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  • 85
    Thermo Fisher nanog hs002387400 g1
    Nanog Hs002387400 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 16 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 301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ABclonal nanog
    Histone crotonylation is enriched in and required for self-renewal of mESCs. (A) WB analysis showing a significantly higher level of histone crotonylation in mESC than in differentiated embryoid bodies (EB). CGR8 mESC were induced to differentiate by suspension culture in dish for 9 days. (B) WB analysis showing induced expression of WT HDAC1 and HDAC1-VRPP in CGR8 cells. HA antibody detected only Dox-induced HA-tagged HDAC1 or HDAC1-VRPP, whereas HDAC1 antibody detected both induced and endogenous HDAC1 proteins. (C) Contrast-phase images of control, WT HDAC1 and HDAC1-VRPP CGR8 colonies cultured for 9 days with or without Dox. (D) WB analysis showing the effect of induced expression of WT HDAC1 and HDAC1-VRPP on the levels of mESC core transcription factors Sox2, Oct4 and <t>Nanog</t> and histone crotonylation and histone acetylation. Note that reduced levels of Sox2, Oct4, Nanog, histone crotonylation and histone acetylation were observed upon 3 days of Dox treatment. (E) Confirmation of induced differentiation upon Dox-induced expression of WT HDAC1 or HDAC1-VRPP by qRT-PCR analysis of indicated differentiation marker genes. (F) Working model illustrating a non-redundant function of histone crotonylation to histone acetylation in transcription. CBP/p300 and MOF catalyze both histone acetylation and crotonylation, which in turn recruit corresponding reader proteins such as <t>BRD4</t> or DPF2 and AF9, and facilitate transcriptional activation (left panel). Selective decrotonylation by HDCR1-VRPP is sufficient to repress transcription, indicating that histone crotonylation is required for transcriptional activation (right panel).
    Nanog, supplied by ABclonal, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc nanog
    Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) <t>NANOG</t> and <t>OCT-4</t> were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.
    Nanog, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore nanog
    Premature activation of <t>RASSF1A</t> impairs embryogenesis via p73. a Indicated gene expression levels in published GEO data sets GDS3599 and GDS2156. b Temporal expression of Oct4 and Rassf1A mRNA in the pre-implantation embryo (% of maximum expression) from published GEO data sets GDS752 (black colour) and GDS814 (red colour). c Nuclear localisation of YAP during early stages of pre-implantation development. d <t>Nanog</t> immunofluorescence and e representative images of embryos microinjected with either control (zsCtrl) or RASSF1A-expressing (zsR1A) vectors stained for stem cell marker expression. Bar graph showing total OCT4 protein levels across all embryos in zsR1A versus zsCtrl. f 'Kill curve' to determine lethal RASSF1A concentration in pre-implantation embryos. The graph expresses percentage (%) of blastocyst-forming embryos at the indicated RASSF1A concentration. g Viability of embryos in response to RASSF1A expression and/or sip73 microinjection, n = 15. BF bright field channel. Scale bars: 10–50 μm. *P
    Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 720 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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    93
    Millipore human 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.
    Human Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Aviva Systems 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 Aviva Systems, 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
    R&D Systems nanog
    Characterisation of <t>hESCs</t> maintained in CMp11 and media conditioned by passage 18 HFFs (CMp18). Representative immunofluorescent images of hESCs maintained for 5 days in CMp11 (n = 5) and CMp18 media (n = 5) (bar = 100 µm, TRA-1.81 bar = 200 µm). Cells cultured in CMp11 presented well defined colonies with morphology characteristic of undifferentiated cells. In contrast, CMp18 cultured cell were morphologically distinct with colonies lacking smooth and defined edges and a clearing effect from the centre. Consistent with an undifferentiated phenotype, CMp11 cultured cells were negative for the early differentiation marker SSEA-1 (green) and positive for the undifferentiation markers SSEA-4, TRA-1.60, TRA-1.81, OCT4 and <t>NANOG.</t> Cells cultured in CMp18 were positive for SSEA-1 and negative for undifferentiation markers. All cells were co-labelled with the nuclear stain DAPI (blue).
    Nanog, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 457 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher nanog
    Modulating <t>NANOG</t> expression did not affect in vitro cell proliferation and colony formation ( A ) Cell viability was measured over 3 days in Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either <t>Renilla</t> luciferase or NANOG . ( B ) Moody cells transfected with Firefly luciferase or NANOG expression construct or in SKOV-3 cells transfected with shRNA targeting either Renilla luciferase or NANOG were grown for 2 weeks before being counted by a colony counter. Only colonies greater than 50 μm in diameter were counted as positive. Error bars, S.D.
    Nanog, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    Histone crotonylation is enriched in and required for self-renewal of mESCs. (A) WB analysis showing a significantly higher level of histone crotonylation in mESC than in differentiated embryoid bodies (EB). CGR8 mESC were induced to differentiate by suspension culture in dish for 9 days. (B) WB analysis showing induced expression of WT HDAC1 and HDAC1-VRPP in CGR8 cells. HA antibody detected only Dox-induced HA-tagged HDAC1 or HDAC1-VRPP, whereas HDAC1 antibody detected both induced and endogenous HDAC1 proteins. (C) Contrast-phase images of control, WT HDAC1 and HDAC1-VRPP CGR8 colonies cultured for 9 days with or without Dox. (D) WB analysis showing the effect of induced expression of WT HDAC1 and HDAC1-VRPP on the levels of mESC core transcription factors Sox2, Oct4 and Nanog and histone crotonylation and histone acetylation. Note that reduced levels of Sox2, Oct4, Nanog, histone crotonylation and histone acetylation were observed upon 3 days of Dox treatment. (E) Confirmation of induced differentiation upon Dox-induced expression of WT HDAC1 or HDAC1-VRPP by qRT-PCR analysis of indicated differentiation marker genes. (F) Working model illustrating a non-redundant function of histone crotonylation to histone acetylation in transcription. CBP/p300 and MOF catalyze both histone acetylation and crotonylation, which in turn recruit corresponding reader proteins such as BRD4 or DPF2 and AF9, and facilitate transcriptional activation (left panel). Selective decrotonylation by HDCR1-VRPP is sufficient to repress transcription, indicating that histone crotonylation is required for transcriptional activation (right panel).

