed by WB with the eBioscience mAb. Lanes 1-6 were regular WB with either cytosol (C) or NE. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain (∼53 kD). Note that the prominent 42 kD protein band was detected on WB (lane 4) and immunoprecipitated down (lane 9) only in NTERA-2 NE. ( B ) The NTERA-2 NE or Du145 cytosol (cyto) or NE was used in IP with the CS anti-Nanog rabbit mAb followed by WB with the R D goat pAb. Lanes 1-3 were regular WB. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain. Note that the 42 kD Nanog protein was detected on WB (lane 1) and immunoprecipitated down (lane 9) only in NTERA-2 NE. ( C ) The NE of NTERA-2 and Du145 cells was used in IP with the SC anti-Nanog rabbit pAb H155 followed by WB with eBioscience mAb. Lanes 1–4 were regular WB using two independent preparations of Du145 or NTERA-2 NE. Red arrowhead, the 42-kD band; black arrowhead, the 35-kD Nanog band; IgH, IgG heavy chain. Note that the 42-kD protein band was detected on WB (lane 3 and 4) and immunoprecipitated down (lane 8) only in NTERA-2 NE. The right-pointing bracket indicates the cluster of Nanog protein bands below the dominant 42 kD band. ( D ) The NE of NTERA-2 cells and MCF7 cells (two independent preparations) was used in IP with the R D anti-Nanog goat pAb (goat IgG used as the control) followed by WB with the eBioscience mAb. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain. Note that the 42-kD protein band was detected on WB (lane 4; input) and immunoprecipitated down (lane 3) only in NTERA-2 NE. ( E–F ) Nanog protein ID by MALDI-TOF/TOF in NTERA-2 NE following IP using the R D goat pAb. Shown are SYPRO Ruby gel image (E; NTRD1-4 refer to the 4 gel slices cut out for protein elution) and the Nanog peptides recovered from each gel slice (F).

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: IP and ID studies with 5 anti-Nanog antibodies in NTERA-2 and cancer cells. ( A ) The NE of NTERA-2 and various cancer cells was used in IP with the Kamiya anti-Nanog Rb pAb followed by WB with the eBioscience mAb. Lanes 1-6 were regular WB with either cytosol (C) or NE. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain (∼53 kD). Note that the prominent 42 kD protein band was detected on WB (lane 4) and immunoprecipitated down (lane 9) only in NTERA-2 NE. ( B ) The NTERA-2 NE or Du145 cytosol (cyto) or NE was used in IP with the CS anti-Nanog rabbit mAb followed by WB with the R D goat pAb. Lanes 1-3 were regular WB. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain. Note that the 42 kD Nanog protein was detected on WB (lane 1) and immunoprecipitated down (lane 9) only in NTERA-2 NE. ( C ) The NE of NTERA-2 and Du145 cells was used in IP with the SC anti-Nanog rabbit pAb H155 followed by WB with eBioscience mAb. Lanes 1–4 were regular WB using two independent preparations of Du145 or NTERA-2 NE. Red arrowhead, the 42-kD band; black arrowhead, the 35-kD Nanog band; IgH, IgG heavy chain. Note that the 42-kD protein band was detected on WB (lane 3 and 4) and immunoprecipitated down (lane 8) only in NTERA-2 NE. The right-pointing bracket indicates the cluster of Nanog protein bands below the dominant 42 kD band. ( D ) The NE of NTERA-2 cells and MCF7 cells (two independent preparations) was used in IP with the R D anti-Nanog goat pAb (goat IgG used as the control) followed by WB with the eBioscience mAb. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain. Note that the 42-kD protein band was detected on WB (lane 4; input) and immunoprecipitated down (lane 3) only in NTERA-2 NE. ( E–F ) Nanog protein ID by MALDI-TOF/TOF in NTERA-2 NE following IP using the R D goat pAb. Shown are SYPRO Ruby gel image (E; NTRD1-4 refer to the 4 gel slices cut out for protein elution) and the Nanog peptides recovered from each gel slice (F).

    Article Snippet: Similarly, when IP was done with another C-terminus directed anti-Nanog Ab, i.e., the R & D goat pAb, followed by WB using eBioscience mAb, we again detected the 42 kD Nanog protein in NTERA-2 NE ( , lane 3).

    Techniques: Western Blot, Immunoprecipitation

    Exogenous NanogP8 expressed in LNCaP cells migrates mainly at 42 kD. ( A ) Exogenous NanogP8 migrates at 42 kD on WB. Whole cell lysate (80 µg/lane) prepared from control LNCaP (pLVX) or Nanog1/NanogP8 overexpressing LNCaP cells [34] in the presence of increasing amounts of doxycycline (Dox) was used in WB with the Kamiya anti-Nanog rabbit pAb. The red and black arrowheads indicate the main 42 kD and minor 35 kD Nanog bands, respectively. Green arrow, a ∼28 kD band that also increased upon Dox induction. Asterisk, a non-specific band. N-tera, NE of NTERA-2 cell; SE, short exposure; LE, long exposure. ( B ) The exogenous 42 kD and 35 kD bands could be immunoprecipitated down by the R D anti-Nanog pAb. Whole-cell lysate (WCL; 500 µg) derived from pLVX, pLVX-NANOG1 and pLVX-NANOGP8 LNCaP cells [34] were used in IP with the R D anti-Nanog goat pAb (goat IgG used as the control) followed by WB with Kamiya anti-Nanog rabbit pAb. The red and black arrowheads indicate the main 42 kD and minor 35 kD Nanog bands, respectively. Asterisk, a non-specific band. NE, nuclear extract; cyto, cytosol protein. Note that the 42-kD and the 35-kD protein bands only from pLVX-NANOG1 and pLVX-NANOGP8 LNCaP cells (but not from LNCaP-pLVX cells) were IP'ed down and detected on WB (lanes 5,6). Goat IgG did not pull down any specific bands. Also, WCL from two batches (1 and 2) of Du145 s and LNCaP cells (80 µg/lane) did not reveal the 42 kD and 35 kD protein bands on WB (lanes 7-10). The 37 kD non-specific band (asterisk) was not immunoprecipitated down by the R D goat pAb (lane 5–6).