    Journal: Cell Research

    Article Title: Class I histone deacetylases are major histone decrotonylases: evidence for critical and broad function of histone crotonylation in transcription

    doi: 10.1038/cr.2017.68

    Figure Lengend Snippet: Histone crotonylation is enriched in and required for self-renewal of mESCs. (A) WB analysis showing a significantly higher level of histone crotonylation in mESC than in differentiated embryoid bodies (EB). CGR8 mESC were induced to differentiate by suspension culture in dish for 9 days. (B) WB analysis showing induced expression of WT HDAC1 and HDAC1-VRPP in CGR8 cells. HA antibody detected only Dox-induced HA-tagged HDAC1 or HDAC1-VRPP, whereas HDAC1 antibody detected both induced and endogenous HDAC1 proteins. (C) Contrast-phase images of control, WT HDAC1 and HDAC1-VRPP CGR8 colonies cultured for 9 days with or without Dox. (D) WB analysis showing the effect of induced expression of WT HDAC1 and HDAC1-VRPP on the levels of mESC core transcription factors Sox2, Oct4 and Nanog and histone crotonylation and histone acetylation. Note that reduced levels of Sox2, Oct4, Nanog, histone crotonylation and histone acetylation were observed upon 3 days of Dox treatment. (E) Confirmation of induced differentiation upon Dox-induced expression of WT HDAC1 or HDAC1-VRPP by qRT-PCR analysis of indicated differentiation marker genes. (F) Working model illustrating a non-redundant function of histone crotonylation to histone acetylation in transcription. CBP/p300 and MOF catalyze both histone acetylation and crotonylation, which in turn recruit corresponding reader proteins such as BRD4 or DPF2 and AF9, and facilitate transcriptional activation (left panel). Selective decrotonylation by HDCR1-VRPP is sufficient to repress transcription, indicating that histone crotonylation is required for transcriptional activation (right panel).

    Article Snippet: The following antibodies were used in this study: pan-Kac (PTM-Biolabs 101), pan-Kcr (PTM-Biolabs 501), H3K4cr (PTM-Biolabs PTM-527), H3K9cr (PTM-Biolabs 516), H3K18cr (PTM-Biolabs 517), H3K23cr (PTM-Biolabs 519), H4K8cr (PTM-Biolabs 522), H4K12cr (PTM-Biolabs 523), H4ac (Millipore 05-1355), H3 (Epitomics M1309-1), Flag (Sigma 7425/1804), HA (Santa Cruz SC-805), Gal4DBD (Santa Cruz SC-510), GAPDH (Abmart ), actin (Huabio M1210-2), HDAC1 (ABclonal A0238), HDAC2 (ABclonal A2084) and HDAC3 (ABclonal A2139), BRD4 (Abcam ab128874), Sox2 (Abcam ab92494), Oct4 (Santa Cruz sc-5279) and Nanog (Abclonal A3232).

    Techniques: Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Marker, Activation Assay

    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

    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

    BMJ targets and downregulates the expression of CSC associated transcription factors in MiaPaCa2 xenografts. Paraffin embedded MiaPaCa2 xenograft tumor sections from control and BMJ treated groups were subjected to immunohistochemical analysis for determining the in vivo effect on the protein levels of PanC-CSC associated transcription factors: SOX2 (A) , OCT4 (B) , NANOG (C) . and PDX1 (D) . The percent positive cells were quantitated by counting the brown stained nuclei for each molecule in control versus the BMJ treated groups. p ≤ 0.05 is denoted by * and p ≤ 0.001 is denoted by ***.

    Journal: Molecular carcinogenesis

    Article Title: Bitter melon juice exerts its efficacy against pancreatic cancer via targeting both bulk and cancer stem cells

    doi: 10.1002/mc.22833

    Figure Lengend Snippet: BMJ targets and downregulates the expression of CSC associated transcription factors in MiaPaCa2 xenografts. Paraffin embedded MiaPaCa2 xenograft tumor sections from control and BMJ treated groups were subjected to immunohistochemical analysis for determining the in vivo effect on the protein levels of PanC-CSC associated transcription factors: SOX2 (A) , OCT4 (B) , NANOG (C) . and PDX1 (D) . The percent positive cells were quantitated by counting the brown stained nuclei for each molecule in control versus the BMJ treated groups. p ≤ 0.05 is denoted by * and p ≤ 0.001 is denoted by ***.