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: Exogenous NanogP8 expressed in LNCaP cells migrates mainly at 42 kD. ( A ) Exogenous NanogP8 migrates at 42 kD on WB. Whole cell lysate (80 µg/lane) prepared from control LNCaP (pLVX) or Nanog1/NanogP8 overexpressing LNCaP cells [34] in the presence of increasing amounts of doxycycline (Dox) was used in WB with the Kamiya anti-Nanog rabbit pAb. The red and black arrowheads indicate the main 42 kD and minor 35 kD Nanog bands, respectively. Green arrow, a ∼28 kD band that also increased upon Dox induction. Asterisk, a non-specific band. N-tera, NE of NTERA-2 cell; SE, short exposure; LE, long exposure. ( B ) The exogenous 42 kD and 35 kD bands could be immunoprecipitated down by the R D anti-Nanog pAb. Whole-cell lysate (WCL; 500 µg) derived from pLVX, pLVX-NANOG1 and pLVX-NANOGP8 LNCaP cells [34] were used in IP with the R D anti-Nanog goat pAb (goat IgG used as the control) followed by WB with Kamiya anti-Nanog rabbit pAb. The red and black arrowheads indicate the main 42 kD and minor 35 kD Nanog bands, respectively. Asterisk, a non-specific band. NE, nuclear extract; cyto, cytosol protein. Note that the 42-kD and the 35-kD protein bands only from pLVX-NANOG1 and pLVX-NANOGP8 LNCaP cells (but not from LNCaP-pLVX cells) were IP'ed down and detected on WB (lanes 5,6). Goat IgG did not pull down any specific bands. Also, WCL from two batches (1 and 2) of Du145 s and LNCaP cells (80 µg/lane) did not reveal the 42 kD and 35 kD protein bands on WB (lanes 7-10). The 37 kD non-specific band (asterisk) was not immunoprecipitated down by the R D goat pAb (lane 5–6).

    Article Snippet: Similarly, when IP was done with another C-terminus directed anti-Nanog Ab, i.e., the R & D goat pAb, followed by WB using eBioscience mAb, we again detected the 42 kD Nanog protein in NTERA-2 NE ( , lane 3).

    Techniques: Western Blot, Immunoprecipitation, Derivative Assay

    Characterization of Nanog proteins in NTERA-2 cells upon siRNA-mediated knockdown. ( A ) Nanog siRNA experiments in NTERA-2 cells. NTERA-2 cells were transfected with a pool of Nanog-specific siRNAs for 48 h, harvested, and used to isolate the NE for WB with the Kamiya Ab. The left and right panels represent the short and long exposure (SE and LE, respectively) films (note that the left bottom panel is the shortest exposed film to illustrate the significant knockdown of the 42 kD band). Lamin A/C was the loading control. UT, untransfected; NC siRNA, negative control (siCONTROL non-targeting) siRNAs; Nanog siRNA, SMARTpool siRNA against Nanog. Red, black, and blue arrowheads indicate the major 42 kD, minor 35 kD, and 48 kD protein bands, respectively, that were reduced upon Nanog knockdown. The green arrows refer to additional bands that also showed reduction. ( B ) The WB was performed with the CS anti-Nanog rabbit pAb.

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: Characterization of Nanog proteins in NTERA-2 cells upon siRNA-mediated knockdown. ( A ) Nanog siRNA experiments in NTERA-2 cells. NTERA-2 cells were transfected with a pool of Nanog-specific siRNAs for 48 h, harvested, and used to isolate the NE for WB with the Kamiya Ab. The left and right panels represent the short and long exposure (SE and LE, respectively) films (note that the left bottom panel is the shortest exposed film to illustrate the significant knockdown of the 42 kD band). Lamin A/C was the loading control. UT, untransfected; NC siRNA, negative control (siCONTROL non-targeting) siRNAs; Nanog siRNA, SMARTpool siRNA against Nanog. Red, black, and blue arrowheads indicate the major 42 kD, minor 35 kD, and 48 kD protein bands, respectively, that were reduced upon Nanog knockdown. The green arrows refer to additional bands that also showed reduction. ( B ) The WB was performed with the CS anti-Nanog rabbit pAb.

    Article Snippet: Similarly, when IP was done with another C-terminus directed anti-Nanog Ab, i.e., the R & D goat pAb, followed by WB using eBioscience mAb, we again detected the 42 kD Nanog protein in NTERA-2 NE ( , lane 3).

    Techniques: Transfection, Western Blot, Negative Control

    Mass spectrometry analysis of Nanog peptides recovered from NTERA-2 NE. ( A ) Flow chart of tandem IP and the sample preparation for LTQ mass spec analysis. ( B ) The gel image of SYPRO Ruby staining. The NE from NTERA-2 cells was used to perform the tandem IP with two anti-Nanog antibodies (i.e., Kamiya rabbit pAb and R D goat pAb). The immunoprecipitates were separated by SDS-PAGE and the gel was stained with SYPRO Ruby. Gel slices as indicated (dashed lines and numbers) were cut out to elute proteins for LTQ mass spectrometry analysis. M.W#1 and M.W#2 were two protein markers. ( C ) Nanog peptides (sequences and total counts indicated) recovered for each gel slice.

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: Mass spectrometry analysis of Nanog peptides recovered from NTERA-2 NE. ( A ) Flow chart of tandem IP and the sample preparation for LTQ mass spec analysis. ( B ) The gel image of SYPRO Ruby staining. The NE from NTERA-2 cells was used to perform the tandem IP with two anti-Nanog antibodies (i.e., Kamiya rabbit pAb and R D goat pAb). The immunoprecipitates were separated by SDS-PAGE and the gel was stained with SYPRO Ruby. Gel slices as indicated (dashed lines and numbers) were cut out to elute proteins for LTQ mass spectrometry analysis. M.W#1 and M.W#2 were two protein markers. ( C ) Nanog peptides (sequences and total counts indicated) recovered for each gel slice.

    Article Snippet: Similarly, when IP was done with another C-terminus directed anti-Nanog Ab, i.e., the R & D goat pAb, followed by WB using eBioscience mAb, we again detected the 42 kD Nanog protein in NTERA-2 NE ( , lane 3).

    Techniques: Mass Spectrometry, Flow Cytometry, Sample Prep, Staining, SDS Page

    WB analysis of endogenous Nanog1 protein species in NTERA-2 cells. ( A ) Schematic of the human Nanog protein and 8 anti-Nanog Abs used in this study. Shown in parentheses are epitopes of individual Abs. ND, N-terminus domain; HD, homeodomain; CD1 and CD2, C-terminus domain 1 and 2; WR, tryptophan-rich domain. The asterisk in ND indicates the Leu61 residue recognized by the eBioscience mAb (arrow) mapped from our present studies. (B–H) WB analysis in NTERA-2 NE (N, two different batches) or cytosol (C) using 8 anti-Nanog Abs. Individual Ab is indicated at the bottom and M.W on the left. Black arrowhead, the predicted 35 kD Nanog protein; red arrowhead, the main 42 kD band; green arrows, additional bands (especially after longer exposures).