    Article Snippet: SOX2 and NANOG antibodies were from Cell signaling (Beverly, CA), and OCT4 and CD44 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Expressing, Immunohistochemistry, In Vivo, Staining

    BMJ decreases the protein expression of CSC associated transcription factors and surface markers in PanC cell monolayers. Immunofluorescence staining of PanC cell monolayers treated with 2% BMJ for 72 hours shows a reduction in SOX2, OCT4, NANOG and CD44 protein expression levels in MiaPaCa2 (A) , PANC1 (B) , and AsPC1 (C) cells. The results from densitometry by Image J software are also provided and correlate to the data from the images. p ≤ 0.05 is denoted by *, p ≤ 0.01 is denoted by ** and p ≤ 0.001 is denoted by ***.

    Journal: Molecular carcinogenesis

    Article Title: Bitter melon juice exerts its efficacy against pancreatic cancer via targeting both bulk and cancer stem cells

    doi: 10.1002/mc.22833

    Figure Lengend Snippet: BMJ decreases the protein expression of CSC associated transcription factors and surface markers in PanC cell monolayers. Immunofluorescence staining of PanC cell monolayers treated with 2% BMJ for 72 hours shows a reduction in SOX2, OCT4, NANOG and CD44 protein expression levels in MiaPaCa2 (A) , PANC1 (B) , and AsPC1 (C) cells. The results from densitometry by Image J software are also provided and correlate to the data from the images. p ≤ 0.05 is denoted by *, p ≤ 0.01 is denoted by ** and p ≤ 0.001 is denoted by ***.

    Article Snippet: SOX2 and NANOG antibodies were from Cell signaling (Beverly, CA), and OCT4 and CD44 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Expressing, Immunofluorescence, Staining, Software

    Overexpression of galetin-3 in lung CSCs (A) The mRNA levels of galectins (Gal) in first and secondary passages of H1299 spheres were detected by RT-qPCR and normalized against mRNA levels of monolayers. (B) The mRNA levels of galectin-3 in NSCLC spheres compared to that in monolayers were detected by RT-qPCR. (C) Galectin-3 (Gal-3) was knocked down in H1299 cell line by lentiviral shGal-3 infection. Luciferase shRNA lentivirus (shLuc) was used as a control. The expression levels of galectin-3 were detected using RT-qPCR and Western blotting. (D) The mRNA levels of stemness-associated genes in monolayers (shLuc monolayer), spheres (shLuc sphere), or shGal-3-infected spheres (shGal-3 sphere) were detected by RT-qPCR. (E) shLacZ- or shGal-3-infected H1299 monolayers or spheres were cotransfected with pGL4-Oct4-luc, pGL4-Sox2-luc or pGL4-Nanog-luc, and pRL-SV40. After 48 h, luciferase reporter assays was conducted to investigate Oct4, Sox2, or Nanog promoter activity regulated by galectin-3. (F) The protein levels of Oct4, Sox2, and Nanog in shLuc- or shGal-3-infected H1299 spheres were detected by using flow cytometry. (G) H1299 cells from shLuc- or shGal-3-infected H1299 spheres were analyzed for CD44/CD133 double staining by flow cytometry. Control: isotype control. Data represents the mean ± SD of at least 3 independent experiments, * p

    Journal: Oncotarget

    Article Title: Galectin-3 augments tumor initiating property and tumorigenicity of lung cancer through interaction with β-catenin

    doi:

    Figure Lengend Snippet: Overexpression of galetin-3 in lung CSCs (A) The mRNA levels of galectins (Gal) in first and secondary passages of H1299 spheres were detected by RT-qPCR and normalized against mRNA levels of monolayers. (B) The mRNA levels of galectin-3 in NSCLC spheres compared to that in monolayers were detected by RT-qPCR. (C) Galectin-3 (Gal-3) was knocked down in H1299 cell line by lentiviral shGal-3 infection. Luciferase shRNA lentivirus (shLuc) was used as a control. The expression levels of galectin-3 were detected using RT-qPCR and Western blotting. (D) The mRNA levels of stemness-associated genes in monolayers (shLuc monolayer), spheres (shLuc sphere), or shGal-3-infected spheres (shGal-3 sphere) were detected by RT-qPCR. (E) shLacZ- or shGal-3-infected H1299 monolayers or spheres were cotransfected with pGL4-Oct4-luc, pGL4-Sox2-luc or pGL4-Nanog-luc, and pRL-SV40. After 48 h, luciferase reporter assays was conducted to investigate Oct4, Sox2, or Nanog promoter activity regulated by galectin-3. (F) The protein levels of Oct4, Sox2, and Nanog in shLuc- or shGal-3-infected H1299 spheres were detected by using flow cytometry. (G) H1299 cells from shLuc- or shGal-3-infected H1299 spheres were analyzed for CD44/CD133 double staining by flow cytometry. Control: isotype control. Data represents the mean ± SD of at least 3 independent experiments, * p

    Article Snippet: Flow cytometry Initially, to examine the protein levels of transcriptional factors Oct4, Sox2, or Nanog in H1299 monolayers or spheres, the cells (5× 10­­­­5 cells/ml) were fixed and permeabilized with BD Cytofix/CytopermTM Fixation/Permeabilization kit (BD Biosciences) for 15 minutes, and incubated with primary anti-Oct4, anti-Sox2, or anti-Nanog antibodies (Cell Signaling) in BD Perm/WashTM buffer for 1 hour at 4°C.