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: WB analysis of endogenous Nanog1 protein species in NTERA-2 cells. ( A ) Schematic of the human Nanog protein and 8 anti-Nanog Abs used in this study. Shown in parentheses are epitopes of individual Abs. ND, N-terminus domain; HD, homeodomain; CD1 and CD2, C-terminus domain 1 and 2; WR, tryptophan-rich domain. The asterisk in ND indicates the Leu61 residue recognized by the eBioscience mAb (arrow) mapped from our present studies. (B–H) WB analysis in NTERA-2 NE (N, two different batches) or cytosol (C) using 8 anti-Nanog Abs. Individual Ab is indicated at the bottom and M.W on the left. Black arrowhead, the predicted 35 kD Nanog protein; red arrowhead, the main 42 kD band; green arrows, additional bands (especially after longer exposures).

    Article Snippet: In the first , we performed IP experiments using 500 µ g of NE from NTERA-2 and somatic cancer cells and with 5 anti-Nanog Abs, i.e., eBioscience mAb, Kamiya pAb, CS Rb mAb, SC pAb H155, and R & D goat pAb ( ; ; data not shown).

    Techniques: Western Blot

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2016.157

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

    Article Snippet: Antibodies were mouse monoclonal anti-EpCAM (HEA125), anti-Twist2 (Abcam, Cambridge, UK), anti-Nanog and anti-β -actin (Sigma-Aldrich) and rabbit monoclonal anti-E-cadherin (24E10, Cell Signalling Technology, Danvers, MA, USA).

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

    H7.S6 OCT4 and NANOG reporter lines created by existing transgenic methods show mosaic expression, highlighting the need for BAC transgenes. ( a ) Stable H7.S6 hESC clones carrying 19 kb OCT4 or 25 kb NANOG reporter transgenes to express mCherry or GFP IRES neo reporter cassettes (see also Supplementary Figure S1 ), exhibited mosaic expression as determined by fluorescent imaging (left panels) or flow cytometry (FACS scans at the right). Fluorescent protein expression from the transgenes fell upon removal of G418 selection (compare +G418 with –G418 FACS panels). ( b ) Immunostaining of H7.S6 OCT4-mCherry and NANOG-mCherry reporter lines (63×, zoom) for endogenous OCT4 or NANOG expression showed that most cells expressed the endogenous proteins but many did not express the fluorescent reporter (arrowheads), indicating that the mosaic expression was due to silencing of the transgene and not differentiation of the cells. ( c ) Double stable reporter H7.S6 OCT4-mCherry/OCT4-GFP and NANOG-mCherry/NANOG-GFP lines were generated after transfecting the single OCT4 and NANOG reporters above. Both OCT4 and NANOG double mCherry/GFP reporters showed partially overlapping mosaicism, indicating random silencing of the reporter. Arrows and asterisk show cells that exhibited only GFP or mCherry fluorescence, respectively.

    Journal: Nucleic Acids Research

    Article Title: Transposon-mediated BAC transgenesis in human ES cells

    doi: 10.1093/nar/gks643

    Figure Lengend Snippet: H7.S6 OCT4 and NANOG reporter lines created by existing transgenic methods show mosaic expression, highlighting the need for BAC transgenes. ( a ) Stable H7.S6 hESC clones carrying 19 kb OCT4 or 25 kb NANOG reporter transgenes to express mCherry or GFP IRES neo reporter cassettes (see also Supplementary Figure S1 ), exhibited mosaic expression as determined by fluorescent imaging (left panels) or flow cytometry (FACS scans at the right). Fluorescent protein expression from the transgenes fell upon removal of G418 selection (compare +G418 with –G418 FACS panels). ( b ) Immunostaining of H7.S6 OCT4-mCherry and NANOG-mCherry reporter lines (63×, zoom) for endogenous OCT4 or NANOG expression showed that most cells expressed the endogenous proteins but many did not express the fluorescent reporter (arrowheads), indicating that the mosaic expression was due to silencing of the transgene and not differentiation of the cells. ( c ) Double stable reporter H7.S6 OCT4-mCherry/OCT4-GFP and NANOG-mCherry/NANOG-GFP lines were generated after transfecting the single OCT4 and NANOG reporters above. Both OCT4 and NANOG double mCherry/GFP reporters showed partially overlapping mosaicism, indicating random silencing of the reporter. Arrows and asterisk show cells that exhibited only GFP or mCherry fluorescence, respectively.

    Article Snippet: Immunostaining and microscopy The incubation with primary antibodies was for 1 h at room temperature with mouse anti-Oct4 (1:50, sc-5279; Santa Cruz), rabbit anti-Nanog (1:30, AB5731; Chemicon) or overnight at +4°C with mouse anti-Pax6 (1:30; Developmental Studies Hybridoma Bank).

    Techniques: Transgenic Assay, Expressing, BAC Assay, Clone Assay, Imaging, Flow Cytometry, Cytometry, FACS, Selection, Immunostaining, Generated, Fluorescence

    HLEC-iPSCs exhibit key HESC markers and HESC morphology. (A) Real-time RT-PCR analysis of ES markers in HLEC-iPS clones and H9 ES clones. Primers were used that specifically recognize endogenous HESC-specific genes in iPS clones (OCT-4, NANOG, REX-1, SOX-2) and vimentin, which is an HLEC-specific gene. (B) A microarray analysis was performed to compare the gene expression profiles of HLEC-iPSCs, a human H9 ES cell line and primary HLECs. A hierarchical cluster analysis of 8030 orthologous human genes was performed based on the signal ratios. The distances between the genetic profiles of the samples are shown. (C-H): Pluripotency marker staining (red) of the iPS1 clone, (C) SSEA-4, (D) SSEA-3, (E) NANOG, (F) TRA-60, (G) TRA-81 and (H) OCT-4.

    Journal: PLoS ONE

    Article Title: Efficient Generation of Lens Progenitor Cells from Cataract Patient-Specific Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0032612

    Figure Lengend Snippet: HLEC-iPSCs exhibit key HESC markers and HESC morphology. (A) Real-time RT-PCR analysis of ES markers in HLEC-iPS clones and H9 ES clones. Primers were used that specifically recognize endogenous HESC-specific genes in iPS clones (OCT-4, NANOG, REX-1, SOX-2) and vimentin, which is an HLEC-specific gene. (B) A microarray analysis was performed to compare the gene expression profiles of HLEC-iPSCs, a human H9 ES cell line and primary HLECs. A hierarchical cluster analysis of 8030 orthologous human genes was performed based on the signal ratios. The distances between the genetic profiles of the samples are shown. (C-H): Pluripotency marker staining (red) of the iPS1 clone, (C) SSEA-4, (D) SSEA-3, (E) NANOG, (F) TRA-60, (G) TRA-81 and (H) OCT-4.

    Article Snippet: Following 3 washes with PBS, the cells were permeabilized in 0.2% Triton X-100 for 30 min, blocked for 1 hour in 10% FCS and 1% BSA in PBS and incubated overnight at 4°C with primary antibodies against NANOG, OCT-4, SSEA-3, SSEA-4, TRA-60, TRA-81, PAX6, α-crystallin andβ-crystallin (Chemicon, USA, 1∶200) in 1% BSA in PBS.

    Techniques: Quantitative RT-PCR, Clone Assay, Microarray, Expressing, Marker, Staining

    Differentiation of hiPSC into cortical neurons and efficient transduction with AAV-TAU-P301L (A) Immunostaining for OCT4 and NANOG shows that iPSC0028 is pluripotent. Scale bar represents 50μm. (B-C) Immunostaining for Nestin and PAX6 revealing NPC stage at DIV25. Scale bar = 50μm for both. (D-F) Immunostaining on DIV70 visualizes the neuronal marker TUBB3 and cortical markers TBR1 and CTIP2 (D) as well as the dendritic marker MAP2 (E-F) together with either vGLUT2 (E) or vGAT (F). Scale bar = 25μm. (G-I) Representative traces of intrinsic neuronal properties of DIV70 neurons showing evoked responses in current clamp (G) as well as sodium and potassium currents (H) in voltage clamp (n = 13 cells). (I) Example of spontaneous EPSCs recorded at a holding of -65mV in the presence of 50μM PTX in voltage clamp mode. (J) Quantitative RTPCR data showing that transduced neurons express both 3R and 4R TAU mRNA, represented by an increased 4R/3R TAU ratio compared to non-transduced control cells (P = 0.04; n≥3 from different experiments). Values were normalized to PGK1 before analyses. * P

    Journal: PLoS ONE

    Article Title: Using Human iPSC-Derived Neurons to Model TAU Aggregation

    doi: 10.1371/journal.pone.0146127

    Figure Lengend Snippet: Differentiation of hiPSC into cortical neurons and efficient transduction with AAV-TAU-P301L (A) Immunostaining for OCT4 and NANOG shows that iPSC0028 is pluripotent. Scale bar represents 50μm. (B-C) Immunostaining for Nestin and PAX6 revealing NPC stage at DIV25. Scale bar = 50μm for both. (D-F) Immunostaining on DIV70 visualizes the neuronal marker TUBB3 and cortical markers TBR1 and CTIP2 (D) as well as the dendritic marker MAP2 (E-F) together with either vGLUT2 (E) or vGAT (F). Scale bar = 25μm. (G-I) Representative traces of intrinsic neuronal properties of DIV70 neurons showing evoked responses in current clamp (G) as well as sodium and potassium currents (H) in voltage clamp (n = 13 cells). (I) Example of spontaneous EPSCs recorded at a holding of -65mV in the presence of 50μM PTX in voltage clamp mode. (J) Quantitative RTPCR data showing that transduced neurons express both 3R and 4R TAU mRNA, represented by an increased 4R/3R TAU ratio compared to non-transduced control cells (P = 0.04; n≥3 from different experiments). Values were normalized to PGK1 before analyses. * P

    Article Snippet: After 30’ blocking, cells were incubated overnight at 4°C with following primary antibodies: mouse anti-β3 tubulin, mouse anti-PAX6 (both Covance), chicken anti-MAP2 (Aves), rabbit anti-Tbr1, rat anti-Ctip2, mouse anti-Nestin (all Abcam), rabbit anti-vGlut2, rabbit anti-vGAT (both Synaptic Systems), mouse anti-OCT4, mouse anti-HuC/D (both Invitrogen), mouse anti-Nanog, mouse anti-RD4 (both Millipore), mouse anti-AT8 (Innogenetics) or AT8 conjugated with Alexa 568.

    Techniques: Transduction, Immunostaining, Marker, Reverse Transcription Polymerase Chain Reaction

    NANOG plays a critical role in liver oncogenesis ( A ) Results of gene expression microarray comparing liver cancers arising from feeding of ethanol or Western diet (WD)-fed in HCV NS5A transgenic mice and WD+ HCV Core transgenic (Tg) mice. Venn diagram shows genes associated with each etiology and those shared among two or more liver cancer models. ( B ) Summary of proteomic analysis of three mouse liver cancer models listed in A. All models showed similar metabolomic properties as shown in the Venn diagram. ( C ) Heat map showing more extensive proteomic signatures in liver cancer models: alcohol+NS5A; alcohol alone, high cholesterol high- fat Western diet (WD); WD+HCV core gene, and alcohol + HCV core gene. Animals used were either wt, transgenic for either NS5A or core, as indicated. ( D ) Western diet (WD) combined with alcohol increased tumor incidence in NS5A Tg mice compared to control. Upper panel-tumor incidence percentage. Lower panel-immunoblot of Nanog expression. Sh-Nanog Tg indicates animals receiving inducible transgene for NANOG silencing. ( E ) Liver tumor formation in NANOG and NS5A Tg mice. Upper panel, liver tumors arising from NS5A and Nanog showing contributions of alcohol+Western diet. Knockdown of NANOG (ΔLi), as indicated, reduced tumor incidence in wt control and NS5A mice. Lower panel-liver histology showing pathology is increased following Nanog knockdown in NS5A Tg mice. ( F ) NANOG ChIP analysis: comparison of promoter fragments from CD133 − and CD133 + cell populations. ( G ) Summary of gene ontology families identified by NANOG ChIP-seq analysis.