    Techniques: Over Expression, Quantitative RT-PCR, Infection, Luciferase, shRNA, Expressing, Western Blot, Activity Assay, Flow Cytometry, Cytometry, Double Staining

    Overexpression of galectin-3 in A549 cells promoted sphere-forming capacity and in vitro tumorigenicity (A) The morphology of galectin-3 (Gal-3)- or empty vector (ev)-transduced spheres. The expression levels of galectin-3 in Gal-3- or ev-transduced monolayers or spheres were detected by using RT-qPCR and Western blot. (B) The mRNA levels of stemness-related genes in Gal-3- or ev-transduced A549 monolayers or spheres were detected by RT-qPCR. (C) Gal-3- or ev-transduced A549 monolayers or spheres were cotransfected with pGL4-Oct4-luc, pGL4-Sox2-luc or pGL4-Nanog-luc, and pRL-SV40. After 48 h, luciferase reporter assays were conducted to investigate Oct4, Sox2, or Nanog promoter activity regulated by galectin-3. (D) The protein levels of Oct4, Sox2, or Nanog in Gal-3- or ev-transduced A549 monolayers or spheres were analyzed by flow cytometry. (E) Gal-3- or ev-transduced A549 monolayers or spheres (2 × 10 4 cells) were seeded onto upper side of Matrigel-coated transwell inserts and incubated for 24 h to determine the invasive ability. (F) Gal-3- or ev-transduced A549 monolayers or spheres (1 × 10 3 cells per 6-well plate) were seeded in soft agar for evaluating anchorage-independent tumor growth. (G) Gal-3- or ev-transduced A549 were seeded onto 96-well plates in serum-free tumor sphere media and cultured for 30 days. The primary spheres were dissociated to single cells and re-seeded to yield the second generation. Data represents the mean ± SD of at least 3 independent experiments, * p

    Journal: Oncotarget

    Article Title: Galectin-3 augments tumor initiating property and tumorigenicity of lung cancer through interaction with β-catenin

    doi:

    Figure Lengend Snippet: Overexpression of galectin-3 in A549 cells promoted sphere-forming capacity and in vitro tumorigenicity (A) The morphology of galectin-3 (Gal-3)- or empty vector (ev)-transduced spheres. The expression levels of galectin-3 in Gal-3- or ev-transduced monolayers or spheres were detected by using RT-qPCR and Western blot. (B) The mRNA levels of stemness-related genes in Gal-3- or ev-transduced A549 monolayers or spheres were detected by RT-qPCR. (C) Gal-3- or ev-transduced A549 monolayers or spheres were cotransfected with pGL4-Oct4-luc, pGL4-Sox2-luc or pGL4-Nanog-luc, and pRL-SV40. After 48 h, luciferase reporter assays were conducted to investigate Oct4, Sox2, or Nanog promoter activity regulated by galectin-3. (D) The protein levels of Oct4, Sox2, or Nanog in Gal-3- or ev-transduced A549 monolayers or spheres were analyzed by flow cytometry. (E) Gal-3- or ev-transduced A549 monolayers or spheres (2 × 10 4 cells) were seeded onto upper side of Matrigel-coated transwell inserts and incubated for 24 h to determine the invasive ability. (F) Gal-3- or ev-transduced A549 monolayers or spheres (1 × 10 3 cells per 6-well plate) were seeded in soft agar for evaluating anchorage-independent tumor growth. (G) Gal-3- or ev-transduced A549 were seeded onto 96-well plates in serum-free tumor sphere media and cultured for 30 days. The primary spheres were dissociated to single cells and re-seeded to yield the second generation. Data represents the mean ± SD of at least 3 independent experiments, * p

    Article Snippet: Flow cytometry Initially, to examine the protein levels of transcriptional factors Oct4, Sox2, or Nanog in H1299 monolayers or spheres, the cells (5× 10­­­­5 cells/ml) were fixed and permeabilized with BD Cytofix/CytopermTM Fixation/Permeabilization kit (BD Biosciences) for 15 minutes, and incubated with primary anti-Oct4, anti-Sox2, or anti-Nanog antibodies (Cell Signaling) in BD Perm/WashTM buffer for 1 hour at 4°C.