    Journal: Cell metabolism

    Article Title: NANOG metabolically reprograms tumor-initiating stem-like cells through tumorigenic changes in oxidative phosphorylation and fatty acid metabolism

    doi: 10.1016/j.cmet.2015.12.004

    Figure Lengend Snippet: NANOG plays a critical role in liver oncogenesis ( A ) Results of gene expression microarray comparing liver cancers arising from feeding of ethanol or Western diet (WD)-fed in HCV NS5A transgenic mice and WD+ HCV Core transgenic (Tg) mice. Venn diagram shows genes associated with each etiology and those shared among two or more liver cancer models. ( B ) Summary of proteomic analysis of three mouse liver cancer models listed in A. All models showed similar metabolomic properties as shown in the Venn diagram. ( C ) Heat map showing more extensive proteomic signatures in liver cancer models: alcohol+NS5A; alcohol alone, high cholesterol high- fat Western diet (WD); WD+HCV core gene, and alcohol + HCV core gene. Animals used were either wt, transgenic for either NS5A or core, as indicated. ( D ) Western diet (WD) combined with alcohol increased tumor incidence in NS5A Tg mice compared to control. Upper panel-tumor incidence percentage. Lower panel-immunoblot of Nanog expression. Sh-Nanog Tg indicates animals receiving inducible transgene for NANOG silencing. ( E ) Liver tumor formation in NANOG and NS5A Tg mice. Upper panel, liver tumors arising from NS5A and Nanog showing contributions of alcohol+Western diet. Knockdown of NANOG (ΔLi), as indicated, reduced tumor incidence in wt control and NS5A mice. Lower panel-liver histology showing pathology is increased following Nanog knockdown in NS5A Tg mice. ( F ) NANOG ChIP analysis: comparison of promoter fragments from CD133 − and CD133 + cell populations. ( G ) Summary of gene ontology families identified by NANOG ChIP-seq analysis.

    Article Snippet: ChIP was performed with NANOG antibody using CD 133(+) as well as CD133(−) cell lines following a standard protocol as suggested by the manufacturer (Millipore).

    Techniques: Expressing, Microarray, Western Blot, Transgenic Assay, Mouse Assay, Chromatin Immunoprecipitation

    NANOG orchestrated TIC oncogenic and therapeutic resistance mechanisms via mitochondrial metabolic reprogramming ( A ) Mitochondrial ROS production increased in sh- Nanog TICs, but total mitochondrial levels were unchanged in TICs compared to sh- Nanog TICs. ( B ) ROS inducer Paraquat (Para), but not ROS scavenger (NAC), inhibited spheroid formation, but minimal cell death induction was observed ( C ). ( D ) Restoration of OXPHOS genes in TICs promoted self-renewal ability. ( E ) Silencing OXPHOS genes and FAO genes inhibited spheroid formation. ( F ) Mitochondrial cytochrome c release was increased by the combination of sorafenib and ETO treatment or overexpression of Cox6a2 in TICs. Cytochrome c release from mitochondria was analyzed by immunoblotting of the cytosol (soluble fraction) and mitochondria-rich (heavy membrane: HM) fractions of the cell lysates. TICs and CD133(−) cells transduced with sh- Nanog were lysed and fractionated into purified heavy membrane (HM) and cytosolic (S) fractions. The fractions were then probed for cytochrome c (Cyt c), VDAC1 and Cu/Zn SOD. ( G ) Overexpression of Cox6a2 and ETO treatment abrogated drug-resistance and reduced tumor growth. ( H ) A summary diagram depicting the proposed roles of TLR4/NANOG for metabolic reprogramming and genesis of TICs in liver oncogenesis due to alcohol and HCV. NANOG-induced chemotherapy-resistance occurred via mitochondrial metabolic reprogramming (suppression of mitochondrial OXPHOS and promotion of FAO).

    Journal: Cell metabolism

    Article Title: NANOG metabolically reprograms tumor-initiating stem-like cells through tumorigenic changes in oxidative phosphorylation and fatty acid metabolism

    doi: 10.1016/j.cmet.2015.12.004

    Figure Lengend Snippet: NANOG orchestrated TIC oncogenic and therapeutic resistance mechanisms via mitochondrial metabolic reprogramming ( A ) Mitochondrial ROS production increased in sh- Nanog TICs, but total mitochondrial levels were unchanged in TICs compared to sh- Nanog TICs. ( B ) ROS inducer Paraquat (Para), but not ROS scavenger (NAC), inhibited spheroid formation, but minimal cell death induction was observed ( C ). ( D ) Restoration of OXPHOS genes in TICs promoted self-renewal ability. ( E ) Silencing OXPHOS genes and FAO genes inhibited spheroid formation. ( F ) Mitochondrial cytochrome c release was increased by the combination of sorafenib and ETO treatment or overexpression of Cox6a2 in TICs. Cytochrome c release from mitochondria was analyzed by immunoblotting of the cytosol (soluble fraction) and mitochondria-rich (heavy membrane: HM) fractions of the cell lysates. TICs and CD133(−) cells transduced with sh- Nanog were lysed and fractionated into purified heavy membrane (HM) and cytosolic (S) fractions. The fractions were then probed for cytochrome c (Cyt c), VDAC1 and Cu/Zn SOD. ( G ) Overexpression of Cox6a2 and ETO treatment abrogated drug-resistance and reduced tumor growth. ( H ) A summary diagram depicting the proposed roles of TLR4/NANOG for metabolic reprogramming and genesis of TICs in liver oncogenesis due to alcohol and HCV. NANOG-induced chemotherapy-resistance occurred via mitochondrial metabolic reprogramming (suppression of mitochondrial OXPHOS and promotion of FAO).

    Article Snippet: ChIP was performed with NANOG antibody using CD 133(+) as well as CD133(−) cell lines following a standard protocol as suggested by the manufacturer (Millipore).

    Techniques: Over Expression, Transduction, Purification

    Downregulation of ST6GAL1 in hPSCs has an impact on signaling networks involved in pluripotency regulation and embryogenesis. ( a ) Left Panel: Western blotting analysis showed that two independent shRNA sequences (shRNA2 and shRNA5) that target ST6GAL1 transcripts led to effective downregulation of ST6GAL1 protein 72 hours after transduction. While the protein level of NANOG was relatively unaffected, the protein level of POU5F1 was decreased in hPSCs that received shRNA2 and shRNA5. Right Panel: SNA-mediated blotting showed that protein samples extracted from hPSCs which received shRNA2 had lower reactivity to SNA, indicating a decreased amount of α-2,6 sialylated glycoconjugates in the cells. pLKO1: the empty factor control for the transduction of shRNA expression vectors. ( b ) Global gene expression profiling followed by differential gene expression analysis revealed a group of genes (~400 genes) that were differentially expressed ( P

    Journal: Scientific Reports

    Article Title: Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells

    doi: 10.1038/srep13317

    Figure Lengend Snippet: Downregulation of ST6GAL1 in hPSCs has an impact on signaling networks involved in pluripotency regulation and embryogenesis. ( a ) Left Panel: Western blotting analysis showed that two independent shRNA sequences (shRNA2 and shRNA5) that target ST6GAL1 transcripts led to effective downregulation of ST6GAL1 protein 72 hours after transduction. While the protein level of NANOG was relatively unaffected, the protein level of POU5F1 was decreased in hPSCs that received shRNA2 and shRNA5. Right Panel: SNA-mediated blotting showed that protein samples extracted from hPSCs which received shRNA2 had lower reactivity to SNA, indicating a decreased amount of α-2,6 sialylated glycoconjugates in the cells. pLKO1: the empty factor control for the transduction of shRNA expression vectors. ( b ) Global gene expression profiling followed by differential gene expression analysis revealed a group of genes (~400 genes) that were differentially expressed ( P

    Article Snippet: For measuring UEA-I binding and the expression of NANOG and POU5F1, aliquots of harvested cell samples were fixed using PBS containing 4% paraformaldehyde, perforated using PBS containing 0.1% Triton X-100, labeled with biotinylated UEA-I lectin (6.5 μg/ml; Vector Laboratories, Burlingame, CA) and specific antibodies targeting NANOG and POU5F1 (Millipore, Billerica, MA), and stained with fluorescein-conjugated streptavidin and secondary antibodies.

    Techniques: Western Blot, shRNA, Transduction, Expressing

    ST6GAL1 knockdown impedes cellular reprogramming and establishment of induced pluripotency in human somatic cells. ( a ) Schematic illustration of the experimental strategy to examine the influence of ST6GAL1 knockdown on cellular reprogramming. Twenty-four hours after HDFs received POU5F1, SOX2, KLF4, and MYC with or without ST6GAL1 shRNA, the transduced HDFs were seeded on X-ray irradiated feeder cells (mouse embryonic fibroblasts, MEFs). Fourteen days later, alkaline phosphatase (AP) staining was used to examine hiPSC colonies formed by transduced HDFs on the feeder cells. ( b ) AP staining showed that dramatically fewer hiPSC colonies with AP activity were obtained from cellular reprogramming under ST6GAL1 knockdown mediated by shRNA2 and shRNA5. ( c ) Quantitative analysis of NANOG expressing cells in the reprogrammed cell population showed that shRNA2 and shRNA5 both led to a significant reduction in NANOG expressing cells in the analyzed cell populations. Left Panel: The histogram representation of flow cytometry analysis. Right panel: the quantitative result of flow cytometry analysis ( n = 3; * P

    Journal: Scientific Reports

    Article Title: Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells

    doi: 10.1038/srep13317

    Figure Lengend Snippet: ST6GAL1 knockdown impedes cellular reprogramming and establishment of induced pluripotency in human somatic cells. ( a ) Schematic illustration of the experimental strategy to examine the influence of ST6GAL1 knockdown on cellular reprogramming. Twenty-four hours after HDFs received POU5F1, SOX2, KLF4, and MYC with or without ST6GAL1 shRNA, the transduced HDFs were seeded on X-ray irradiated feeder cells (mouse embryonic fibroblasts, MEFs). Fourteen days later, alkaline phosphatase (AP) staining was used to examine hiPSC colonies formed by transduced HDFs on the feeder cells. ( b ) AP staining showed that dramatically fewer hiPSC colonies with AP activity were obtained from cellular reprogramming under ST6GAL1 knockdown mediated by shRNA2 and shRNA5. ( c ) Quantitative analysis of NANOG expressing cells in the reprogrammed cell population showed that shRNA2 and shRNA5 both led to a significant reduction in NANOG expressing cells in the analyzed cell populations. Left Panel: The histogram representation of flow cytometry analysis. Right panel: the quantitative result of flow cytometry analysis ( n = 3; * P

    Article Snippet: For measuring UEA-I binding and the expression of NANOG and POU5F1, aliquots of harvested cell samples were fixed using PBS containing 4% paraformaldehyde, perforated using PBS containing 0.1% Triton X-100, labeled with biotinylated UEA-I lectin (6.5 μg/ml; Vector Laboratories, Burlingame, CA) and specific antibodies targeting NANOG and POU5F1 (Millipore, Billerica, MA), and stained with fluorescein-conjugated streptavidin and secondary antibodies.

    Techniques: shRNA, Irradiation, Staining, Activity Assay, Expressing, Flow Cytometry, Cytometry

    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

    SKM FK 2i-06 cell line expresses pluripotency markers SSEA1, OCT4, and NANOG in feeder-free conditions. SKM FK 2i-06 was transitioned to serum without small molecules at two passages postisolation. SKM FK 2i-06 is shown eight passages postisolation and

    Journal: Molecular Therapy

    Article Title: cAMP and EPAC Signaling Functionally Replace OCT4 During Induced Pluripotent Stem Cell Reprogramming

    doi: 10.1038/mt.2015.28

    Figure Lengend Snippet: SKM FK 2i-06 cell line expresses pluripotency markers SSEA1, OCT4, and NANOG in feeder-free conditions. SKM FK 2i-06 was transitioned to serum without small molecules at two passages postisolation. SKM FK 2i-06 is shown eight passages postisolation and

    Article Snippet: The fixed reprogrammed cells were stained for OCT4 and NANOG expression: an anti-OCT4 antibody (Santa Cruz Biotechnology, sc-5279, 1 : 100) and an anti-NANOG antibody (Abcam, ab70482, 1:250) were incubated for 1 hour at room temperature.

    Techniques:

    Cell lines created with the EPAC agonist are pluripotent and form three germ layers in vitro . ( a ) CSKM-06 cells express pluripotency markers OCT4, NANOG, and SSEA1. Cells were imaged two passages after isolation from feeder cultures. ( b ) CSKM-06 cells

    Journal: Molecular Therapy

    Article Title: cAMP and EPAC Signaling Functionally Replace OCT4 During Induced Pluripotent Stem Cell Reprogramming

    doi: 10.1038/mt.2015.28

    Figure Lengend Snippet: Cell lines created with the EPAC agonist are pluripotent and form three germ layers in vitro . ( a ) CSKM-06 cells express pluripotency markers OCT4, NANOG, and SSEA1. Cells were imaged two passages after isolation from feeder cultures. ( b ) CSKM-06 cells

    Article Snippet: The fixed reprogrammed cells were stained for OCT4 and NANOG expression: an anti-OCT4 antibody (Santa Cruz Biotechnology, sc-5279, 1 : 100) and an anti-NANOG antibody (Abcam, ab70482, 1:250) were incubated for 1 hour at room temperature.