    Techniques: Over Expression, In Vitro, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Flow Cytometry, Cytometry, Incubation, Cell Culture

    Galectin-3 maintained the stemness properties of lung CSCs Initially, H1299 cells were incubated in tumor sphere medium and grown as spheres. H1299 spheres were then dissociated to single cells and infected with shLuc- or shGal-3 lentivirus. (A) The mRNA levels of galectin-3 were detected by RT-qPCR. (B) The mRNA levels of Oct4, Sox2, Nanog or CXCR4 genes were detected by RT-qPCR. (C) H1299 cells (2 × 10 4 cells) were analyzed for their invasive ability using Matrigel-coated Transwell assay. (D) After lentivirus infection, H1299 cells (500 cells per 6-well plate) were seeded in soft agar for evaluating anchorage-independent tumor growth. (E) After lentivirus infection, H1299 cells were evaluated the sphere forming ability. Data represents the mean SD of at least 3 independent experiments, * p

    Journal: Oncotarget

    Article Title: Galectin-3 augments tumor initiating property and tumorigenicity of lung cancer through interaction with β-catenin

    doi:

    Figure Lengend Snippet: Galectin-3 maintained the stemness properties of lung CSCs Initially, H1299 cells were incubated in tumor sphere medium and grown as spheres. H1299 spheres were then dissociated to single cells and infected with shLuc- or shGal-3 lentivirus. (A) The mRNA levels of galectin-3 were detected by RT-qPCR. (B) The mRNA levels of Oct4, Sox2, Nanog or CXCR4 genes were detected by RT-qPCR. (C) H1299 cells (2 × 10 4 cells) were analyzed for their invasive ability using Matrigel-coated Transwell assay. (D) After lentivirus infection, H1299 cells (500 cells per 6-well plate) were seeded in soft agar for evaluating anchorage-independent tumor growth. (E) After lentivirus infection, H1299 cells were evaluated the sphere forming ability. Data represents the mean SD of at least 3 independent experiments, * p

    Article Snippet: Flow cytometry Initially, to examine the protein levels of transcriptional factors Oct4, Sox2, or Nanog in H1299 monolayers or spheres, the cells (5× 10­­­­5 cells/ml) were fixed and permeabilized with BD Cytofix/CytopermTM Fixation/Permeabilization kit (BD Biosciences) for 15 minutes, and incubated with primary anti-Oct4, anti-Sox2, or anti-Nanog antibodies (Cell Signaling) in BD Perm/WashTM buffer for 1 hour at 4°C.

    Techniques: Incubation, Infection, Quantitative RT-PCR, Transwell Assay

    Lung tumor spheres exhibited CSC features (A) The mRNA levels of stemness-related genes in secondary spheres were detected by RT-qPCR and normalized against mRNA levels of parental cells of NSCLC cell lines. (B) The mRNA levels of stemness-related genes in the first and second passages of spheres from H1299 cells were compared with those in monolayer cells. (C) The protein levels of Oct4, Sox2, and Nanog in monolayers (H1299) and sphere (H1299-S) were detected using flow cytometry. (D) Immunofluorescence analysis was performed to detect the expression levels of Oct4, Sox2, Nanog, and CD133 in monolayers (H1299) and spheres (H1299-S). (E) H1299 cells from monolayers (H1299) and spheres (H1299-S) were analyzed for CD44/CD133 double staining by flow cytometry. Control: isotype control. Data represents the mean ± SD of at least 3 independent experiments, * p

    Journal: Oncotarget

    Article Title: Galectin-3 augments tumor initiating property and tumorigenicity of lung cancer through interaction with β-catenin

    doi:

    Figure Lengend Snippet: Lung tumor spheres exhibited CSC features (A) The mRNA levels of stemness-related genes in secondary spheres were detected by RT-qPCR and normalized against mRNA levels of parental cells of NSCLC cell lines. (B) The mRNA levels of stemness-related genes in the first and second passages of spheres from H1299 cells were compared with those in monolayer cells. (C) The protein levels of Oct4, Sox2, and Nanog in monolayers (H1299) and sphere (H1299-S) were detected using flow cytometry. (D) Immunofluorescence analysis was performed to detect the expression levels of Oct4, Sox2, Nanog, and CD133 in monolayers (H1299) and spheres (H1299-S). (E) H1299 cells from monolayers (H1299) and spheres (H1299-S) were analyzed for CD44/CD133 double staining by flow cytometry. Control: isotype control. Data represents the mean ± SD of at least 3 independent experiments, * p

    Article Snippet: Flow cytometry Initially, to examine the protein levels of transcriptional factors Oct4, Sox2, or Nanog in H1299 monolayers or spheres, the cells (5× 10­­­­5 cells/ml) were fixed and permeabilized with BD Cytofix/CytopermTM Fixation/Permeabilization kit (BD Biosciences) for 15 minutes, and incubated with primary anti-Oct4, anti-Sox2, or anti-Nanog antibodies (Cell Signaling) in BD Perm/WashTM buffer for 1 hour at 4°C.

    Techniques: Quantitative RT-PCR, Flow Cytometry, Cytometry, Immunofluorescence, Expressing, Double Staining

    DDX3 suppresses stemness gene signature. ( a ) Lower expression of DDX3 along with overexpression of stemness markers was observed in the poorly differentiated cell line. Cell lysates (50 μg) of HepG2, Hep3B, HuH-7 and SK-Hep-1 cells were analyzed by western blotting with antibodies against DDX3, Nanog, Oct4, c-Myc, Sox2, KLF4, Bmi1, CK19 and β-actin. ( b ) Knockdown of DDX3 led to up-regulation of stemness markers. Cell lysates (50 μg) of stable shLuc, shDDX3 #2 and shDDX3 #3 HepG2 cells were analyzed by western blotting with antibodies described in ( a ). ( c ) DDX3 overexpression suppressed stemness markers. Plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 was transfected into SK-Hep-1 cells. At 48 h post transfection, cell lysates were prepared and subjected to immunoblotting with antibodies described in ( a ) and anti-FLAG antibody. β-actin was used as internal control in ( a – c ). ( d ) DDX3 knockdown was correlated with up-regulation of hepatic CSC surface markers. mRNA expressions of DDX3, CD133, CD13, EpCAM, CD90 and GAPDH in shLuc, shDDX3 #2 and shDDX3 #3 cells were detected by qRT-PCR. GAPDH was used as internal control. Fold change of each mRNA transcript in shDDX3 #2 and shDDX3 #3 cells was relative to that of shLuc cells. ( e ) DDX3 overexpression resulted in suppression of hepatic CSC surface markers. SK-Hep-1 cells were transfected with plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 as described in ( c ). At 48 h post transfection, total RNA was extracted and subjected to qRT-PCR analysis. GAPDH was used as internal control. Fold change of each mRNA transcript in FLAG-DDX3-expressing cells was relative to that of vector control cells. All experiments were performed at least three times, and the error bar indicates ± 1 s.d. of the mean. Statistical analyses were carried out using t test (* p

    Journal: Scientific Reports

    Article Title: DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma

    doi: 10.1038/srep28637

    Figure Lengend Snippet: DDX3 suppresses stemness gene signature. ( a ) Lower expression of DDX3 along with overexpression of stemness markers was observed in the poorly differentiated cell line. Cell lysates (50 μg) of HepG2, Hep3B, HuH-7 and SK-Hep-1 cells were analyzed by western blotting with antibodies against DDX3, Nanog, Oct4, c-Myc, Sox2, KLF4, Bmi1, CK19 and β-actin. ( b ) Knockdown of DDX3 led to up-regulation of stemness markers. Cell lysates (50 μg) of stable shLuc, shDDX3 #2 and shDDX3 #3 HepG2 cells were analyzed by western blotting with antibodies described in ( a ). ( c ) DDX3 overexpression suppressed stemness markers. Plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 was transfected into SK-Hep-1 cells. At 48 h post transfection, cell lysates were prepared and subjected to immunoblotting with antibodies described in ( a ) and anti-FLAG antibody. β-actin was used as internal control in ( a – c ). ( d ) DDX3 knockdown was correlated with up-regulation of hepatic CSC surface markers. mRNA expressions of DDX3, CD133, CD13, EpCAM, CD90 and GAPDH in shLuc, shDDX3 #2 and shDDX3 #3 cells were detected by qRT-PCR. GAPDH was used as internal control. Fold change of each mRNA transcript in shDDX3 #2 and shDDX3 #3 cells was relative to that of shLuc cells. ( e ) DDX3 overexpression resulted in suppression of hepatic CSC surface markers. SK-Hep-1 cells were transfected with plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 as described in ( c ). At 48 h post transfection, total RNA was extracted and subjected to qRT-PCR analysis. GAPDH was used as internal control. Fold change of each mRNA transcript in FLAG-DDX3-expressing cells was relative to that of vector control cells. All experiments were performed at least three times, and the error bar indicates ± 1 s.d. of the mean. Statistical analyses were carried out using t test (* p

    Article Snippet: Antibodies against Nanog (#3580), Oct4 (#2840), c-Myc (#9402), Bmi1 (#2830) and CK19 (#4558) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Over Expression, Western Blot, Plasmid Preparation, Transfection, Quantitative RT-PCR

    Premature activation of RASSF1A impairs embryogenesis via p73. a Indicated gene expression levels in published GEO data sets GDS3599 and GDS2156. b Temporal expression of Oct4 and Rassf1A mRNA in the pre-implantation embryo (% of maximum expression) from published GEO data sets GDS752 (black colour) and GDS814 (red colour). c Nuclear localisation of YAP during early stages of pre-implantation development. d Nanog immunofluorescence and e representative images of embryos microinjected with either control (zsCtrl) or RASSF1A-expressing (zsR1A) vectors stained for stem cell marker expression. Bar graph showing total OCT4 protein levels across all embryos in zsR1A versus zsCtrl. f 'Kill curve' to determine lethal RASSF1A concentration in pre-implantation embryos. The graph expresses percentage (%) of blastocyst-forming embryos at the indicated RASSF1A concentration. g Viability of embryos in response to RASSF1A expression and/or sip73 microinjection, n = 15. BF bright field channel. Scale bars: 10–50 μm. *P

    Journal: Nature Communications

    Article Title: RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

    doi: 10.1038/s41467-017-02786-5

    Figure Lengend Snippet: Premature activation of RASSF1A impairs embryogenesis via p73. a Indicated gene expression levels in published GEO data sets GDS3599 and GDS2156. b Temporal expression of Oct4 and Rassf1A mRNA in the pre-implantation embryo (% of maximum expression) from published GEO data sets GDS752 (black colour) and GDS814 (red colour). c Nuclear localisation of YAP during early stages of pre-implantation development. d Nanog immunofluorescence and e representative images of embryos microinjected with either control (zsCtrl) or RASSF1A-expressing (zsR1A) vectors stained for stem cell marker expression. Bar graph showing total OCT4 protein levels across all embryos in zsR1A versus zsCtrl. f 'Kill curve' to determine lethal RASSF1A concentration in pre-implantation embryos. The graph expresses percentage (%) of blastocyst-forming embryos at the indicated RASSF1A concentration. g Viability of embryos in response to RASSF1A expression and/or sip73 microinjection, n = 15. BF bright field channel. Scale bars: 10–50 μm. *P

    Article Snippet: The following primary antibodies were used at a concentration of 1:1000; TEF-4 (sc-67115), β-catenin (sc-7199), p73 (ep436Y), YAP (sc-15404 and sc-101199), Gapdh (abcam;2251-1; 1/10000), Tcf-3 (sc-166411), FLAG (M2; Agilent; 200472-21), Rassf1a (sc-58470), HA (Millipore;05-904), Nanog (ab80892), Oct4 (ab19857), TEF-1 (BD Laboratories;610922), Tcf-4 (Cell Signaling; C48H11/2569), LATS1 (Bethyl Laboratories; A300-477A-1), pS127 YAP (Cell Signaling; 4911), pY357 YAP (ab62751), pSer (ab9332), GATA-1 (sc-13053), MST2 (ab52641) and HRP conjugated anti-mouse and anti-rabbit secondary antibodies were used at a concentration of 1:5000 (Jackson Immunoresearch).

    Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Marker, Concentration Assay

    RASSF1A is a barrier to somatic cell reprogramming and iPS cell self-renewal. a Experimental scheme for iPSC generation from MEFs. b Top: example images of Nanog/alkaline phosphatase (AP)-positive round iPSC colonies and quantification of reprogramming efficiency in the respective conditions. c qPCR in MEFs and iPSC for core stem cell marker expression. d . e . f Neural differentiation of Rassf1A +/+ and −/− iPSC in N2B27 medium and retinoic acid (RA). Differentiation capacity of iPSC into neural progenitors is assessed via Nestin and Pax3 expression g Model. Scale bars: 25–50 μm. *P

    Journal: Nature Communications

    Article Title: RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

    doi: 10.1038/s41467-017-02786-5

    Figure Lengend Snippet: RASSF1A is a barrier to somatic cell reprogramming and iPS cell self-renewal. a Experimental scheme for iPSC generation from MEFs. b Top: example images of Nanog/alkaline phosphatase (AP)-positive round iPSC colonies and quantification of reprogramming efficiency in the respective conditions. c qPCR in MEFs and iPSC for core stem cell marker expression. d . e . f Neural differentiation of Rassf1A +/+ and −/− iPSC in N2B27 medium and retinoic acid (RA). Differentiation capacity of iPSC into neural progenitors is assessed via Nestin and Pax3 expression g Model. Scale bars: 25–50 μm. *P

    Article Snippet: The following primary antibodies were used at a concentration of 1:1000; TEF-4 (sc-67115), β-catenin (sc-7199), p73 (ep436Y), YAP (sc-15404 and sc-101199), Gapdh (abcam;2251-1; 1/10000), Tcf-3 (sc-166411), FLAG (M2; Agilent; 200472-21), Rassf1a (sc-58470), HA (Millipore;05-904), Nanog (ab80892), Oct4 (ab19857), TEF-1 (BD Laboratories;610922), Tcf-4 (Cell Signaling; C48H11/2569), LATS1 (Bethyl Laboratories; A300-477A-1), pS127 YAP (Cell Signaling; 4911), pY357 YAP (ab62751), pSer (ab9332), GATA-1 (sc-13053), MST2 (ab52641) and HRP conjugated anti-mouse and anti-rabbit secondary antibodies were used at a concentration of 1:5000 (Jackson Immunoresearch).

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

    RASSF1A regulates the ESC core pluripotency network. a Representative fluorescent images of Nanog and zsRASSF1A (zsR1A)-expressing cells in mouse ESC colonies. Bar graph and western blotting represent quantification of Nanog and RASSF1A levels, respectively. b Nanog immunofluorescence in siNT versus siRASSF1A-transfected ESC. Additional expression of siRASSF1A-resistant zsRASSF1A rescues the phenotype in siRASSF1A-transfected ESC, quantified in the displayed bar graphs and additionally demonstrated by Western blotting. Zoom in displays RASSF1A localisation peripherally to the nucleus at the microtubule organising centre, in zsRASSF1A-expressing cells. Validation using a second siRNA to Rassf1A . c qPCR for core stem cell markers from ESC in b . d qPCR for germ layer-specific differentiation markers in ESC subject to LIF withdrawal. e RNAseq analysis in shRNA-expressing ESC versus control (shGFP) reveals establishment of self-renewal and pluripotency signatures in the absence of Rassf1A . Scale bars: 25 and 50 μm. *P

    Journal: Nature Communications

    Article Title: RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

    doi: 10.1038/s41467-017-02786-5

    Figure Lengend Snippet: RASSF1A regulates the ESC core pluripotency network. a Representative fluorescent images of Nanog and zsRASSF1A (zsR1A)-expressing cells in mouse ESC colonies. Bar graph and western blotting represent quantification of Nanog and RASSF1A levels, respectively. b Nanog immunofluorescence in siNT versus siRASSF1A-transfected ESC. Additional expression of siRASSF1A-resistant zsRASSF1A rescues the phenotype in siRASSF1A-transfected ESC, quantified in the displayed bar graphs and additionally demonstrated by Western blotting. Zoom in displays RASSF1A localisation peripherally to the nucleus at the microtubule organising centre, in zsRASSF1A-expressing cells. Validation using a second siRNA to Rassf1A . c qPCR for core stem cell markers from ESC in b . d qPCR for germ layer-specific differentiation markers in ESC subject to LIF withdrawal. e RNAseq analysis in shRNA-expressing ESC versus control (shGFP) reveals establishment of self-renewal and pluripotency signatures in the absence of Rassf1A . Scale bars: 25 and 50 μm. *P

    Article Snippet: The following primary antibodies were used at a concentration of 1:1000; TEF-4 (sc-67115), β-catenin (sc-7199), p73 (ep436Y), YAP (sc-15404 and sc-101199), Gapdh (abcam;2251-1; 1/10000), Tcf-3 (sc-166411), FLAG (M2; Agilent; 200472-21), Rassf1a (sc-58470), HA (Millipore;05-904), Nanog (ab80892), Oct4 (ab19857), TEF-1 (BD Laboratories;610922), Tcf-4 (Cell Signaling; C48H11/2569), LATS1 (Bethyl Laboratories; A300-477A-1), pS127 YAP (Cell Signaling; 4911), pY357 YAP (ab62751), pSer (ab9332), GATA-1 (sc-13053), MST2 (ab52641) and HRP conjugated anti-mouse and anti-rabbit secondary antibodies were used at a concentration of 1:5000 (Jackson Immunoresearch).

    Techniques: Expressing, Western Blot, Immunofluorescence, Transfection, Real-time Polymerase Chain Reaction, shRNA

    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

    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

    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

    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

    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

    Characterisation of hESCs maintained in CMp11 and media conditioned by passage 18 HFFs (CMp18). Representative immunofluorescent images of hESCs maintained for 5 days in CMp11 (n = 5) and CMp18 media (n = 5) (bar = 100 µm, TRA-1.81 bar = 200 µm). Cells cultured in CMp11 presented well defined colonies with morphology characteristic of undifferentiated cells. In contrast, CMp18 cultured cell were morphologically distinct with colonies lacking smooth and defined edges and a clearing effect from the centre. Consistent with an undifferentiated phenotype, CMp11 cultured cells were negative for the early differentiation marker SSEA-1 (green) and positive for the undifferentiation markers SSEA-4, TRA-1.60, TRA-1.81, OCT4 and NANOG. Cells cultured in CMp18 were positive for SSEA-1 and negative for undifferentiation markers. All cells were co-labelled with the nuclear stain DAPI (blue).

    Journal: PLoS ONE

    Article Title: Characterisation of Human Embryonic Stem Cells Conditioning Media by 1H-Nuclear Magnetic Resonance Spectroscopy

    doi: 10.1371/journal.pone.0016732

    Figure Lengend Snippet: Characterisation of hESCs maintained in CMp11 and media conditioned by passage 18 HFFs (CMp18). Representative immunofluorescent images of hESCs maintained for 5 days in CMp11 (n = 5) and CMp18 media (n = 5) (bar = 100 µm, TRA-1.81 bar = 200 µm). Cells cultured in CMp11 presented well defined colonies with morphology characteristic of undifferentiated cells. In contrast, CMp18 cultured cell were morphologically distinct with colonies lacking smooth and defined edges and a clearing effect from the centre. Consistent with an undifferentiated phenotype, CMp11 cultured cells were negative for the early differentiation marker SSEA-1 (green) and positive for the undifferentiation markers SSEA-4, TRA-1.60, TRA-1.81, OCT4 and NANOG. Cells cultured in CMp18 were positive for SSEA-1 and negative for undifferentiation markers. All cells were co-labelled with the nuclear stain DAPI (blue).

    Article Snippet: Briefly, hESCs were characterized by immunohistochemistry using antibodies against a cassette of undifferentiated hESCs markers including SSEA4, TRA-1-60, TRA-1-81, OCT-4 (Millipore, Billerica, MA) and NANOG (R & D systems, Minneapolis, MN).

    Techniques: Cell Culture, Marker, Staining

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

    Journal: Bioscience Reports

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

    doi: 10.1042/BSR20160247

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

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

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

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

    Journal: Bioscience Reports

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

    doi: 10.1042/BSR20160247

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

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

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