    Techniques: In Vitro, Isolation

    Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.

    Article Snippet: However, different Abs detect different patterns of reactive protein bands; 4 ) With respect to sensitivity, the CS mAb and CS pAb are the most sensitive followed by the Kamiya Ab whereas the eBioscience mAb is the least sensitive; 5 ) The BioLegend anti-Nanog Ab does not detect the 42 kD as the major protein band in the NTERA-2 NE; and 6 ) Different antibodies may preferentially recognize different protein bands.

    Techniques: Western Blot, Clone Assay

    The time window for induced Epi lineage conversion can be narrowed to a 4 hours time period prior E3.75. ( A ) Schematic of the time schedule of inhibitor treatment. Orange and grey lines indicate the culture periods in the presence of inhibitors or DMSO (vehicle), respectively. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. Error bars indicate SEM. n , number of embryos analyzed.

    Journal: Scientific Reports

    Article Title: ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

    doi: 10.1038/s41598-017-12120-0

    Figure Lengend Snippet: The time window for induced Epi lineage conversion can be narrowed to a 4 hours time period prior E3.75. ( A ) Schematic of the time schedule of inhibitor treatment. Orange and grey lines indicate the culture periods in the presence of inhibitors or DMSO (vehicle), respectively. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. Error bars indicate SEM. n , number of embryos analyzed.

    Article Snippet: In this study, anti-GATA6 (1/100, AF1700, R & D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R & D systems) were used.

    Techniques: Immunodetection, Cell Culture, Expressing

    Kinetics of lineage marker expression during blastocyst formation. ( A ) Schematic representation of the culture periods in KSOM + DMSO (vehicle) before analysis. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. ( D ) Immunodetection of NANOG (green) and SOX17 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( E ) Number of SOX17+, NANOG− (S17+, N−; blue), SOX17+, NANOG + (S17+, N+; green) and SOX17−, NANOG− (S17−, N−; yellow) ICM cells in embryos cultured for the indicated period of times. Statistical Mann–Whitney tests are indicated when significant (*p

    Journal: Scientific Reports

    Article Title: ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

    doi: 10.1038/s41598-017-12120-0

    Figure Lengend Snippet: Kinetics of lineage marker expression during blastocyst formation. ( A ) Schematic representation of the culture periods in KSOM + DMSO (vehicle) before analysis. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. ( D ) Immunodetection of NANOG (green) and SOX17 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( E ) Number of SOX17+, NANOG− (S17+, N−; blue), SOX17+, NANOG + (S17+, N+; green) and SOX17−, NANOG− (S17−, N−; yellow) ICM cells in embryos cultured for the indicated period of times. Statistical Mann–Whitney tests are indicated when significant (*p

    Article Snippet: In this study, anti-GATA6 (1/100, AF1700, R & D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R & D systems) were used.

    Techniques: Marker, Expressing, Immunodetection, Cell Culture, MANN-WHITNEY

    The conversion of ICM into Epi cell lineage requires a 8 hours time period of FGF/ERK inhibition starting from E3.25. ( A ) Schematic of the time schedule of inhibitor treatment. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. Error bars indicate SEM. n , number of embryos analyzed.

    Journal: Scientific Reports

    Article Title: ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

    doi: 10.1038/s41598-017-12120-0

    Figure Lengend Snippet: The conversion of ICM into Epi cell lineage requires a 8 hours time period of FGF/ERK inhibition starting from E3.25. ( A ) Schematic of the time schedule of inhibitor treatment. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured for the indicated periods of time. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in embryos cultured for the indicated period of times. Error bars indicate SEM. n , number of embryos analyzed.

    Article Snippet: In this study, anti-GATA6 (1/100, AF1700, R & D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R & D systems) were used.

    Techniques: Inhibition, Immunodetection, Cell Culture, Expressing

    Effect of modulating transcription and proteasome activity during ICM to Epi conversion. ( A ) Schematic of the time schedule of inhibitor treatment. Orange box indicates the 4 hours treatment with FGF/ERK inhibitors prior E3.75. Green, purple and grey lines indicate the culture periods in the presence of flavopiridol, MG132 and DMSO (vehicle), respectively. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured in presence/absence drug treatment. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. Red arrowheads: pyknotic nuclei; light green arrows: metaphase. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in cultured embryos. Error bars indicate SEM. n , number of embryos analyzed. ( D ) Quantification of NANOG levels in Epi cells (NANOG-positive). ( E ) Quantification of GATA6 levels in PrE cells (GATA6-positive). Error bars indicate SEM. n , number of cells analyzed. Statistical Mann–Whitney tests are indicated when significant (*p

    Journal: Scientific Reports

    Article Title: ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

    doi: 10.1038/s41598-017-12120-0

    Figure Lengend Snippet: Effect of modulating transcription and proteasome activity during ICM to Epi conversion. ( A ) Schematic of the time schedule of inhibitor treatment. Orange box indicates the 4 hours treatment with FGF/ERK inhibitors prior E3.75. Green, purple and grey lines indicate the culture periods in the presence of flavopiridol, MG132 and DMSO (vehicle), respectively. ( B ) Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured in presence/absence drug treatment. Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. Red arrowheads: pyknotic nuclei; light green arrows: metaphase. ( C ) Distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey) in cultured embryos. Error bars indicate SEM. n , number of embryos analyzed. ( D ) Quantification of NANOG levels in Epi cells (NANOG-positive). ( E ) Quantification of GATA6 levels in PrE cells (GATA6-positive). Error bars indicate SEM. n , number of cells analyzed. Statistical Mann–Whitney tests are indicated when significant (*p

    Article Snippet: In this study, anti-GATA6 (1/100, AF1700, R & D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R & D systems) were used.

    Techniques: Activity Assay, Immunodetection, Cell Culture, Expressing, MANN-WHITNEY

    Modulation of FGF/ERK signaling during early mouse development. Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured from E2.75 to E3.25 ( A ), E3.25 to E3.75 ( B ), E3.75 to E4.5 ( C ) and distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey). Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. Statistical Mann–Whitney tests are indicated when significant (*p

    Journal: Scientific Reports

    Article Title: ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

    doi: 10.1038/s41598-017-12120-0

    Figure Lengend Snippet: Modulation of FGF/ERK signaling during early mouse development. Immunodetection of NANOG (green) and GATA6 (red) in embryos cultured from E2.75 to E3.25 ( A ), E3.25 to E3.75 ( B ), E3.75 to E4.5 ( C ) and distribution of ICM cells expressing NANOG (N+, red), GATA6 (G6+, blue) or both markers (Coexp., grey). Pictures correspond to a projection of 5 confocal optical slices. Scale bar: 20 µm. Statistical Mann–Whitney tests are indicated when significant (*p

    Article Snippet: In this study, anti-GATA6 (1/100, AF1700, R & D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R & D systems) were used.

    Techniques: Immunodetection, Cell Culture, Expressing, MANN-WHITNEY

    Model of temporal dynamics of ICM cell specification. Specification into Epi (red) or PrE (blue) is a progressive and asynchronous process that occurs for a majority of ICM cells (grey) between E3.25 and E3.75. The formation of Epi progenitors precedes that of PrE progenitors. ICM cell responsiveness to the modulation of FGF/ERK signaling varies over time. First, between E2.5 and E3.25, exogenous FGF4 treatment efficiently diverts unspecified ICM cell from Epi fate. Then, between E3.25 and E3.5, ICM cells are globally insensitive to modulation of FGF/ERK signaling. Between E3.5 and E3.75, remaining unspecified ICM cells but not already specified Epi progenitors are able to respond to exogenous FGF4 leading to a moderate shift in Epi/PrE specification upon treatment. FGF signaling and ERK phosphorylation increases in ICM cells and PrE progenitors during this time window. Accordingly, embryos become highly sensitive to FGF/ERK inhibition leading to the complete ICM conversion to Epi. During that period, proteasome degradation and transcription control NANOG and GATA6 levels in Epi and PrE progenitors. Downregulation of GATA6 levels in PrE progenitors upon FGF/ERK inhibition is partially mediated by the proteasome. After E3.75, responsiveness to exogenous FGF4 is lost when all ICM cells become specified while PrE progenitors are still able to respond to FGF/ERK inhibition.

    Journal: Scientific Reports

    Article Title: ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

    doi: 10.1038/s41598-017-12120-0

    Figure Lengend Snippet: Model of temporal dynamics of ICM cell specification. Specification into Epi (red) or PrE (blue) is a progressive and asynchronous process that occurs for a majority of ICM cells (grey) between E3.25 and E3.75. The formation of Epi progenitors precedes that of PrE progenitors. ICM cell responsiveness to the modulation of FGF/ERK signaling varies over time. First, between E2.5 and E3.25, exogenous FGF4 treatment efficiently diverts unspecified ICM cell from Epi fate. Then, between E3.25 and E3.5, ICM cells are globally insensitive to modulation of FGF/ERK signaling. Between E3.5 and E3.75, remaining unspecified ICM cells but not already specified Epi progenitors are able to respond to exogenous FGF4 leading to a moderate shift in Epi/PrE specification upon treatment. FGF signaling and ERK phosphorylation increases in ICM cells and PrE progenitors during this time window. Accordingly, embryos become highly sensitive to FGF/ERK inhibition leading to the complete ICM conversion to Epi. During that period, proteasome degradation and transcription control NANOG and GATA6 levels in Epi and PrE progenitors. Downregulation of GATA6 levels in PrE progenitors upon FGF/ERK inhibition is partially mediated by the proteasome. After E3.75, responsiveness to exogenous FGF4 is lost when all ICM cells become specified while PrE progenitors are still able to respond to FGF/ERK inhibition.

    Article Snippet: In this study, anti-GATA6 (1/100, AF1700, R & D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R & D systems) were used.

    Techniques: Inhibition

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

    Journal: Neurochemistry International

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

    doi: 10.1016/j.neuint.2018.10.014

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

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

    Techniques: Immunocytochemistry, Staining, Expressing

    Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.

    Journal: PLoS ONE

    Article Title: Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    doi: 10.1371/journal.pone.0090615

    Figure Lengend Snippet: Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs. WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see Methods ). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.

    Article Snippet: Also, different rNanog proteins displayed differential reactivity to different anti-Nanog Abs.

    Techniques: Western Blot, Clone Assay

    The expression of reprogramming transcription factors in 2D- and 3D-grown A549 cells. ( A ) qRT-PCR for the expression of reprogramming transcription factors OCT4, SOX2, NANOG, c-MYC and LIN28 in 2D- and 3D-grown A549 cells. ( B ) Western blotting for expression of OCT4, SOX2 and NANOG in 2D- and 3D-grown A549 cells. ( C ) Gray analysis the result of Figure 3 B. ( D ) qRT-PCR for the expression of reprogramming factor miR-302a expression in 2D- and 3D-grown A549 cells. ( E ) Western blotting for the expression of β-catenin in 2D- and 3D-grown A549 cells. Data are presented as mean ± SE. Experiments were independently repeated at least three times.

    Journal: Journal of Radiation Research

    Article Title: Reprogramming mediated radio-resistance of 3D-grown cancer cells

    doi: 10.1093/jrr/rrv018

    Figure Lengend Snippet: The expression of reprogramming transcription factors in 2D- and 3D-grown A549 cells. ( A ) qRT-PCR for the expression of reprogramming transcription factors OCT4, SOX2, NANOG, c-MYC and LIN28 in 2D- and 3D-grown A549 cells. ( B ) Western blotting for expression of OCT4, SOX2 and NANOG in 2D- and 3D-grown A549 cells. ( C ) Gray analysis the result of Figure 3 B. ( D ) qRT-PCR for the expression of reprogramming factor miR-302a expression in 2D- and 3D-grown A549 cells. ( E ) Western blotting for the expression of β-catenin in 2D- and 3D-grown A549 cells. Data are presented as mean ± SE. Experiments were independently repeated at least three times.

    Article Snippet: The primary antibodies included: OCT4 (1:1000, Abcam, USA), SOX2 (1:1000, CST, USA), NANOG (1:1000, ABGENT, USA), β-catenin (1:1000, CST, USA) and GAPDH (1:1000, ZSGB-BIO, Beijing, China).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot