n2b27 medium  (Thermo Fisher)


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    DMEM F 12
    Description:
    DMEM F 12 Dulbecco s Modified Eagle Medium Nutrient Mixture F 12 is a widely used basal medium for supporting the growth of many different mammalian cells Cells successfully cultured in DMEM F 12 include MDCK glial cells fibroblasts human endothelial cells and rat fibroblasts We offer a variety of DMEM F 12 modifications for a range of cell culture applications Find the right formulation using the media selector tool This DMEM F 12 is modified as follows WithWithout• L glutamine• HEPES• Phenol RedThe complete formulation is available Using DMEM F 12DMEM F 12 is a 1 1 mixture of DMEM and Ham s F 12 This formulation combines DMEM s high concentrations of glucose amino acids and vitamins with F 12 s wide variety of components DMEM F 12 contains no proteins lipids or growth factors Therefore DMEM F 12 may require supplementation commonly with 10 Fetal Bovine Serum FBS DMEM F 12 uses a sodium bicarbonate buffer system and therefore requires a 5 10 CO2 environment to maintain physiological pH cGMP manufacturing and quality systemDMEM F 12 is manufactured at a cGMP compliant facility located in Grand Island New York The facility is registered with the FDA as a medical device manufacturer and is certified to ISO 13485 standards For supply chain continuity we offer a comparable DMEM F 12 product made in our Scotland facility 11320 074 This facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standard
    Catalog Number:
    11320033
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    Cell Culture Transfection Reagents
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    Cell Culture|Mammalian Cell Culture
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    Structured Review

    Thermo Fisher n2b27 medium
    Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in <t>N2B27</t> conditions with selection for geneticin (G418)
    DMEM F 12 Dulbecco s Modified Eagle Medium Nutrient Mixture F 12 is a widely used basal medium for supporting the growth of many different mammalian cells Cells successfully cultured in DMEM F 12 include MDCK glial cells fibroblasts human endothelial cells and rat fibroblasts We offer a variety of DMEM F 12 modifications for a range of cell culture applications Find the right formulation using the media selector tool This DMEM F 12 is modified as follows WithWithout• L glutamine• HEPES• Phenol RedThe complete formulation is available Using DMEM F 12DMEM F 12 is a 1 1 mixture of DMEM and Ham s F 12 This formulation combines DMEM s high concentrations of glucose amino acids and vitamins with F 12 s wide variety of components DMEM F 12 contains no proteins lipids or growth factors Therefore DMEM F 12 may require supplementation commonly with 10 Fetal Bovine Serum FBS DMEM F 12 uses a sodium bicarbonate buffer system and therefore requires a 5 10 CO2 environment to maintain physiological pH cGMP manufacturing and quality systemDMEM F 12 is manufactured at a cGMP compliant facility located in Grand Island New York The facility is registered with the FDA as a medical device manufacturer and is certified to ISO 13485 standards For supply chain continuity we offer a comparable DMEM F 12 product made in our Scotland facility 11320 074 This facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standard
    https://www.bioz.com/result/n2b27 medium/product/Thermo Fisher
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    Images

    1) Product Images from "A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages"

    Article Title: A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages

    Journal: Nature cell biology

    doi: 10.1038/ncb2742

    Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in N2B27 conditions with selection for geneticin (G418)
    Figure Legend Snippet: Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in N2B27 conditions with selection for geneticin (G418)

    Techniques Used: Cell Culture, Selection

    2) Product Images from "Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3"

    Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.012

    Flcn Regulates ESC Commitment (A) Flow cytometry profile of Rex1GFPd2 expression in Flcn shRNA knockdown clones (Flcn.2,4) and controls (ctrl.1,2) in 2i conditions (left panel) and after 24 hr of 2i withdrawal (right panel). (B) Flcn shRNA cell lines and controls kept in 2i (10-fold fewer cells) or differentiated for 72 hr in N2B27, or N2B27 supplemented with 10% FBS or 25 ng/ml FGF4, 10 ng/ml BMP4, and 20 ng/ml Activin A, were replated in 2i and selected in blasticidin for Rex1 expression. Resulting ESC colonies were visualized by AP staining. (C and D) Differentiating Flcn shRNA cell lines (C) or Flcn shRNA cell lines expressing an empty vector or shRNA-resistant Flcn transgene (rescue) (D) and resp ective controls were replated at clonal density, and colonies arising from uncommitted cells stained for AP. Average clonogenicity and SD are relative to number of plated cells of two independent experiments. (E) CreERT2-expressing clones of indicated genotypes were treated with Tam, differentiated for 3 days, and uncommitted cells quantified in 2i/LIF. Average and SD are of at least three independent biological replicates. See also Figure S2 .
    Figure Legend Snippet: Flcn Regulates ESC Commitment (A) Flow cytometry profile of Rex1GFPd2 expression in Flcn shRNA knockdown clones (Flcn.2,4) and controls (ctrl.1,2) in 2i conditions (left panel) and after 24 hr of 2i withdrawal (right panel). (B) Flcn shRNA cell lines and controls kept in 2i (10-fold fewer cells) or differentiated for 72 hr in N2B27, or N2B27 supplemented with 10% FBS or 25 ng/ml FGF4, 10 ng/ml BMP4, and 20 ng/ml Activin A, were replated in 2i and selected in blasticidin for Rex1 expression. Resulting ESC colonies were visualized by AP staining. (C and D) Differentiating Flcn shRNA cell lines (C) or Flcn shRNA cell lines expressing an empty vector or shRNA-resistant Flcn transgene (rescue) (D) and resp ective controls were replated at clonal density, and colonies arising from uncommitted cells stained for AP. Average clonogenicity and SD are relative to number of plated cells of two independent experiments. (E) CreERT2-expressing clones of indicated genotypes were treated with Tam, differentiated for 3 days, and uncommitted cells quantified in 2i/LIF. Average and SD are of at least three independent biological replicates. See also Figure S2 .

    Techniques Used: Flow Cytometry, Cytometry, Expressing, shRNA, Clone Assay, Staining, Plasmid Preparation

    Flcn Acts downstream of or in Parallel to mTOR and Interacts with Fnip1/2 (A) Rex1GFPd2 cells were differentiated in N2B27 with and without 20 nM Rapa (left panel) or in N2B27/10%FBS (right panel), and cell lysates probed with indicated antibodies. (B) Rex1GFPd2 cells differentiated in N2B27 with and without 20 nM Rapa were replated at single-cell density in 2i including Rex1-expression selection at the indicated time points. The average percentage of uncommitted cells forming AP-positive colonies relative to the number of cells plated and SD are of two technical replicates. (C) Rex1GFPd2 cells transfected with indicated siRNAs were differentiated for 72 hr in N2B27 with and without 20 nM Rapa and replated in 2i with Rex1-expression selection, and resulting colonies were stained for AP. (D) Proteins were immunoprecipitated with FLAG antibodies from stably transfected Rex1GFPd2 cells cultured in 2i or differentiated for 40 hr and probed with indicated antibodies. (E) mRNA levels were quantified during differentiation and normalized to 2i-cultured cells. Average and SD are of two cell lines. (F) O4GIP ESCs were transfected with indicated siRNAs, and after differentiation for 3 days, exit from pluripotency quantified with a cell-viability assay and normalized to no siRNA transfection controls. Average and SD are of two technical replicates. See also Figure S3 .
    Figure Legend Snippet: Flcn Acts downstream of or in Parallel to mTOR and Interacts with Fnip1/2 (A) Rex1GFPd2 cells were differentiated in N2B27 with and without 20 nM Rapa (left panel) or in N2B27/10%FBS (right panel), and cell lysates probed with indicated antibodies. (B) Rex1GFPd2 cells differentiated in N2B27 with and without 20 nM Rapa were replated at single-cell density in 2i including Rex1-expression selection at the indicated time points. The average percentage of uncommitted cells forming AP-positive colonies relative to the number of cells plated and SD are of two technical replicates. (C) Rex1GFPd2 cells transfected with indicated siRNAs were differentiated for 72 hr in N2B27 with and without 20 nM Rapa and replated in 2i with Rex1-expression selection, and resulting colonies were stained for AP. (D) Proteins were immunoprecipitated with FLAG antibodies from stably transfected Rex1GFPd2 cells cultured in 2i or differentiated for 40 hr and probed with indicated antibodies. (E) mRNA levels were quantified during differentiation and normalized to 2i-cultured cells. Average and SD are of two cell lines. (F) O4GIP ESCs were transfected with indicated siRNAs, and after differentiation for 3 days, exit from pluripotency quantified with a cell-viability assay and normalized to no siRNA transfection controls. Average and SD are of two technical replicates. See also Figure S3 .

    Techniques Used: Expressing, Selection, Transfection, Staining, Immunoprecipitation, Stable Transfection, Cell Culture, Viability Assay

    Related to Figure 7 (A) Flow cytometry of Rex1GFPd2 TET cells after Tam withdrawal. (B) Expression levels of indicated mRNAs in O4GIP TET and EpiSCs normalized to 2i. Average and SD are from two independent experiments. (n.d.) indicates not detectable. (C) Expression levels of indicated mRNAs in Flcn knockout cells maintained in N2B27 without 2i or LIF normalized to wild-type ESCs in 2i/LIF. Average and SD are of three independent experiments. (D) Immunohistochemistry for GATA4 in Rex1GFPd2 TET cells. (E) Rex1GFPd2 TET cells were sorted for GFP expression and expression of indicated mRNAs determined. Fold changes relative to sorted Rex1GFPd2 ESCs maintained in 2i and SD are from two technical replicates. (F) Flcn knockout cells maintained in N2B27 without 2i or LIF were plated at single-cell density in indicated culture conditions. Resulting colonies were stained for AP and quantified. Average relative to number of plated cells and SD are of three independent experiments (left bar: Flcn (−/−) (a), right bar: Flcn (−/−) (b)). (G) Rex1GPPd2 TET cells were transfected with indicated siRNAs and GFP expression monitored by flow cytometry 2 days after transfection. (H) Passage 8 Rex1GFPd2 TET cells were injected into C57BL/6 blastocysts without 2i preculture. Contribution of the TET cell agouti gene to coat color is visible against black host fur.
    Figure Legend Snippet: Related to Figure 7 (A) Flow cytometry of Rex1GFPd2 TET cells after Tam withdrawal. (B) Expression levels of indicated mRNAs in O4GIP TET and EpiSCs normalized to 2i. Average and SD are from two independent experiments. (n.d.) indicates not detectable. (C) Expression levels of indicated mRNAs in Flcn knockout cells maintained in N2B27 without 2i or LIF normalized to wild-type ESCs in 2i/LIF. Average and SD are of three independent experiments. (D) Immunohistochemistry for GATA4 in Rex1GFPd2 TET cells. (E) Rex1GFPd2 TET cells were sorted for GFP expression and expression of indicated mRNAs determined. Fold changes relative to sorted Rex1GFPd2 ESCs maintained in 2i and SD are from two technical replicates. (F) Flcn knockout cells maintained in N2B27 without 2i or LIF were plated at single-cell density in indicated culture conditions. Resulting colonies were stained for AP and quantified. Average relative to number of plated cells and SD are of three independent experiments (left bar: Flcn (−/−) (a), right bar: Flcn (−/−) (b)). (G) Rex1GPPd2 TET cells were transfected with indicated siRNAs and GFP expression monitored by flow cytometry 2 days after transfection. (H) Passage 8 Rex1GFPd2 TET cells were injected into C57BL/6 blastocysts without 2i preculture. Contribution of the TET cell agouti gene to coat color is visible against black host fur.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Knock-Out, Immunohistochemistry, Staining, Transfection, Injection

    3) Product Images from "Inhibition of Transforming Growth Factor β (TGF-β) Signaling can Substitute for Oct4 Protein in Reprogramming and Maintain Pluripotency *"

    Article Title: Inhibition of Transforming Growth Factor β (TGF-β) Signaling can Substitute for Oct4 Protein in Reprogramming and Maintain Pluripotency *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.609016

    Inhibition of the TGF-β signaling pathway substitutes for Oct4 in reprogramming. A–D , MEFs were infected with lentiviruses encoding for OSKM or SKM. After 6 days under serum conditions with factors, these cells were cultured under serum-free N2B27 conditions supplemented with factors. On day 24 of induction, AP staining was performed. A , AP-positive colonies were induced from MEFs transduced with SKM in combination with VSCF. B , the effect of withdrawal of individual factors from the VSCF pool on the formation of AP-positive colonies in MEFs transduced with SKM. C , AP-positive colonies were generated from MEFs transduced with SKM in combination with VPA and SB431542. DMSO , dimethyl sulfoxide. D , AP-positive colony numbers induced from 1 × 10 5 MEFs by SKM in combination with different factors. V , VPA; S , SB431542; C , CHIR99021; F , FGF2; P , PD0325901. Three independent experiments were performed, and the results are presented as the mean ± S.D. *, p
    Figure Legend Snippet: Inhibition of the TGF-β signaling pathway substitutes for Oct4 in reprogramming. A–D , MEFs were infected with lentiviruses encoding for OSKM or SKM. After 6 days under serum conditions with factors, these cells were cultured under serum-free N2B27 conditions supplemented with factors. On day 24 of induction, AP staining was performed. A , AP-positive colonies were induced from MEFs transduced with SKM in combination with VSCF. B , the effect of withdrawal of individual factors from the VSCF pool on the formation of AP-positive colonies in MEFs transduced with SKM. C , AP-positive colonies were generated from MEFs transduced with SKM in combination with VPA and SB431542. DMSO , dimethyl sulfoxide. D , AP-positive colony numbers induced from 1 × 10 5 MEFs by SKM in combination with different factors. V , VPA; S , SB431542; C , CHIR99021; F , FGF2; P , PD0325901. Three independent experiments were performed, and the results are presented as the mean ± S.D. *, p

    Techniques Used: Inhibition, Infection, Cell Culture, Staining, Transduction, Generated

    4) Product Images from "Activated ?-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma"

    Article Title: Activated ?-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.3520

    BIO promotes N2A cell-derived neuron morphological changes. (A) N2A cells were induced to differentiate into neurons as previously described (b). Four days later, N2B27 medium was replaced by 10% FBS (c). The induced neurite outgrowth was shown by brightfield images. Sclar bar = 200μm. (B) After induction of N2A cells for 4 days, the cells were treated with 10% FBS (a) or 10% FBS+DKK1 (100ng/ml) (b). Sclar bar = 100μm. (C) After induction of N2A cells for 4 days, the cells were treated with DMSO (a), 2μM (b), 5μM (c) or 10μM (d) BIO for 12 hours, respectively. Sclar bar = 100μm
    Figure Legend Snippet: BIO promotes N2A cell-derived neuron morphological changes. (A) N2A cells were induced to differentiate into neurons as previously described (b). Four days later, N2B27 medium was replaced by 10% FBS (c). The induced neurite outgrowth was shown by brightfield images. Sclar bar = 200μm. (B) After induction of N2A cells for 4 days, the cells were treated with 10% FBS (a) or 10% FBS+DKK1 (100ng/ml) (b). Sclar bar = 100μm. (C) After induction of N2A cells for 4 days, the cells were treated with DMSO (a), 2μM (b), 5μM (c) or 10μM (d) BIO for 12 hours, respectively. Sclar bar = 100μm

    Techniques Used: Derivative Assay

    5) Product Images from "SALL4 controls cell fate in response to DNA base composition"

    Article Title: SALL4 controls cell fate in response to DNA base composition

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2020.11.046

    Phenotypic effects of SALL4 ZFC4 mutation on neuronal differentiation (A) TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/leukemia inhibitory factor (LIF) medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars, 100 μm. (B) qRT-PCR analysis of the neuronal markers Tuj1, Ascl1, and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcript levels were normalized to TATA-binding protein (TBP) and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation. See also Figure S4 .
    Figure Legend Snippet: Phenotypic effects of SALL4 ZFC4 mutation on neuronal differentiation (A) TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/leukemia inhibitory factor (LIF) medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars, 100 μm. (B) qRT-PCR analysis of the neuronal markers Tuj1, Ascl1, and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcript levels were normalized to TATA-binding protein (TBP) and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation. See also Figure S4 .

    Techniques Used: Mutagenesis, Immunofluorescence, Cell Culture, Staining, Quantitative RT-PCR, Binding Assay, Standard Deviation

    Effects of SALL4 ZFC1 and ZFC2 deletion in ESCs on chromatin binding, gene expression, and differentiation (A) Diagram showing the in-frame deletion within the Sall4 coding sequence, generated by CRISPR-Cas9. (B) SALL4 ZFC1-2Δ localization determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars, 3 μm. (C) Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. (D) Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit ±250 bp) in WT (blue) and ZFC1-2Δ (purple) ESCs. (E) Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2Δ ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple. (F) Correlation between gene mis-regulation (log2 fold change versus WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT content. (G) TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars, 100 μm. See also Figure S6 and Table S2 .
    Figure Legend Snippet: Effects of SALL4 ZFC1 and ZFC2 deletion in ESCs on chromatin binding, gene expression, and differentiation (A) Diagram showing the in-frame deletion within the Sall4 coding sequence, generated by CRISPR-Cas9. (B) SALL4 ZFC1-2Δ localization determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars, 3 μm. (C) Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. (D) Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit ±250 bp) in WT (blue) and ZFC1-2Δ (purple) ESCs. (E) Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2Δ ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple. (F) Correlation between gene mis-regulation (log2 fold change versus WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT content. (G) TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars, 100 μm. See also Figure S6 and Table S2 .

    Techniques Used: Binding Assay, Expressing, Sequencing, Generated, CRISPR, Immunofluorescence, Staining, Chromatin Immunoprecipitation, RNA Sequencing Assay, Mutagenesis, Cell Culture

    6) Product Images from "SALL4 controls cell fate in response to DNA base composition"

    Article Title: SALL4 controls cell fate in response to DNA base composition

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.179481

    Characterisation of SALL4 C2H2 zinc-finger clusters 1 and 2 in ESCs A. Diagram showing the in frame deletion of SALL4 within the Sall4 coding sequence, generated by CRISPR/Cas9. B. SALL4 ZFC1-2Δ localisation determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars: 3μm. C. Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. D. Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit +/- 250bp) in WT (blue) and ZFC1-2 Δ (purple) ESCs. E. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2 Δ ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple. F. Correlation between gene mis-regulation (log2 fold-change vs WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT-content. G. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm.
    Figure Legend Snippet: Characterisation of SALL4 C2H2 zinc-finger clusters 1 and 2 in ESCs A. Diagram showing the in frame deletion of SALL4 within the Sall4 coding sequence, generated by CRISPR/Cas9. B. SALL4 ZFC1-2Δ localisation determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars: 3μm. C. Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. D. Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit +/- 250bp) in WT (blue) and ZFC1-2 Δ (purple) ESCs. E. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2 Δ ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple. F. Correlation between gene mis-regulation (log2 fold-change vs WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT-content. G. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm.

    Techniques Used: Sequencing, Generated, CRISPR, Immunofluorescence, Staining, Chromatin Immunoprecipitation, RNA Sequencing Assay, Mutagenesis, Cell Culture

    Phenotypic characterisation of SALL4 ZFC4 mutation during neuronal differentiation A. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm. B. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.
    Figure Legend Snippet: Phenotypic characterisation of SALL4 ZFC4 mutation during neuronal differentiation A. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm. B. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.

    Techniques Used: Mutagenesis, Immunofluorescence, Cell Culture, Staining, Quantitative RT-PCR, Standard Deviation

    A. SALL4 co-immunoprecipitation with SALL1 and NuRD components in WT, S4KO (negative control) and ZFC1-2 Δ ESCs. B. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between independent replicate experiments in ZFC1-2 Δ ESCs. C. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between WT, ZFC1-2 Δ and ZFC4mut ESCs. D, E. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-regulated (D) and ZFC1/2-regulated (E) genes (see Figure 6E ) divided into five equal categories according to AT-content. F. Statistical analysis of AT-dependent gene expression changes (coefficient estimates with 99% confidence intervals) observed with ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes (see Figure 6E ). Significance is attributed by F-test. Empty circles represent non-significant model fits ( > 0.01 FDR) and filled circles represent significant model fit. G. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.
    Figure Legend Snippet: A. SALL4 co-immunoprecipitation with SALL1 and NuRD components in WT, S4KO (negative control) and ZFC1-2 Δ ESCs. B. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between independent replicate experiments in ZFC1-2 Δ ESCs. C. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between WT, ZFC1-2 Δ and ZFC4mut ESCs. D, E. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-regulated (D) and ZFC1/2-regulated (E) genes (see Figure 6E ) divided into five equal categories according to AT-content. F. Statistical analysis of AT-dependent gene expression changes (coefficient estimates with 99% confidence intervals) observed with ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes (see Figure 6E ). Significance is attributed by F-test. Empty circles represent non-significant model fits ( > 0.01 FDR) and filled circles represent significant model fit. G. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.

    Techniques Used: Immunoprecipitation, Negative Control, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR, Standard Deviation

    7) Product Images from "Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells"

    Article Title: Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

    Journal: Nature cell biology

    doi: 10.1038/ncb2314

    Wnt3a protein is sufficient to inhibit the differentiation of ESCs into EpiSCs. ( a ) FACS plots of EpiSCs and of R1 cells passaged every 3 days in N2B27 supplemented with LIF, bFGF and ActivinA, in the presence of Wnt3a protein (240 ng ml −1 ) or
    Figure Legend Snippet: Wnt3a protein is sufficient to inhibit the differentiation of ESCs into EpiSCs. ( a ) FACS plots of EpiSCs and of R1 cells passaged every 3 days in N2B27 supplemented with LIF, bFGF and ActivinA, in the presence of Wnt3a protein (240 ng ml −1 ) or

    Techniques Used: FACS

    LIF and Wnt3a are sufficient to support ESC self-renewal. ( a , b ) Expansion over multiple passages of alkaline phosphatase-positive (AP + ) R1 ESC colonies in medium containing serum and LIF ( a ) or in N2B27 containing LIF ( b ) (mean + s.e.m., n = 3). ( c , d
    Figure Legend Snippet: LIF and Wnt3a are sufficient to support ESC self-renewal. ( a , b ) Expansion over multiple passages of alkaline phosphatase-positive (AP + ) R1 ESC colonies in medium containing serum and LIF ( a ) or in N2B27 containing LIF ( b ) (mean + s.e.m., n = 3). ( c , d

    Techniques Used:

    8) Product Images from "Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2"

    Article Title: Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2

    Journal: The EMBO journal

    doi: 10.15252/embj.2019102591

    Zfp281 drives exit from naïve pluripotency independent of Tet enzymes. ( A , B , E ) Self-renewal in RGd2 ESCs of specified genotypes expressing indicated transgenes ( B ) after differentiation in indicated conditions ( A ) or 72h in N2B27 ( B , E ). Average and SD of 2 experiments performed in duplicates. Note that control cells were lost during continuous passaging in N2B27 ( A ). Not determined (n.d.). ( C ) Representative flow cytometry profiles of WT and Zfp281 KO.1 cells after 32h of 2i withdrawal before (unsorted) and after purification of cells with indicated GFP expression (top panel). Self-renewal of undifferentiated (2i) and sorted GFP high,sort and GFP low,sort cells of indicated genotypes (bottom panel). Average and SD of 3 experiments performed in duplicates. ( D ) Representative flow cytometry profiles of control and Zfp281-inducible ESCs (top panel) and quantification of GFP low cells (bottom panel) after 2d in 2i and in the presence (green) or absence (black) of Dox. Average and SD of 2 experiments. ( F ) (right). Average and SD of 2 technical replicates (left).
    Figure Legend Snippet: Zfp281 drives exit from naïve pluripotency independent of Tet enzymes. ( A , B , E ) Self-renewal in RGd2 ESCs of specified genotypes expressing indicated transgenes ( B ) after differentiation in indicated conditions ( A ) or 72h in N2B27 ( B , E ). Average and SD of 2 experiments performed in duplicates. Note that control cells were lost during continuous passaging in N2B27 ( A ). Not determined (n.d.). ( C ) Representative flow cytometry profiles of WT and Zfp281 KO.1 cells after 32h of 2i withdrawal before (unsorted) and after purification of cells with indicated GFP expression (top panel). Self-renewal of undifferentiated (2i) and sorted GFP high,sort and GFP low,sort cells of indicated genotypes (bottom panel). Average and SD of 3 experiments performed in duplicates. ( D ) Representative flow cytometry profiles of control and Zfp281-inducible ESCs (top panel) and quantification of GFP low cells (bottom panel) after 2d in 2i and in the presence (green) or absence (black) of Dox. Average and SD of 2 experiments. ( F ) (right). Average and SD of 2 technical replicates (left).

    Techniques Used: Expressing, Passaging, Flow Cytometry, Purification

    Zfp281 directs sequential gene expression despite stable occupancy of target sites. ( A ) mRNA log2 fold changes (log2FC) in WT 16h , WT 32h , Zfp281 16h and Zfp281 32h samples relative to WT 2i cells, and in EpiSCs relative to WT 2i/Lif ). Zfp281 2i , Zfp281 16h , Zfp281 32h and WT 16h and WT 32h samples were used for k-means clustering. ( B , C ) Quantification of ( A ) including mRNA log2FC in EpiLCs relative to WT 2i/Lif ) and as indicated ( C ). ( D ) Representative immunofluorescence staining of spheroids in Matrigel derived from WT or Zfp281 KO.1 ESCs grown in 2i or N2B27 for 3d. Blue: DNA. Red: F-actin. Scale bar is 10μm. ( E ) Scatter plot comparing Zfp281 log2 ChIP enrichment relative to matched inputs in WT 2i and WT 32h cells. ( F ) Same as in ( E ) with dots colored according to H3K27ac ChIP log2FC at the same peaks (top left), and to gene expression log2FC associated with peaks by nearest distance to TSS (bottom left) in WT 32h relative to WT 2i cells. Quantification of H3K27ac ChIP (top right) and mRNA (bottom right) log2FC at top 1000 Zfp281 peaks with increased (red) or decreased (blue) Zfp281 binding during ESC differentiation. ( G ) Quantification of Zfp281 (left) and H3K27ac (right) ChIP log2FC in WT 32h compared to WT 2i cells at Zfp281 peaks assigned to gene clusters 1-6.
    Figure Legend Snippet: Zfp281 directs sequential gene expression despite stable occupancy of target sites. ( A ) mRNA log2 fold changes (log2FC) in WT 16h , WT 32h , Zfp281 16h and Zfp281 32h samples relative to WT 2i cells, and in EpiSCs relative to WT 2i/Lif ). Zfp281 2i , Zfp281 16h , Zfp281 32h and WT 16h and WT 32h samples were used for k-means clustering. ( B , C ) Quantification of ( A ) including mRNA log2FC in EpiLCs relative to WT 2i/Lif ) and as indicated ( C ). ( D ) Representative immunofluorescence staining of spheroids in Matrigel derived from WT or Zfp281 KO.1 ESCs grown in 2i or N2B27 for 3d. Blue: DNA. Red: F-actin. Scale bar is 10μm. ( E ) Scatter plot comparing Zfp281 log2 ChIP enrichment relative to matched inputs in WT 2i and WT 32h cells. ( F ) Same as in ( E ) with dots colored according to H3K27ac ChIP log2FC at the same peaks (top left), and to gene expression log2FC associated with peaks by nearest distance to TSS (bottom left) in WT 32h relative to WT 2i cells. Quantification of H3K27ac ChIP (top right) and mRNA (bottom right) log2FC at top 1000 Zfp281 peaks with increased (red) or decreased (blue) Zfp281 binding during ESC differentiation. ( G ) Quantification of Zfp281 (left) and H3K27ac (right) ChIP log2FC in WT 32h compared to WT 2i cells at Zfp281 peaks assigned to gene clusters 1-6.

    Techniques Used: Expressing, Immunofluorescence, Staining, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay

    9) Product Images from "Derivation of Transgene‐Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells"

    Article Title: Derivation of Transgene‐Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2015-0390

    Establishment of riPS cells and ES cells. (A): Derivation of rat ES cells from blastocyst outgrowth. We flushed E4.5 blastocysts from the uterus of pregnant Sprague Dawley rats (left). Scale bar = 100 μm. The blastocysts were plated on feeders in the 3i/Lif medium until the primary outgrowth attached at day 4. ES cells were established from blastocyst outgrowth after three passages and showed typical ES cell morphology. Scale bar = 200 μm. (B): Schematic diagram of the EBNA/oriP‐based reprogramming vector. Plasmid pMaster3 contains 7 human transcription factor genes of OCT4/POU5f1 , SOX2 , KLF4 , C‐MYC , NANOG , LIN28 , and NR5A2 for reprogramming and two drug resistance genes, neo and HSVtk , for positive/negative selection. Plasmid pMaster12 and pMaster22 with additional human and rat miR‐302/367 gene cluster, respectively. (C): Flowchart of transgene‐free riPS cell derivation. Rat fibroblast cells were seeded on mouse embryonic fibroblast feeders and cultured in serum medium after electrotransfection with pMaster3/pMaster12/pMaster22, followed by G418 drug selection for 5 days to ensure successful plasmid transfection. After the eighth day, 3i/Lif medium was used for riPS cell maintenance and primary clones were picked and propagated. To obtain transgene‐free subclones, we plated 20,000 cells into a 10‐cm dish to carry out FIAU negative selection to exclude cells carrying transgenes after the second day. Clones that passed negative selection were picked and expanded for further verification. G418, geneticin, a derivative of gentamycin; serum medium, Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and Lif; and 3i/Lif medium, N2B27 based serum‐free medium with additional CHIR99021, PD0325901, A83‐01, and Lif. (D): Cell morphology during different reprogramming stages. Rat embryonic fibroblasts at passage 1 were used for reprogramming. Primary riPS cells have rat ES‐cell like morphology at day 10 after electrotransfection under hypoxic culture conditions in 3i/Lif medium. The established iPS clones showed round and smooth border morphology, which is a typical characteristic of ES cells. Scale bar = 200 μm. Abbreviations: ES, embryonic stem; FIAU, 1‐(2‐deoxy‐2‐fluoro‐1‐D‐arabinofuranosyl)‐5‐iodoracil; iPS, induced pluripotent stem; Lif, leukemia inhibitory factor; riPS, rat induced pluripotent stem.
    Figure Legend Snippet: Establishment of riPS cells and ES cells. (A): Derivation of rat ES cells from blastocyst outgrowth. We flushed E4.5 blastocysts from the uterus of pregnant Sprague Dawley rats (left). Scale bar = 100 μm. The blastocysts were plated on feeders in the 3i/Lif medium until the primary outgrowth attached at day 4. ES cells were established from blastocyst outgrowth after three passages and showed typical ES cell morphology. Scale bar = 200 μm. (B): Schematic diagram of the EBNA/oriP‐based reprogramming vector. Plasmid pMaster3 contains 7 human transcription factor genes of OCT4/POU5f1 , SOX2 , KLF4 , C‐MYC , NANOG , LIN28 , and NR5A2 for reprogramming and two drug resistance genes, neo and HSVtk , for positive/negative selection. Plasmid pMaster12 and pMaster22 with additional human and rat miR‐302/367 gene cluster, respectively. (C): Flowchart of transgene‐free riPS cell derivation. Rat fibroblast cells were seeded on mouse embryonic fibroblast feeders and cultured in serum medium after electrotransfection with pMaster3/pMaster12/pMaster22, followed by G418 drug selection for 5 days to ensure successful plasmid transfection. After the eighth day, 3i/Lif medium was used for riPS cell maintenance and primary clones were picked and propagated. To obtain transgene‐free subclones, we plated 20,000 cells into a 10‐cm dish to carry out FIAU negative selection to exclude cells carrying transgenes after the second day. Clones that passed negative selection were picked and expanded for further verification. G418, geneticin, a derivative of gentamycin; serum medium, Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and Lif; and 3i/Lif medium, N2B27 based serum‐free medium with additional CHIR99021, PD0325901, A83‐01, and Lif. (D): Cell morphology during different reprogramming stages. Rat embryonic fibroblasts at passage 1 were used for reprogramming. Primary riPS cells have rat ES‐cell like morphology at day 10 after electrotransfection under hypoxic culture conditions in 3i/Lif medium. The established iPS clones showed round and smooth border morphology, which is a typical characteristic of ES cells. Scale bar = 200 μm. Abbreviations: ES, embryonic stem; FIAU, 1‐(2‐deoxy‐2‐fluoro‐1‐D‐arabinofuranosyl)‐5‐iodoracil; iPS, induced pluripotent stem; Lif, leukemia inhibitory factor; riPS, rat induced pluripotent stem.

    Techniques Used: Plasmid Preparation, Selection, Cell Culture, Transfection, Clone Assay, Modification

    10) Product Images from "High-throughput micro-patterning platform reveals Nodal-dependent dissection of peri-gastrulation-associated versus pre-neurulation associated fate patterning"

    Article Title: High-throughput micro-patterning platform reveals Nodal-dependent dissection of peri-gastrulation-associated versus pre-neurulation associated fate patterning

    Journal: bioRxiv

    doi: 10.1101/465039

    RD-like spatial oscillations of pre-neurulation-like fates detected when large geometrically confined hPSC colonies are treated with 200ng/ml BMP4 and SB in N2B27 medium. Representative immunofluorescent images of geometrically confined hPSC colonies of 3mm diameter stained for SOX2, and GATA3. The colonies were treated with 200ng/ml of BMP4 and SB for 48h. Scale bar represents 1mm.
    Figure Legend Snippet: RD-like spatial oscillations of pre-neurulation-like fates detected when large geometrically confined hPSC colonies are treated with 200ng/ml BMP4 and SB in N2B27 medium. Representative immunofluorescent images of geometrically confined hPSC colonies of 3mm diameter stained for SOX2, and GATA3. The colonies were treated with 200ng/ml of BMP4 and SB for 48h. Scale bar represents 1mm.

    Techniques Used: Staining

    Treatment of large geometrically confined hPSC colonies with 200ng/ml BMP4 and SB in N2B27 medium results in spatial oscillations of pSMAD1. A-B) Spatial oscillations of pSMAD1 expression detected with geometrically confined hPSC colonies of 3mm diameter were treated with 200ng/ml of BMP4 and SB for 24h in N2B27 medium. A) Stitched images of the entire colony stained for pSMAD1 shown in greyscale for ease of visibility. B) Enlarged fields that are indicated by white squares in A. White arrows indicate regions that contain cells with positive pSMAD1 expression. Scale bar represents 1mm.
    Figure Legend Snippet: Treatment of large geometrically confined hPSC colonies with 200ng/ml BMP4 and SB in N2B27 medium results in spatial oscillations of pSMAD1. A-B) Spatial oscillations of pSMAD1 expression detected with geometrically confined hPSC colonies of 3mm diameter were treated with 200ng/ml of BMP4 and SB for 24h in N2B27 medium. A) Stitched images of the entire colony stained for pSMAD1 shown in greyscale for ease of visibility. B) Enlarged fields that are indicated by white squares in A. White arrows indicate regions that contain cells with positive pSMAD1 expression. Scale bar represents 1mm.

    Techniques Used: Expressing, Staining

    Additional replicates of immunofluorescent images demonstrating oscillatory pSMAD1 expression in the center of large geometrically confined hPSC colonies treated with 200ng/ml BMP4 and SB in N2B27 medium. Additional representative images of 3mm diameter geometrically confined hPSC colonies treated with 200ng/ml BMP4 and SB in N2B27 medium for 24h and stained for pSMAD1. Zoomed-in images of fields contained within white squares shown adjacent to the stitched images. White arrows indicate regions that contain cells with positive pSMAD1 expression. Scale bar represents 1mm.
    Figure Legend Snippet: Additional replicates of immunofluorescent images demonstrating oscillatory pSMAD1 expression in the center of large geometrically confined hPSC colonies treated with 200ng/ml BMP4 and SB in N2B27 medium. Additional representative images of 3mm diameter geometrically confined hPSC colonies treated with 200ng/ml BMP4 and SB in N2B27 medium for 24h and stained for pSMAD1. Zoomed-in images of fields contained within white squares shown adjacent to the stitched images. White arrows indicate regions that contain cells with positive pSMAD1 expression. Scale bar represents 1mm.

    Techniques Used: Expressing, Staining

    Minor spatial oscillations of pre-neurulation-like fates detected when large geometrically confined hPSC colonies are treated with 50ng/ml BMP4 and SB in N2B27 medium. AB) Treatment of geometrically confined-hPSC colonies with 200ng/ml of BMP4 and SB for 48h results in RD-like periodic spatial oscillations of SOX2 and GATA3 expression. i) Representative stitched images of 3mm diameter hPSC colonies differentiated with 200ng/ml of BMP4 for 48h. Scale bar represents 1mm. ii) Zoomed section outlined by the white square in (i). The experiment was repeated two times.
    Figure Legend Snippet: Minor spatial oscillations of pre-neurulation-like fates detected when large geometrically confined hPSC colonies are treated with 50ng/ml BMP4 and SB in N2B27 medium. AB) Treatment of geometrically confined-hPSC colonies with 200ng/ml of BMP4 and SB for 48h results in RD-like periodic spatial oscillations of SOX2 and GATA3 expression. i) Representative stitched images of 3mm diameter hPSC colonies differentiated with 200ng/ml of BMP4 for 48h. Scale bar represents 1mm. ii) Zoomed section outlined by the white square in (i). The experiment was repeated two times.

    Techniques Used: Expressing

    Marginal spatial oscillations of pSMAD1 detected when large geometrically confined hPSC colonies are treated with 50ng/ml BMP4 and SB in N2B27 medium. A-B) Marginal spatial oscillations of pSMAD1 expression detected with geometrically confined hPSC colonies of 3mm diameter were treated with 50ng/ml of BMP4 and SB for 24h in N2B27 medium. A) Stitched images of the entire colony stained for pSMAD1 shown in greyscale for ease of visibility. B) Enlarged fields that are indicated by white squares in A. White arrows indicate regions that contain cells with positive pSMAD1 expression. Scale bar represents 1mm.
    Figure Legend Snippet: Marginal spatial oscillations of pSMAD1 detected when large geometrically confined hPSC colonies are treated with 50ng/ml BMP4 and SB in N2B27 medium. A-B) Marginal spatial oscillations of pSMAD1 expression detected with geometrically confined hPSC colonies of 3mm diameter were treated with 50ng/ml of BMP4 and SB for 24h in N2B27 medium. A) Stitched images of the entire colony stained for pSMAD1 shown in greyscale for ease of visibility. B) Enlarged fields that are indicated by white squares in A. White arrows indicate regions that contain cells with positive pSMAD1 expression. Scale bar represents 1mm.

    Techniques Used: Expressing, Staining

    11) Product Images from "A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages"

    Article Title: A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages

    Journal: Nature cell biology

    doi: 10.1038/ncb2742

    Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in N2B27 conditions with selection for geneticin (G418)
    Figure Legend Snippet: Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in N2B27 conditions with selection for geneticin (G418)

    Techniques Used: Cell Culture, Selection

    12) Product Images from "Pluripotent state transitions coordinate morphogenesis in mouse and human embryos"

    Article Title: Pluripotent state transitions coordinate morphogenesis in mouse and human embryos

    Journal: Nature

    doi: 10.1038/nature24675

    Exit from naive pluripotency is required for lumenogenesis in hES cells. a, Immunostaining of WIBR3 Δ PE–OCT4–GFP cells cultured in primed FBS/KSR/bFGF2 medium or N2B27 2i/hLIF with or without DOX. DOX addition induces the expression of NANOG and KLF2. Scale bars, 20 μm. b, c, mRNA levels of pluripotency genes in cells from a. n = 3 independent samples per condition. ANOVA; * * * P
    Figure Legend Snippet: Exit from naive pluripotency is required for lumenogenesis in hES cells. a, Immunostaining of WIBR3 Δ PE–OCT4–GFP cells cultured in primed FBS/KSR/bFGF2 medium or N2B27 2i/hLIF with or without DOX. DOX addition induces the expression of NANOG and KLF2. Scale bars, 20 μm. b, c, mRNA levels of pluripotency genes in cells from a. n = 3 independent samples per condition. ANOVA; * * * P

    Techniques Used: Immunostaining, Cell Culture, Expressing

    13) Product Images from "Dissecting signalling hierarchies in the patterning of the mouse primitive streak using micro-patterned EpiLC colonies"

    Article Title: Dissecting signalling hierarchies in the patterning of the mouse primitive streak using micro-patterned EpiLC colonies

    Journal: bioRxiv

    doi: 10.1101/2020.11.30.404418

    Characterization of EpiLC and differentiation of EpiSC on micropatterns A - EpiLC characterization 48h after replacement of 2i+LIF by in ACTIVIN+FGF homogenous stain for OCT4, ECAD, NANOG and SOX2. Representative image of n=7 pictures. B – qRT-PCR characterization of the expression of markers characteristic of naïve and formative pluripotency, for mESC maintained in N2B27+2i+LIF medium and for micropatterned EpiLC colonies 48h after initiation of their conversion in either N2B27+ACTIVIN+FGF+KSR (EpiLC). The EpiLC conversion is characterized by down-regulation of core pluripotency markers Nanog and Sox2 but not Pou5fl , upregulation of epiblast markers Fgf5 and Otx2 , while primitive endoderm markers such as Gata4 remains low, as previously reported in ( Hayashi et al., 2011 ) error bars: standart deviation of n=2 separate experiments C - EpiSC maintained in growth medium containing ACTIVIN and FGF (AF) are heterogeneous and display cluster of cells expressing anterior primitive streak markers BRA and SOX17. (top row) (patches of variable size were observed in 10/10 randomly selected fields). This heterogeneity is not observed after 3 passages if an inhibitor of WNT secretion (IWP2) is added to the growth medium (bottom row) (no BRA or SOX17 patches observed in 10/10 randomly selected fields) D - EpiSC maintained in ACTIVN +FGF medium display patchy differentiation after 48h of BMP4 differentiation on micro-patterned substrates (n=60 colonies, 2 replicates) E - EpiSC maintained in AF+IWP2 for 3 passages before being seeded on micropatterned substrates and differentiated for 48h in 50ng/ml BMP4 stimulation display a radial pattern of differentiation (left column). Right column: the ring of BRACHYURY is lost upon ACTIVIN/NODAL pathway inhibition with the small molecule inhibitor SB431542 (SB). Representative pictures of n=4 colonies for each condition. F - Comparison of gene expression temporal dynamics between EpiSC and EpiLC during BMP4 induced differentiation on micro-patterns. (Gene expression matrix and temporal trajectory along the first 2 principal components). Scale bars: 500μm
    Figure Legend Snippet: Characterization of EpiLC and differentiation of EpiSC on micropatterns A - EpiLC characterization 48h after replacement of 2i+LIF by in ACTIVIN+FGF homogenous stain for OCT4, ECAD, NANOG and SOX2. Representative image of n=7 pictures. B – qRT-PCR characterization of the expression of markers characteristic of naïve and formative pluripotency, for mESC maintained in N2B27+2i+LIF medium and for micropatterned EpiLC colonies 48h after initiation of their conversion in either N2B27+ACTIVIN+FGF+KSR (EpiLC). The EpiLC conversion is characterized by down-regulation of core pluripotency markers Nanog and Sox2 but not Pou5fl , upregulation of epiblast markers Fgf5 and Otx2 , while primitive endoderm markers such as Gata4 remains low, as previously reported in ( Hayashi et al., 2011 ) error bars: standart deviation of n=2 separate experiments C - EpiSC maintained in growth medium containing ACTIVIN and FGF (AF) are heterogeneous and display cluster of cells expressing anterior primitive streak markers BRA and SOX17. (top row) (patches of variable size were observed in 10/10 randomly selected fields). This heterogeneity is not observed after 3 passages if an inhibitor of WNT secretion (IWP2) is added to the growth medium (bottom row) (no BRA or SOX17 patches observed in 10/10 randomly selected fields) D - EpiSC maintained in ACTIVN +FGF medium display patchy differentiation after 48h of BMP4 differentiation on micro-patterned substrates (n=60 colonies, 2 replicates) E - EpiSC maintained in AF+IWP2 for 3 passages before being seeded on micropatterned substrates and differentiated for 48h in 50ng/ml BMP4 stimulation display a radial pattern of differentiation (left column). Right column: the ring of BRACHYURY is lost upon ACTIVIN/NODAL pathway inhibition with the small molecule inhibitor SB431542 (SB). Representative pictures of n=4 colonies for each condition. F - Comparison of gene expression temporal dynamics between EpiSC and EpiLC during BMP4 induced differentiation on micro-patterns. (Gene expression matrix and temporal trajectory along the first 2 principal components). Scale bars: 500μm

    Techniques Used: Staining, Quantitative RT-PCR, Expressing, Inhibition

    characterization of reproducibility of developmental trajectories (A) Gene expression matrix of multiple replicates obtained by qPCR 0/7/24/48/72h after BMP stimulation. Duplicates 1 2 belong to the same experiment. The replicate belongs to and independent experiment. Genes were clustered according to the similarity of their expression patterns in all replicates and 3 groups were defined based on the clustering. (B,) Temporal trajectory of the replicates along the first 2 principal components. (C,D) replicate experiment of figure 2 . (C) Gene expression matrix obtained by qRT-PCR 0/6/29/52/94h after various stimulations: B: BMP4, A:ACTIVIN, F:FGF2, W: WNT3a. BAFn and BAFo are two replicates of the same protocol but with cells with not the same passage number (n: 4 passages in N2B27+2i+LIF, o:27 passages in N2B27+2i+LIF) all other conditions are done with early passage cells
    Figure Legend Snippet: characterization of reproducibility of developmental trajectories (A) Gene expression matrix of multiple replicates obtained by qPCR 0/7/24/48/72h after BMP stimulation. Duplicates 1 2 belong to the same experiment. The replicate belongs to and independent experiment. Genes were clustered according to the similarity of their expression patterns in all replicates and 3 groups were defined based on the clustering. (B,) Temporal trajectory of the replicates along the first 2 principal components. (C,D) replicate experiment of figure 2 . (C) Gene expression matrix obtained by qRT-PCR 0/6/29/52/94h after various stimulations: B: BMP4, A:ACTIVIN, F:FGF2, W: WNT3a. BAFn and BAFo are two replicates of the same protocol but with cells with not the same passage number (n: 4 passages in N2B27+2i+LIF, o:27 passages in N2B27+2i+LIF) all other conditions are done with early passage cells

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    14) Product Images from "The Distinct Role of Tcfs and Lef1 in the Self-Renewal or Differentiation of Mouse Embryonic Stem Cells"

    Article Title: The Distinct Role of Tcfs and Lef1 in the Self-Renewal or Differentiation of Mouse Embryonic Stem Cells

    Journal: International Journal of Stem Cells

    doi: 10.15283/ijsc20044

    Ectopic expression of Tcf1 maintains self-renewal and delays differentiation of ES cells. (A) mRNA expressions for Oct4, Sox2 and Nanog at ES cells stage and Day 4 of differentiation upon TCF1 overexpression were determined by using qPCR. RNA was isolated from cells expressing empty vector (EV) or Tcf1-FL (TCF1) on specified stage. GAPDH was used for normalization. (B) The amount of Nanog transcript was measured by RT-PCR analysis. RNA was isolated from ES cells expressing control vector or Tcf1-FL on the days specified. 18s rRNA was used as a loading control. (C) ChIP assay was performed to examine the binding of Tcf1, Lef1, and Tcf3 on Nanog promoter. Samples for ChIP were isolated from ES cells grown with LIF and from ES cells expressing control vector or Tcf1-FL cultured for 2 days without LIF. For immunoprecipitiation, endogenous antibody against Tcf1, Lef1, or Tcf3 was used. (D) To differentiate into neural precursors, 46C ES cells expressing control vector, Tcf1-FL, or Tcf1-DN were plated for 6 days in N2B27 medium. GFP fluorescence showed that 46C ES cells expressing control vector or Tcf1-DN, but not Tcf1-FL effectively differentiated into neural precursor (upper panel). The amount of Nanog or Sox1 transcript on the days indicated was measured via RT-PCR analysis (lower panel). β -actin was used as a loading control. (E) RNA samples were isolated from A6P10 ES cells expressing control vector or Tcf1-FL on the days indicated after EB formation. Self-renewal (Nanog), mesoderm (Brachyury), and cardiomyocyte (Islet1, α MHC) markers were used for RT-PCR analysis.
    Figure Legend Snippet: Ectopic expression of Tcf1 maintains self-renewal and delays differentiation of ES cells. (A) mRNA expressions for Oct4, Sox2 and Nanog at ES cells stage and Day 4 of differentiation upon TCF1 overexpression were determined by using qPCR. RNA was isolated from cells expressing empty vector (EV) or Tcf1-FL (TCF1) on specified stage. GAPDH was used for normalization. (B) The amount of Nanog transcript was measured by RT-PCR analysis. RNA was isolated from ES cells expressing control vector or Tcf1-FL on the days specified. 18s rRNA was used as a loading control. (C) ChIP assay was performed to examine the binding of Tcf1, Lef1, and Tcf3 on Nanog promoter. Samples for ChIP were isolated from ES cells grown with LIF and from ES cells expressing control vector or Tcf1-FL cultured for 2 days without LIF. For immunoprecipitiation, endogenous antibody against Tcf1, Lef1, or Tcf3 was used. (D) To differentiate into neural precursors, 46C ES cells expressing control vector, Tcf1-FL, or Tcf1-DN were plated for 6 days in N2B27 medium. GFP fluorescence showed that 46C ES cells expressing control vector or Tcf1-DN, but not Tcf1-FL effectively differentiated into neural precursor (upper panel). The amount of Nanog or Sox1 transcript on the days indicated was measured via RT-PCR analysis (lower panel). β -actin was used as a loading control. (E) RNA samples were isolated from A6P10 ES cells expressing control vector or Tcf1-FL on the days indicated after EB formation. Self-renewal (Nanog), mesoderm (Brachyury), and cardiomyocyte (Islet1, α MHC) markers were used for RT-PCR analysis.

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Isolation, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Cell Culture, Fluorescence

    The pattern of Tcfs and Lef1 expression in ES cells. (A) A6P10 ES cells were differentiated in the absence of LIF (left), in N2B27 medium without LIF (middle), or via EB formation without LIF (right). The differentiated ES cells were harvested on the days indicated. The amount of Tcfs and Lef1 transcripts was measured by RT-PCR analysis. The bands for Tcfs and Lef1 represent wild-type transcripts including β -catenin-binding domain. Self-renewal marker (Nanog), neural precursor markers (Sox1 and Fgf5) for N2B27 differentiation and mesodermal markers (Brachyury, Nkx2.5, α MHC) for EB differentiation were used to confirm proper differentiation; 18s rRNA was used as a loading control. (B) The endogenous protein levels of Tcfs/Lef1 were detected by western blotting. β -actin was used as a loading control.
    Figure Legend Snippet: The pattern of Tcfs and Lef1 expression in ES cells. (A) A6P10 ES cells were differentiated in the absence of LIF (left), in N2B27 medium without LIF (middle), or via EB formation without LIF (right). The differentiated ES cells were harvested on the days indicated. The amount of Tcfs and Lef1 transcripts was measured by RT-PCR analysis. The bands for Tcfs and Lef1 represent wild-type transcripts including β -catenin-binding domain. Self-renewal marker (Nanog), neural precursor markers (Sox1 and Fgf5) for N2B27 differentiation and mesodermal markers (Brachyury, Nkx2.5, α MHC) for EB differentiation were used to confirm proper differentiation; 18s rRNA was used as a loading control. (B) The endogenous protein levels of Tcfs/Lef1 were detected by western blotting. β -actin was used as a loading control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Marker, Western Blot

    Transient expression of Lef1 is required for proper differentiation. (A) A6P10 ES cells were infected with retroviruses expressing control or Lef1 shRNA, followed by selection using puromycin. After 2 days without LIF, the selected ES cells were harvested and analyzed by western blotting with anti-Lef1 antibody. β -actin was used as a loading control. (B) 46C ES cells expressing control shRNA or Lef1 shRNA were plated for 7 days in N2B27 medium. GFP fluorescence indicating a neural precursor was not induced in shLef1-46C cells (left panel). The amount of Nanog or Sox1 transcript was measured by RT-PCR analysis (right panel). (C) 46C ES cells were transfected with control vector, Lef1-FL, or Lef1-DN and then selected using Zeocin. 46C ES cells expressing each vector were plated for 7 days in N2B27 medium and GFP fluorescence was captured. (D) RNA samples were isolated from A6P10 ES cells expressing shGFP or shLef1 on the days indicated post-EB formation. Cardiomyocyte ( α -MHC) and endoderm (GATA4) markers and loading control (18s rRNA) were used for RT-PCR analysis.
    Figure Legend Snippet: Transient expression of Lef1 is required for proper differentiation. (A) A6P10 ES cells were infected with retroviruses expressing control or Lef1 shRNA, followed by selection using puromycin. After 2 days without LIF, the selected ES cells were harvested and analyzed by western blotting with anti-Lef1 antibody. β -actin was used as a loading control. (B) 46C ES cells expressing control shRNA or Lef1 shRNA were plated for 7 days in N2B27 medium. GFP fluorescence indicating a neural precursor was not induced in shLef1-46C cells (left panel). The amount of Nanog or Sox1 transcript was measured by RT-PCR analysis (right panel). (C) 46C ES cells were transfected with control vector, Lef1-FL, or Lef1-DN and then selected using Zeocin. 46C ES cells expressing each vector were plated for 7 days in N2B27 medium and GFP fluorescence was captured. (D) RNA samples were isolated from A6P10 ES cells expressing shGFP or shLef1 on the days indicated post-EB formation. Cardiomyocyte ( α -MHC) and endoderm (GATA4) markers and loading control (18s rRNA) were used for RT-PCR analysis.

    Techniques Used: Expressing, Infection, shRNA, Selection, Western Blot, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Isolation

    15) Product Images from "Pluripotent state transitions coordinate morphogenesis in mouse and human embryos"

    Article Title: Pluripotent state transitions coordinate morphogenesis in mouse and human embryos

    Journal: Nature

    doi: 10.1038/nature24675

    Exit from naive pluripotency is required for lumenogenesis in hES cells. a, Immunostaining of WIBR3 Δ PE–OCT4–GFP cells cultured in primed FBS/KSR/bFGF2 medium or N2B27 2i/hLIF with or without DOX. DOX addition induces the expression of NANOG and KLF2. Scale bars, 20 μm. b, c, mRNA levels of pluripotency genes in cells from a. n = 3 independent samples per condition. ANOVA; * * * P
    Figure Legend Snippet: Exit from naive pluripotency is required for lumenogenesis in hES cells. a, Immunostaining of WIBR3 Δ PE–OCT4–GFP cells cultured in primed FBS/KSR/bFGF2 medium or N2B27 2i/hLIF with or without DOX. DOX addition induces the expression of NANOG and KLF2. Scale bars, 20 μm. b, c, mRNA levels of pluripotency genes in cells from a. n = 3 independent samples per condition. ANOVA; * * * P

    Techniques Used: Immunostaining, Cell Culture, Expressing

    16) Product Images from "An alternative pluripotent state confers interspecies chimaeric competency"

    Article Title: An alternative pluripotent state confers interspecies chimaeric competency

    Journal: Nature

    doi: 10.1038/nature14413

    Mechanistic studies of rsEpiSC derivation Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a , In N2B27 media with indicated treatments. NT, no treatment. After 4 days, outgrowths of plated epiblasts were stained with endodermal marker FOXA2, mesodermal marker T, neuroectodermal marker SOX2 and pluripotent marker OCT4. b , In N2B27 media supplemented with either 20% FBS (top) or 20% KnockOut serum replacement (KSR; bottom). Day 4 outgrowths were fixed and stained with mesodermal marker T and pluripotency marker OCT4. Nuclei were counterstained with DAPI. Scale bar, 125 µm.
    Figure Legend Snippet: Mechanistic studies of rsEpiSC derivation Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a , In N2B27 media with indicated treatments. NT, no treatment. After 4 days, outgrowths of plated epiblasts were stained with endodermal marker FOXA2, mesodermal marker T, neuroectodermal marker SOX2 and pluripotent marker OCT4. b , In N2B27 media supplemented with either 20% FBS (top) or 20% KnockOut serum replacement (KSR; bottom). Day 4 outgrowths were fixed and stained with mesodermal marker T and pluripotency marker OCT4. Nuclei were counterstained with DAPI. Scale bar, 125 µm.

    Techniques Used: Isolation, Staining, Marker, Knock-Out

    The effects of culture parameters on epiblast explants Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a , In EpiSC derivation medium containing 20% KnockOut serum replacement (KSR), 20 ng ml −1 Activin-A and 12 ng ml −1 FGF2. Day 3 outgrowth was stained with pluripotency markers OCT4 and SSEA-1. b , In N2B27 media supplemented with indicated growth factors and small molecules. After 4 days, outgrowths of plated epiblasts were stained forOCT4 and SSEA-1. c , Percentages of SSEA-1 + /OCT4 + cells in day 4 epiblast outgrowths in N2B27 KSR+F/A and N2B27 F/R1 culture conditions. A simple randomization method was applied to randomly pick the microscope fields of views for counting the number of SSEA-1 + /OCT4 + cells and total cell numbers. PC, phase contrast. For examining different culture parameter effects, all isolated E5.75 epiblasts were pooled together and randomly allocated to each condition.
    Figure Legend Snippet: The effects of culture parameters on epiblast explants Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a , In EpiSC derivation medium containing 20% KnockOut serum replacement (KSR), 20 ng ml −1 Activin-A and 12 ng ml −1 FGF2. Day 3 outgrowth was stained with pluripotency markers OCT4 and SSEA-1. b , In N2B27 media supplemented with indicated growth factors and small molecules. After 4 days, outgrowths of plated epiblasts were stained forOCT4 and SSEA-1. c , Percentages of SSEA-1 + /OCT4 + cells in day 4 epiblast outgrowths in N2B27 KSR+F/A and N2B27 F/R1 culture conditions. A simple randomization method was applied to randomly pick the microscope fields of views for counting the number of SSEA-1 + /OCT4 + cells and total cell numbers. PC, phase contrast. For examining different culture parameter effects, all isolated E5.75 epiblasts were pooled together and randomly allocated to each condition.

    Techniques Used: Isolation, Knock-Out, Staining, Microscopy

    Mechanistic studies of rsEpiSCs self-renewal a , Phase-contrast images showing colony morphologies of rsEpiSCs after 4 days of indicated treatments. Left, N2B27 media alone; right, N2B27 F/R1 . b , Quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), endodermal ( Sox17 and Gsc ), mesodermal ( Eomes and Mixl1 ) and neuroectodermal ( Sox1 and Pax6 ) markers after indicated treatments for 4 days in culture. Error bars indicate s.d. ( n = 3, biological replicates). c , Schematic representation of how different signalling pathways are involved in promoting or inhibiting self-renewal of rsEpiSCs.
    Figure Legend Snippet: Mechanistic studies of rsEpiSCs self-renewal a , Phase-contrast images showing colony morphologies of rsEpiSCs after 4 days of indicated treatments. Left, N2B27 media alone; right, N2B27 F/R1 . b , Quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), endodermal ( Sox17 and Gsc ), mesodermal ( Eomes and Mixl1 ) and neuroectodermal ( Sox1 and Pax6 ) markers after indicated treatments for 4 days in culture. Error bars indicate s.d. ( n = 3, biological replicates). c , Schematic representation of how different signalling pathways are involved in promoting or inhibiting self-renewal of rsEpiSCs.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    17) Product Images from "Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells"

    Article Title: Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells

    Journal: Stem Cells International

    doi: 10.1155/2015/459301

    Decrease in neural differentiation of precursor cells by an Axin stabilizer (IWR-1-endo) from days 4 to 6 in N2B27 medium. (a)–(f) GFP expression of 46C cells was elevated by BIO treatment (0.75 μ M) from days 4 to 6, whereas it was reduced by IWR-1-endo (0.75 μ M) treatment from days 4 to 6. Cells were cultured in N2B27 medium for 6 days. (g) FACS analysis of Sox1 -GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1 -GFP expressing cells as determined by FACS. Scale bars, 100 μ m.
    Figure Legend Snippet: Decrease in neural differentiation of precursor cells by an Axin stabilizer (IWR-1-endo) from days 4 to 6 in N2B27 medium. (a)–(f) GFP expression of 46C cells was elevated by BIO treatment (0.75 μ M) from days 4 to 6, whereas it was reduced by IWR-1-endo (0.75 μ M) treatment from days 4 to 6. Cells were cultured in N2B27 medium for 6 days. (g) FACS analysis of Sox1 -GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1 -GFP expressing cells as determined by FACS. Scale bars, 100 μ m.

    Techniques Used: Expressing, Cell Culture, FACS

    Increase in canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β -catenin) and β -actin antibodies. Active β -catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4- Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μ M). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2 p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μ m.
    Figure Legend Snippet: Increase in canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β -catenin) and β -actin antibodies. Active β -catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4- Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μ M). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2 p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μ m.

    Techniques Used: Luciferase, Western Blot, Expressing, Stable Transfection, Cell Culture

    Increase in neural differentiation of precursor cells by GSK3 β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μ M) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μ M) from days 0 to 3. (g) FACS analysis of Sox1 -GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1 -GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μ M) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μ m.
    Figure Legend Snippet: Increase in neural differentiation of precursor cells by GSK3 β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μ M) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μ M) from days 0 to 3. (g) FACS analysis of Sox1 -GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1 -GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μ M) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μ m.

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

    Neuronal differentiation of mouse embryonic stem cells and expression of Wnt and Wnt component genes. (a)–(e) Using N2B27 medium, 46C ES cells ( Sox1 -promoter-GFP) were differentiated to neuronal cells for 14 days. GFP in 46C ES cells was expressed during neuronal differentiation for 6 days ((a) phase contrast, (b) Sox1-GFP), and neurite outgrowth increased during neuronal differentiation for 8 days (c) and 14 days ((d) phase contrast, (e) MAPII (red), DAPI (blue)). (f) RT-PCR results of stem cell marker, Wnt, and Wnt signaling components during neuronal differentiation. During neuronal differentiation, RNA levels of many Wnt and Wnt signaling components were changed. Scale bars, 50 μ m.
    Figure Legend Snippet: Neuronal differentiation of mouse embryonic stem cells and expression of Wnt and Wnt component genes. (a)–(e) Using N2B27 medium, 46C ES cells ( Sox1 -promoter-GFP) were differentiated to neuronal cells for 14 days. GFP in 46C ES cells was expressed during neuronal differentiation for 6 days ((a) phase contrast, (b) Sox1-GFP), and neurite outgrowth increased during neuronal differentiation for 8 days (c) and 14 days ((d) phase contrast, (e) MAPII (red), DAPI (blue)). (f) RT-PCR results of stem cell marker, Wnt, and Wnt signaling components during neuronal differentiation. During neuronal differentiation, RNA levels of many Wnt and Wnt signaling components were changed. Scale bars, 50 μ m.

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

    18) Product Images from "YAP Induces Human Naive Pluripotency"

    Article Title: YAP Induces Human Naive Pluripotency

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.02.036

    LPA Can Activate YAP and Promotes a Naive State of Pluripotency (A) LPA increases the level of YAP in human H9 ESCs, as shown by immunofluorescence. Immunostaining was performed 2 days after adding LPA. Green, YAP; blue, DAPI. Scale bar, 50 μm. (B) Western blotting confirming that LPA increases YAP protein levels. Tubulin was used as loading control. (C) H9 cells in N2B27+2iFL+LPA naive medium have a naive-specific dome-like colony morphology and show strong positive immunostaining for pluripotency markers SSEA3, SSEA4, TRA-1-60, and TRA-1-81. Black scale bar, 500 μm; white scale bar, 150 μm. (D) H9-YAP cells in N2B27+2iFL are able to form teratomas comprising tissues derived from all three germ layers. (a) Gut-like epithelium (endoderm). (b) Cartilage (mesoderm). (c) Neural tissue (ectoderm). White scale bar, 200 μm; black scale bar, 50 μm.
    Figure Legend Snippet: LPA Can Activate YAP and Promotes a Naive State of Pluripotency (A) LPA increases the level of YAP in human H9 ESCs, as shown by immunofluorescence. Immunostaining was performed 2 days after adding LPA. Green, YAP; blue, DAPI. Scale bar, 50 μm. (B) Western blotting confirming that LPA increases YAP protein levels. Tubulin was used as loading control. (C) H9 cells in N2B27+2iFL+LPA naive medium have a naive-specific dome-like colony morphology and show strong positive immunostaining for pluripotency markers SSEA3, SSEA4, TRA-1-60, and TRA-1-81. Black scale bar, 500 μm; white scale bar, 150 μm. (D) H9-YAP cells in N2B27+2iFL are able to form teratomas comprising tissues derived from all three germ layers. (a) Gut-like epithelium (endoderm). (b) Cartilage (mesoderm). (c) Neural tissue (ectoderm). White scale bar, 200 μm; black scale bar, 50 μm.

    Techniques Used: Immunofluorescence, Immunostaining, Western Blot, Derivative Assay

    YAP Overexpression Promotes the Generation of Human Naive ESCs (A) YAP protein is localized to the nucleus in both the trophectoderm and the ICM of human blastocysts, as shown by immunofluorescence. White, DAPI; green, OCT4 (ICM); yellow, CDX2 (trophectoderm); red, YAP. Representative image of n = 16 human blastocysts. Scale bar, 20 μm. (B) In naive medium N2B27+2iFL, human H9 ESCs with YAP overexpression (H9-YAP) maintain expression of the pluripotency marker AP, while control H9 cells differentiate and do not express AP. Scale bar, 500 μm. (C) H9-YAP cells in N2B27+2iFL have a naive-specific dome-like colony morphology and show strong positive immunostaining for pluripotency markers SSEA3, SSEA4, TRA-1-60, TRA-1-81, OCT4, and NANOG. Black scale bar, 500 mm; white scale bar, 150 μm. (D) Growth curves of H9 in DF12+bFGF primed medium, H9-YAP in DF12+bFGF primed medium, and H9-YAP in N2B27+2iFL naive medium after trypsinization to single cells. Only H9-YAP cells in naive medium have a high proliferate rate. Error bars represent SD. (E) H9-YAP cells in N2B27+2iFL have a normal female karyotype (46, XX), as evaluated after 20 passages in naive culture conditions. (F) H9-YAP cells in N2B27+2iFL are able to form teratomas comprising tissues derived from all three germ layers. (a) Adipocytes (mesoderm). (b) Neural tissue (ectoderm). (c) Epithelium (endoderm). White scale bar, 200 μm; black scale bar, 50 μm.
    Figure Legend Snippet: YAP Overexpression Promotes the Generation of Human Naive ESCs (A) YAP protein is localized to the nucleus in both the trophectoderm and the ICM of human blastocysts, as shown by immunofluorescence. White, DAPI; green, OCT4 (ICM); yellow, CDX2 (trophectoderm); red, YAP. Representative image of n = 16 human blastocysts. Scale bar, 20 μm. (B) In naive medium N2B27+2iFL, human H9 ESCs with YAP overexpression (H9-YAP) maintain expression of the pluripotency marker AP, while control H9 cells differentiate and do not express AP. Scale bar, 500 μm. (C) H9-YAP cells in N2B27+2iFL have a naive-specific dome-like colony morphology and show strong positive immunostaining for pluripotency markers SSEA3, SSEA4, TRA-1-60, TRA-1-81, OCT4, and NANOG. Black scale bar, 500 mm; white scale bar, 150 μm. (D) Growth curves of H9 in DF12+bFGF primed medium, H9-YAP in DF12+bFGF primed medium, and H9-YAP in N2B27+2iFL naive medium after trypsinization to single cells. Only H9-YAP cells in naive medium have a high proliferate rate. Error bars represent SD. (E) H9-YAP cells in N2B27+2iFL have a normal female karyotype (46, XX), as evaluated after 20 passages in naive culture conditions. (F) H9-YAP cells in N2B27+2iFL are able to form teratomas comprising tissues derived from all three germ layers. (a) Adipocytes (mesoderm). (b) Neural tissue (ectoderm). (c) Epithelium (endoderm). White scale bar, 200 μm; black scale bar, 50 μm.

    Techniques Used: Over Expression, Immunofluorescence, Expressing, Marker, Immunostaining, Derivative Assay

    Yin-PSCs and Lin-PSCs Have a Naive-like Transcriptional Profile Distinct from that of Primed PSCs (A) Primed PSCs and Yin- and Lin-PSCs have distinct gene expression profiles, as shown by unsupervised hierarchical clustering. The top 1,000 genes with the highest coefficient of variation were used to cluster samples using Pearson correlation coefficients. Six human primed PSC samples include H9, H1, IMR-90 iPSCs, H9-YAP, H1-YAP, and IMR-90 iPSC-YAP cells. Six human naive PSC samples include H9-YAP in N2B27+2iFL (H9-YAP N naive), H9-YAP in mTeSR+2iFL (H9-YAP T naive), H1-YAP in mTeSR+2iFL (H1-YAP T naive), IMR-90 iPSC-YAP cells in mTeSR+2iFL (iPSC-YAP T naive), H9 in mTeSR+2iFL+LPA (H9 T+LPA naive), and WIBR3 in mTeSR+2iFL+LPA (WIBR3 T+LPA naive). Primed cells are indicated in blue. H9-YAP in N2B27+2iFL naive medium is in pink, and all other naive cells are in red. (B) GSEA reveals that Yin-PSCs and Lin-PSCs have gene expression profiles concordant with naive PSCs from other studies. The upper panel is the enrichment plot for the gene set of Theunissen et al. (2014) upregulated in naive by Log 2 FC > 3.0. The lower panel is the enrichment plot for the gene set of Takahashi et al. (2014) upregulated in naive by Log 2 FC > 2.0. Vertical black bars represent the position of genes upregulated in naive cells in the Takashima et al. (2014) or Theunissen et al. (2014) studies, distributed along the differential expression values for the entire transcriptome in this study, and ranked from upregulated in Yin-PSCs and Lin-PSCs (red, left) to upregulated in parental primed PSCs (blue, right). (C) Yin-PSCs and Lin-PSCs express markers specific for naive pluripotency, as confirmed by qRT-PCR. Values were normalized to GAPDH and UBB and then compared to H9 in primed medium (left panel) or H1 in primed medium (right panel). Data are averages of triplicate PCR reactions, and error bars represent SD. (D) Hierarchical clustering shows that naive Yin-PSCs and Lin-PSCs are similar to naive cells from two other studies ( Takashima et al., 2014 ; Theunissen et al., 2014 ) and to human pre-implantation embryos. In vivo states at various developmental stages ( Yan et al., 2013 ) are included in the clustering. (E) Volcano plot of significantly differentially expressed genes among the six human naive PSC samples and the six human primed PSC samples. Highlighted in red are genes with aLog 2 FC > 0.7 in naive and p
    Figure Legend Snippet: Yin-PSCs and Lin-PSCs Have a Naive-like Transcriptional Profile Distinct from that of Primed PSCs (A) Primed PSCs and Yin- and Lin-PSCs have distinct gene expression profiles, as shown by unsupervised hierarchical clustering. The top 1,000 genes with the highest coefficient of variation were used to cluster samples using Pearson correlation coefficients. Six human primed PSC samples include H9, H1, IMR-90 iPSCs, H9-YAP, H1-YAP, and IMR-90 iPSC-YAP cells. Six human naive PSC samples include H9-YAP in N2B27+2iFL (H9-YAP N naive), H9-YAP in mTeSR+2iFL (H9-YAP T naive), H1-YAP in mTeSR+2iFL (H1-YAP T naive), IMR-90 iPSC-YAP cells in mTeSR+2iFL (iPSC-YAP T naive), H9 in mTeSR+2iFL+LPA (H9 T+LPA naive), and WIBR3 in mTeSR+2iFL+LPA (WIBR3 T+LPA naive). Primed cells are indicated in blue. H9-YAP in N2B27+2iFL naive medium is in pink, and all other naive cells are in red. (B) GSEA reveals that Yin-PSCs and Lin-PSCs have gene expression profiles concordant with naive PSCs from other studies. The upper panel is the enrichment plot for the gene set of Theunissen et al. (2014) upregulated in naive by Log 2 FC > 3.0. The lower panel is the enrichment plot for the gene set of Takahashi et al. (2014) upregulated in naive by Log 2 FC > 2.0. Vertical black bars represent the position of genes upregulated in naive cells in the Takashima et al. (2014) or Theunissen et al. (2014) studies, distributed along the differential expression values for the entire transcriptome in this study, and ranked from upregulated in Yin-PSCs and Lin-PSCs (red, left) to upregulated in parental primed PSCs (blue, right). (C) Yin-PSCs and Lin-PSCs express markers specific for naive pluripotency, as confirmed by qRT-PCR. Values were normalized to GAPDH and UBB and then compared to H9 in primed medium (left panel) or H1 in primed medium (right panel). Data are averages of triplicate PCR reactions, and error bars represent SD. (D) Hierarchical clustering shows that naive Yin-PSCs and Lin-PSCs are similar to naive cells from two other studies ( Takashima et al., 2014 ; Theunissen et al., 2014 ) and to human pre-implantation embryos. In vivo states at various developmental stages ( Yan et al., 2013 ) are included in the clustering. (E) Volcano plot of significantly differentially expressed genes among the six human naive PSC samples and the six human primed PSC samples. Highlighted in red are genes with aLog 2 FC > 0.7 in naive and p

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, In Vivo

    YAP Regulates the Human Naive State and Acts in Part by Modulating Wnt Signaling (A) H3K9me3 is strongly reduced in naive H9 ESCs, as seen by immunofluorescence. Upper panel: H9 and H9-YAP in DF12+bFGF primed medium. Lower panel: H9-YAP in N2B27+2iFL naive medium, H9-YAP in mTeSR+2iFL naive medium, and H9 in mTeSR+2iFL+LPA naive medium. Scale bar, 20 μm. (B) Decreased total amount of H3K9me3 in Yin-PSCs and Lin-PSCs was confirmed by western blotting. Tubulin was used as loading control. Values indicate densitometry analysis of the H3K9me3 level normalized to tubulin. (C) Flow cytometric analysis of the proportion of OCT4-ΔPE-GFP+ WIBR3 cells with or without YAP overexpression in 5i/L/A (upper panels) or mTeSR+2iFL (lower panels) media. Cells were expanded in bulk and analyzed at passage 3, in the absence of colony picking. YAP overexpression increases the ratio of OCT4-ΔPE-GFP + cells in both media. (D) YAP overexpression decreases levels of unphosphorylated (active) β-catenin, as shown by western blotting. Tubulin was used as loading control. (E) YAP overexpression decreases the expression of Wnt target genes, as shown by qRT-PCR. Values were normalized to GAPDH and UBB and then compared to H9. Data are averages of triplicate PCR reactions, and error bars represent SD. (F) YAP knockout impairs the ability of ESCs to form naive colonies. H9 controls and four clones of CRISPR/Cas9-generated YAP −/− cells cultured in DF12+bFGF (primed), mTeSR+2iFL+LPA (naive), and 5i/L/A (naive, Theunissen et al., 2014 ) were trypsinized to single cells, counted, and plated onto MEFs in the presence of ROCK inhibitor. Seven days later, AP staining was performed and colony numbers were counted. Error bars represent SD. *p
    Figure Legend Snippet: YAP Regulates the Human Naive State and Acts in Part by Modulating Wnt Signaling (A) H3K9me3 is strongly reduced in naive H9 ESCs, as seen by immunofluorescence. Upper panel: H9 and H9-YAP in DF12+bFGF primed medium. Lower panel: H9-YAP in N2B27+2iFL naive medium, H9-YAP in mTeSR+2iFL naive medium, and H9 in mTeSR+2iFL+LPA naive medium. Scale bar, 20 μm. (B) Decreased total amount of H3K9me3 in Yin-PSCs and Lin-PSCs was confirmed by western blotting. Tubulin was used as loading control. Values indicate densitometry analysis of the H3K9me3 level normalized to tubulin. (C) Flow cytometric analysis of the proportion of OCT4-ΔPE-GFP+ WIBR3 cells with or without YAP overexpression in 5i/L/A (upper panels) or mTeSR+2iFL (lower panels) media. Cells were expanded in bulk and analyzed at passage 3, in the absence of colony picking. YAP overexpression increases the ratio of OCT4-ΔPE-GFP + cells in both media. (D) YAP overexpression decreases levels of unphosphorylated (active) β-catenin, as shown by western blotting. Tubulin was used as loading control. (E) YAP overexpression decreases the expression of Wnt target genes, as shown by qRT-PCR. Values were normalized to GAPDH and UBB and then compared to H9. Data are averages of triplicate PCR reactions, and error bars represent SD. (F) YAP knockout impairs the ability of ESCs to form naive colonies. H9 controls and four clones of CRISPR/Cas9-generated YAP −/− cells cultured in DF12+bFGF (primed), mTeSR+2iFL+LPA (naive), and 5i/L/A (naive, Theunissen et al., 2014 ) were trypsinized to single cells, counted, and plated onto MEFs in the presence of ROCK inhibitor. Seven days later, AP staining was performed and colony numbers were counted. Error bars represent SD. *p

    Techniques Used: Immunofluorescence, Western Blot, Flow Cytometry, Over Expression, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Knock-Out, Clone Assay, CRISPR, Generated, Cell Culture, Staining

    19) Product Images from "A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning"

    Article Title: A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.149658

    Nodal signaling is required for primitive streak specification, but does not induce differentiation and fate patterning in geometrically confined hPSC colonies. (A) Percentage of cells expressing BRA, SOX2 and CDX2 in N2B27 with 100 ng/ml of NODAL ( n =73), N2B27+BMP ( n =100), N2B27+BMP+LDN ( n =101), N2B27+BMP+SB ( n =67), N2B27+100 ng/ml Nodal ( n =68) or N2B27+200 ng/ml Nodal ( n =65). The experiment was performed twice. Data are mean±s.d. and individual data points indicate identified colonies. **** P
    Figure Legend Snippet: Nodal signaling is required for primitive streak specification, but does not induce differentiation and fate patterning in geometrically confined hPSC colonies. (A) Percentage of cells expressing BRA, SOX2 and CDX2 in N2B27 with 100 ng/ml of NODAL ( n =73), N2B27+BMP ( n =100), N2B27+BMP+LDN ( n =101), N2B27+BMP+SB ( n =67), N2B27+100 ng/ml Nodal ( n =68) or N2B27+200 ng/ml Nodal ( n =65). The experiment was performed twice. Data are mean±s.d. and individual data points indicate identified colonies. **** P

    Techniques Used: Expressing

    Defined peri-gastrulation-like patterning induction in differentiating hPSC colonies. (A,B) Representative immunofluorescence images of fate patterning of SOX2, BRA and CDX2 in BMP4-supplemented CM (A) as previously reported ( Warmflash et al., 2014 ; Etoc et al., 2016 ), and fate patterning in BMP4-supplemented N2B27 medium stained for SOX2, BRA, CDX2, SOX17 and EOMES (B). (C) Spatial trends for intensity of expression of SOX2, BRA (T), CDX2 and SOX17 in regions marked by white rectangles in B and C (average trends of replicates shown in Figs S1 and S2 ). (D) Representative images of SNAIL and EPCAM staining in a micro-patterned colony differentiated in N2B27 shows that mesenchymal marker-expressing cells in the primitive streak region are located underneath an epithelial layer. Scale bars: 50 µm.
    Figure Legend Snippet: Defined peri-gastrulation-like patterning induction in differentiating hPSC colonies. (A,B) Representative immunofluorescence images of fate patterning of SOX2, BRA and CDX2 in BMP4-supplemented CM (A) as previously reported ( Warmflash et al., 2014 ; Etoc et al., 2016 ), and fate patterning in BMP4-supplemented N2B27 medium stained for SOX2, BRA, CDX2, SOX17 and EOMES (B). (C) Spatial trends for intensity of expression of SOX2, BRA (T), CDX2 and SOX17 in regions marked by white rectangles in B and C (average trends of replicates shown in Figs S1 and S2 ). (D) Representative images of SNAIL and EPCAM staining in a micro-patterned colony differentiated in N2B27 shows that mesenchymal marker-expressing cells in the primitive streak region are located underneath an epithelial layer. Scale bars: 50 µm.

    Techniques Used: Immunofluorescence, Staining, Expressing, Marker

    20) Product Images from "Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics"

    Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2003127

    Characterization of MN differentiation from ESCs. (A) Scheme outlining the differentiation protocol. ESCs are plated in N2B27 + FGF for 2 days before being exposed to N2B27 + FGF/CHIR, resulting in the production of NMPs at day 3. Cells are subsequently exposed to RA and SAG to promote differentiation into ventral NPs and MNs. (B, C) Expression of NP (Pax6, Olig2, Nkx2.2, Sox1) and MN (Isl1/2) markers between day 4 and day 7 in differentiating ESCs. (D) RT-qPCR analysis of Irx3 , Pax6 , Nkx6 . 1 , Olig2 , and Nkx2 . 2 expression from day 3 to day 7 reveals progressive ventralization in response to increasing duration of Shh signaling. Underlying data are provided in S1 Data . (E) MN induction after day 5, revealed by RT-qPCR analysis of Sox1 , Ngn2 , Isl1 , and Tubb3 . Underlying data are provided in S1 Data . Scale bars = 40 μm. CHIR, Wnt pathway activator CHIR99021; ESC, embryonic stem cell; FGF, fibroblast growth factor 2; MN, motor neuron; NMP, neuromesodermal progenitor; NP, neural progenitor; N2B27, N2 and B27 media supplements; RA, retinoic acid; RT-qPCR, real-time quantitative polymerase chain reaction; SAG, Smoothened/Shh signalling agonist.
    Figure Legend Snippet: Characterization of MN differentiation from ESCs. (A) Scheme outlining the differentiation protocol. ESCs are plated in N2B27 + FGF for 2 days before being exposed to N2B27 + FGF/CHIR, resulting in the production of NMPs at day 3. Cells are subsequently exposed to RA and SAG to promote differentiation into ventral NPs and MNs. (B, C) Expression of NP (Pax6, Olig2, Nkx2.2, Sox1) and MN (Isl1/2) markers between day 4 and day 7 in differentiating ESCs. (D) RT-qPCR analysis of Irx3 , Pax6 , Nkx6 . 1 , Olig2 , and Nkx2 . 2 expression from day 3 to day 7 reveals progressive ventralization in response to increasing duration of Shh signaling. Underlying data are provided in S1 Data . (E) MN induction after day 5, revealed by RT-qPCR analysis of Sox1 , Ngn2 , Isl1 , and Tubb3 . Underlying data are provided in S1 Data . Scale bars = 40 μm. CHIR, Wnt pathway activator CHIR99021; ESC, embryonic stem cell; FGF, fibroblast growth factor 2; MN, motor neuron; NMP, neuromesodermal progenitor; NP, neural progenitor; N2B27, N2 and B27 media supplements; RA, retinoic acid; RT-qPCR, real-time quantitative polymerase chain reaction; SAG, Smoothened/Shh signalling agonist.

    Techniques Used: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    21) Product Images from "Efficient Generation of A9 Midbrain Dopaminergic Neurons by Lentiviral Delivery of LMX1A in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells"

    Article Title: Efficient Generation of A9 Midbrain Dopaminergic Neurons by Lentiviral Delivery of LMX1A in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2011.054

    Differentiation protocol implemented for the generation of DA neurons and validation of specific expression of LMX1A in neural precursor cells. (a) Schematic representation of the different stages for the in vitro differentiation of pluripotent stem cells toward DA neurons. At Stage 1, EBs are formed by aggregation. At Stage 2, NPCs are obtained culturing the EBs in suspension with N2B27 medium supplemented with FGF2, FGF8, and SHH. At Stage 3, NPCs are cocultured with PA6 for 3 weeks and DA neurons are generated. (b) NPCs formed at Stage 2 express the neural precursor markers NESTIN (c) and SOX2 (d) . (e) At Stage 3, some of the TUJ1-positive cells generated coexpress TH. (f) Omitting the EB generation step or the coculture with PA6 cells results in
    Figure Legend Snippet: Differentiation protocol implemented for the generation of DA neurons and validation of specific expression of LMX1A in neural precursor cells. (a) Schematic representation of the different stages for the in vitro differentiation of pluripotent stem cells toward DA neurons. At Stage 1, EBs are formed by aggregation. At Stage 2, NPCs are obtained culturing the EBs in suspension with N2B27 medium supplemented with FGF2, FGF8, and SHH. At Stage 3, NPCs are cocultured with PA6 for 3 weeks and DA neurons are generated. (b) NPCs formed at Stage 2 express the neural precursor markers NESTIN (c) and SOX2 (d) . (e) At Stage 3, some of the TUJ1-positive cells generated coexpress TH. (f) Omitting the EB generation step or the coculture with PA6 cells results in

    Techniques Used: Expressing, In Vitro, Generated

    22) Product Images from "Extracellular vesicles from neuronal cells promote neural induction of mESCs through cyclinD1"

    Article Title: Extracellular vesicles from neuronal cells promote neural induction of mESCs through cyclinD1

    Journal: bioRxiv

    doi: 10.1101/2021.05.09.443321

    Differentiated neuronal EVs taken up by mESCs (A) Schematic of mESC treated by PKH6 dye-labeled EVs. (B) Confocal images of differentiated mESCs incubated without EV (No-EV) or PKH6 dye-labeled N6 or N9 EV. Nuclei were stained with DAPI. Scale bar denotes 10 μm. (C) Immunoblot of GFP and CD9 in EVs (GFP-EV) purified from GFP-expressing cells. mESCs (2×10 5 ) in 35mm dish were incubated in 2 ml of N2B27 medium for 24 h with EVs purified from control or GFP expressing cells. (D) Immunoblot of GFP from whole cell lysate of mESCs treated with PBS or GFP-EV for 24 h. (E) Immunoblot of GFP from whole cell lysate of mESCs treated with indicated doses of GFP-EV for 24 h. (F) Immunoblot of GFP from whole cell lysate of mESCs treated with indicated time of incubation with GFP-EV. (G) Immunoblot of GFP from whole cell lysate of mESCs treated with 10 ng GFP protein for indicated time or treated with GFP-EV that contained 10 ng GFP for 24 h. The GFP protein amount within the GFP-EV was detected by quantitative immunoblot.
    Figure Legend Snippet: Differentiated neuronal EVs taken up by mESCs (A) Schematic of mESC treated by PKH6 dye-labeled EVs. (B) Confocal images of differentiated mESCs incubated without EV (No-EV) or PKH6 dye-labeled N6 or N9 EV. Nuclei were stained with DAPI. Scale bar denotes 10 μm. (C) Immunoblot of GFP and CD9 in EVs (GFP-EV) purified from GFP-expressing cells. mESCs (2×10 5 ) in 35mm dish were incubated in 2 ml of N2B27 medium for 24 h with EVs purified from control or GFP expressing cells. (D) Immunoblot of GFP from whole cell lysate of mESCs treated with PBS or GFP-EV for 24 h. (E) Immunoblot of GFP from whole cell lysate of mESCs treated with indicated doses of GFP-EV for 24 h. (F) Immunoblot of GFP from whole cell lysate of mESCs treated with indicated time of incubation with GFP-EV. (G) Immunoblot of GFP from whole cell lysate of mESCs treated with 10 ng GFP protein for indicated time or treated with GFP-EV that contained 10 ng GFP for 24 h. The GFP protein amount within the GFP-EV was detected by quantitative immunoblot.

    Techniques Used: Labeling, Incubation, Staining, Purification, Expressing

    23) Product Images from "Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37442-5

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

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

    24) Product Images from "Cell-cell interaction modulates neuroectodermal specification of embryonic stem cells"

    Article Title: Cell-cell interaction modulates neuroectodermal specification of embryonic stem cells

    Journal: Neuroscience letters

    doi: 10.1016/j.neulet.2008.03.094

    Morphological maturation and temporal expression of cell adhesion molecules during neuroectodermal differentiation Phase contrast images ES cells differentiated in N2B27 medium for (A) 5, (B) 11 or (C) 17 days. (D) Detection of mRNA transcripts by endpoint RT-PCR in ES cells after 0, 3, 7, 11, 14 and 17 days of differentiation in N2B27 medium. Mouse brain tissue (Br) served as an external control and 18S and primers only served as internal controls. Quantitative RT-PCR of Cx-43 (E) expression, relative to an internal housekeeping gene, normalized to expression of Day 0 ES cells. Error bars represent s.e.m. of three independent experiments.
    Figure Legend Snippet: Morphological maturation and temporal expression of cell adhesion molecules during neuroectodermal differentiation Phase contrast images ES cells differentiated in N2B27 medium for (A) 5, (B) 11 or (C) 17 days. (D) Detection of mRNA transcripts by endpoint RT-PCR in ES cells after 0, 3, 7, 11, 14 and 17 days of differentiation in N2B27 medium. Mouse brain tissue (Br) served as an external control and 18S and primers only served as internal controls. Quantitative RT-PCR of Cx-43 (E) expression, relative to an internal housekeeping gene, normalized to expression of Day 0 ES cells. Error bars represent s.e.m. of three independent experiments.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    25) Product Images from "Efficient Feeder-Free Episomal Reprogramming with Small Molecules"

    Article Title: Efficient Feeder-Free Episomal Reprogramming with Small Molecules

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017557

    Developing a feeder-free condition for episomal reprogramming. ( A ) Effects of MEF feeder cells, matrigel and culture media on episomal reprogramming. Transfected human foreskin fibroblasts were plated to MEF feeder cell-seeded or matrigel-coated 10-cm dishes, and subjected to different reprogramming culture conditions. Alkaline phosphatase positive (AP+) colonies were counted on day 18–21 post-transfection. The number of AP+ colonies was from ∼0.33×10 6 input cells. Data shown are mean ± SEM (n = 3). N2B27 : DMEM/F12 medium supplemented with N-2 and B-27; N2B27-100 : N2B27 medium supplemented with 100 ng/ml bFGF. ( B ) Bright-field images of a piPSC colony from test 2 and a typical human ESC-like iPSC colony from test 1, 3 or 4. Scale bars: 100 µm. ( C ) Quantitative RT-PCR analysis of OCT4 and NANOG expression in piPSC clone 1 to 4 (c1 to c4, p3). Total: both endogenous and transgene expression. Human H1 ESCs (H1ESC, p32) were used as a control. Data shown are mean ± SEM (n = 3). ( D ) Temporal requirement of small molecule treatment for feeder-free episomal reprogramming (test 4) ( Fig. 2A ). Transfected human foreskin fibroblasts were plated to matrigel-coated 10-cm dishes. PCALH was added to the N2B27-100 medium for different days at stage 2 of reprogramming. Alkaline phosphatase positive iPSC colonies were counted on day 22 post-transfection. The number of iPSC colonies was from ∼0.33×10 6 input cells. Data shown are mean ± SEM (n = 3).
    Figure Legend Snippet: Developing a feeder-free condition for episomal reprogramming. ( A ) Effects of MEF feeder cells, matrigel and culture media on episomal reprogramming. Transfected human foreskin fibroblasts were plated to MEF feeder cell-seeded or matrigel-coated 10-cm dishes, and subjected to different reprogramming culture conditions. Alkaline phosphatase positive (AP+) colonies were counted on day 18–21 post-transfection. The number of AP+ colonies was from ∼0.33×10 6 input cells. Data shown are mean ± SEM (n = 3). N2B27 : DMEM/F12 medium supplemented with N-2 and B-27; N2B27-100 : N2B27 medium supplemented with 100 ng/ml bFGF. ( B ) Bright-field images of a piPSC colony from test 2 and a typical human ESC-like iPSC colony from test 1, 3 or 4. Scale bars: 100 µm. ( C ) Quantitative RT-PCR analysis of OCT4 and NANOG expression in piPSC clone 1 to 4 (c1 to c4, p3). Total: both endogenous and transgene expression. Human H1 ESCs (H1ESC, p32) were used as a control. Data shown are mean ± SEM (n = 3). ( D ) Temporal requirement of small molecule treatment for feeder-free episomal reprogramming (test 4) ( Fig. 2A ). Transfected human foreskin fibroblasts were plated to matrigel-coated 10-cm dishes. PCALH was added to the N2B27-100 medium for different days at stage 2 of reprogramming. Alkaline phosphatase positive iPSC colonies were counted on day 22 post-transfection. The number of iPSC colonies was from ∼0.33×10 6 input cells. Data shown are mean ± SEM (n = 3).

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing

    Optimizing episomal vector and transgene combinations for small molecule-aided reprogramming of different human somatic cells. ( A ) Effects of different episomal vector combinations and transformation-deficient LMYC on the reprogramming of human fibroblasts. Transfected human foreskin fibroblasts (HFFs) were plated to matrigel-coated 10-cm dishes. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 13 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ∼0.33×10 6 input cells) was counted on day 21 post-transfection. Data shown are mean ± SEM (n = 3). ( B ) Effects of different transgene combinations on the reprogramming of human adult adipose tissue-derived stem cells (AdSCs). Transfected adipose tissue-derived stem cells were plated to matrigel-coated 10-cm dishes. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 11 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ∼0.35×10 6 input cells) was counted on day 21 post-transfection. ( C ) Effects of transformation-deficient LMYC on the reprogramming of human cord blood (CB) cells. Purified human cord blood CD34+ cells were expanded in culture for 7 days (∼75 fold expansion). Following expansion, the cord blood cells were nucleofected with episomal vectors and plated to fibronectin/matrigel-coated 6-well plates. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 11 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ∼0.17×10 6 post-expansion input cells) was counted on day 17 post-transfection. Data shown are mean ± SEM (n = 6).
    Figure Legend Snippet: Optimizing episomal vector and transgene combinations for small molecule-aided reprogramming of different human somatic cells. ( A ) Effects of different episomal vector combinations and transformation-deficient LMYC on the reprogramming of human fibroblasts. Transfected human foreskin fibroblasts (HFFs) were plated to matrigel-coated 10-cm dishes. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 13 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ∼0.33×10 6 input cells) was counted on day 21 post-transfection. Data shown are mean ± SEM (n = 3). ( B ) Effects of different transgene combinations on the reprogramming of human adult adipose tissue-derived stem cells (AdSCs). Transfected adipose tissue-derived stem cells were plated to matrigel-coated 10-cm dishes. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 11 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ∼0.35×10 6 input cells) was counted on day 21 post-transfection. ( C ) Effects of transformation-deficient LMYC on the reprogramming of human cord blood (CB) cells. Purified human cord blood CD34+ cells were expanded in culture for 7 days (∼75 fold expansion). Following expansion, the cord blood cells were nucleofected with episomal vectors and plated to fibronectin/matrigel-coated 6-well plates. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 11 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ∼0.17×10 6 post-expansion input cells) was counted on day 17 post-transfection. Data shown are mean ± SEM (n = 6).

    Techniques Used: Plasmid Preparation, Transformation Assay, Transfection, Derivative Assay, Purification

    26) Product Images from "Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells"

    Article Title: Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093663

    Developmental regulation of NCoA’s gene expression. (A) Representative images of an ES cell colony and resulting neural stem cells after plating during 7 days in N2B27 medium (clearly visible are the rosette formation indicative of neural stem cell differentiation). (B) qPCR quantification of Nestin (cell fate marker) and Nanog (pluripotency marker) mRNA expression during neural conversion. Data is shown as average of three biological replicates and the error bars indicate the standard deviation. (C) qPCR quantification of all three members of the NCoA family mRNA expression during neural conversion. Data is shown as average of three biological replicates and the error bars indicate the standard deviation. (D) qPCR quantification of NCoA1 specific splice variants SRC1A and SRC1E expression during neural conversion. Primers were designed to specifically determine transcript levels of both splice variants. Data is shown as average of three biological replicates and the error bars indicate the standard deviation.
    Figure Legend Snippet: Developmental regulation of NCoA’s gene expression. (A) Representative images of an ES cell colony and resulting neural stem cells after plating during 7 days in N2B27 medium (clearly visible are the rosette formation indicative of neural stem cell differentiation). (B) qPCR quantification of Nestin (cell fate marker) and Nanog (pluripotency marker) mRNA expression during neural conversion. Data is shown as average of three biological replicates and the error bars indicate the standard deviation. (C) qPCR quantification of all three members of the NCoA family mRNA expression during neural conversion. Data is shown as average of three biological replicates and the error bars indicate the standard deviation. (D) qPCR quantification of NCoA1 specific splice variants SRC1A and SRC1E expression during neural conversion. Primers were designed to specifically determine transcript levels of both splice variants. Data is shown as average of three biological replicates and the error bars indicate the standard deviation.

    Techniques Used: Expressing, Cell Differentiation, Real-time Polymerase Chain Reaction, Marker, Standard Deviation

    27) Product Images from "Geminin Regulates the Transcriptional and Epigenetic Status of Neuronal Fate-Promoting Genes during Mammalian Neurogenesis"

    Article Title: Geminin Regulates the Transcriptional and Epigenetic Status of Neuronal Fate-Promoting Genes during Mammalian Neurogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00737-12

    ES cells undergoing neurogenesis and neuronal differentiation in N2B27 monolayer culture. (A to D) By 5 days of culture in N2B27 medium, most ES cells have lost pluripotency marker expression (fewer than 20% Oct4-positive cells) and a majority of cells
    Figure Legend Snippet: ES cells undergoing neurogenesis and neuronal differentiation in N2B27 monolayer culture. (A to D) By 5 days of culture in N2B27 medium, most ES cells have lost pluripotency marker expression (fewer than 20% Oct4-positive cells) and a majority of cells

    Techniques Used: Marker, Expressing

    28) Product Images from "Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37442-5

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

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

    29) Product Images from "Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency"

    Article Title: Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency

    Journal: Journal of Assisted Reproduction and Genetics

    doi: 10.1007/s10815-018-1360-9

    Morphology of outgrowths and mESC colonies derived from whole blastocysts and single 1/8 blastomeres in N2B27 medium without treatment. a Outgrowth from a B6CBAF2 blastocyst cultured in N2B27 medium consisting of a clump of pluripotent cells and swollen refractive cells around, highlighted with arrowheads. b Outgrowth from a B6CBAF2 blastomere cultured in N2B27 medium. c mESC colonies at passage 5 cultured in N2B27 medium, presenting defined edges and peripheral differentiation signs, highlighted with dashed ellipses. The scale bar corresponds to 100 μm
    Figure Legend Snippet: Morphology of outgrowths and mESC colonies derived from whole blastocysts and single 1/8 blastomeres in N2B27 medium without treatment. a Outgrowth from a B6CBAF2 blastocyst cultured in N2B27 medium consisting of a clump of pluripotent cells and swollen refractive cells around, highlighted with arrowheads. b Outgrowth from a B6CBAF2 blastomere cultured in N2B27 medium. c mESC colonies at passage 5 cultured in N2B27 medium, presenting defined edges and peripheral differentiation signs, highlighted with dashed ellipses. The scale bar corresponds to 100 μm

    Techniques Used: Derivative Assay, Cell Culture

    Morphology of outgrowths and mESC colonies derived from whole blastocysts and single 1/8 blastomeres in either KSR medium or N2B27 medium with 2i. a Outgrowth from a 129B6F1 blastocyst cultured in N2B27 medium containing 2i presenting defined edges. b Outgrowth from a 129B6F1 blastomere cultured in KSR medium with 2i. c Outgrowth from a CBA blastomere cultured in KSR medium with 2i. d Outgrowth from a B6CBAF2 blastomere cultured in N2B27 medium with 2i. e Non-progressive outgrowth from a CBA blastomere cultured in N2B27 medium with 2i. f mESC colony at passage 6 cultured in N2B27 medium with 2i, presenting defined edges and a dome shape. g mESC colony cultured in KSR medium with 2i (passage 4), showing defined edges and a flat shape. The scale bar indicates 100 μm
    Figure Legend Snippet: Morphology of outgrowths and mESC colonies derived from whole blastocysts and single 1/8 blastomeres in either KSR medium or N2B27 medium with 2i. a Outgrowth from a 129B6F1 blastocyst cultured in N2B27 medium containing 2i presenting defined edges. b Outgrowth from a 129B6F1 blastomere cultured in KSR medium with 2i. c Outgrowth from a CBA blastomere cultured in KSR medium with 2i. d Outgrowth from a B6CBAF2 blastomere cultured in N2B27 medium with 2i. e Non-progressive outgrowth from a CBA blastomere cultured in N2B27 medium with 2i. f mESC colony at passage 6 cultured in N2B27 medium with 2i, presenting defined edges and a dome shape. g mESC colony cultured in KSR medium with 2i (passage 4), showing defined edges and a flat shape. The scale bar indicates 100 μm

    Techniques Used: Derivative Assay, Cell Culture, Crocin Bleaching Assay

    30) Product Images from "Wnt Signaling Regulates the Lineage Differentiation Potential of Mouse Embryonic Stem Cells through Tcf3 Down-Regulation"

    Article Title: Wnt Signaling Regulates the Lineage Differentiation Potential of Mouse Embryonic Stem Cells through Tcf3 Down-Regulation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003424

    Wnt signaling regulates the differentiation potential of mouse ESCs in a dosage-dependent manner. A. β-catenin/TCF reporter assay in wild type and Apc -mutant ESCs. Measurements are reported as the average luciferase units performed in triplicate for the TOP (filled bars) and FOP (empty bars) reporter constructs (data reported is mean±SD). Numbers in the histogram represent the calculated TOP/FOP ratios. B. Table summarizing the results obtained by teratoma differentiation assay from different Apc -mutant ESCs and their wild type controls. Tissue sections were stained with hematoxylin and eosin (H E) or used in immunohistochemical analysis using specific antibodies against the neural markers: GFAP, neurofilaments and synaptic vesicles. Adult myosin was used as a mesodermal marker to stain the striated muscle differentiation. Cartilage differentiation was assessed either by H E or theonin staining. Two independent clones were used for each genotype and differentiation was scored as: (−) not present, (+) weakly present, and (++) present. C. Histogram showing the percent of colonies formed after plating 500 FACS-sorted cells in N2B27 medium supplemented with different combinations of LIF, Mek inhibitor (PD) and GSK-inhibitor (CHIRON). Bars represent mean ± SD, n = 3. D. Dendrogram derived from unsupervised hierarchical clustering of global gene expression in wild type, Apc TT, Apc NT and Apc NN ES cells. Pearson's correlation coefficient and Ward's method were used after MAS 5.0 normalization of all probe sets.
    Figure Legend Snippet: Wnt signaling regulates the differentiation potential of mouse ESCs in a dosage-dependent manner. A. β-catenin/TCF reporter assay in wild type and Apc -mutant ESCs. Measurements are reported as the average luciferase units performed in triplicate for the TOP (filled bars) and FOP (empty bars) reporter constructs (data reported is mean±SD). Numbers in the histogram represent the calculated TOP/FOP ratios. B. Table summarizing the results obtained by teratoma differentiation assay from different Apc -mutant ESCs and their wild type controls. Tissue sections were stained with hematoxylin and eosin (H E) or used in immunohistochemical analysis using specific antibodies against the neural markers: GFAP, neurofilaments and synaptic vesicles. Adult myosin was used as a mesodermal marker to stain the striated muscle differentiation. Cartilage differentiation was assessed either by H E or theonin staining. Two independent clones were used for each genotype and differentiation was scored as: (−) not present, (+) weakly present, and (++) present. C. Histogram showing the percent of colonies formed after plating 500 FACS-sorted cells in N2B27 medium supplemented with different combinations of LIF, Mek inhibitor (PD) and GSK-inhibitor (CHIRON). Bars represent mean ± SD, n = 3. D. Dendrogram derived from unsupervised hierarchical clustering of global gene expression in wild type, Apc TT, Apc NT and Apc NN ES cells. Pearson's correlation coefficient and Ward's method were used after MAS 5.0 normalization of all probe sets.

    Techniques Used: Reporter Assay, Mutagenesis, Luciferase, Construct, Differentiation Assay, Staining, Immunohistochemistry, Marker, Clone Assay, FACS, Derivative Assay, Expressing

    Characterization of Tcf3 over expressing ESCs. A–B. qRT-PCR (A) and western blot (B) analysis of Tcf3 in Apc NN ESCs stably expressing Tcf3. Wild type and Tcf3 −/− ESCs were used for comparison. Actb was used as an internal control. C. Histogram showing reduction of β-catenin/Tcf reporter activity in Apc NN cells stably expressing Tcf3 (Tcf3 OE) compared to parental Apc NN cells and cells expressing the corresponding empty vector. Luciferase signal from TOP or FOP reporter constructs were measured and TOP/FOP ratios are shown in the graph. Bars represent n = 3 ± SD. D. Histogram showing the percent of alkaline phosphatase (AP) positive colonies formed by plating 500 FACS-sorted cells in N2B27 medium after 7 days. N2B27 medium was supplemented with different combinations of LIF, PD and CHIRON. Two independent Apc NN ESC clones (parental clone and transfected with empty vector) and three independent Apc NN ESC clones expressing Tcf3 (Tcf3 OE) were used. Bars represent n = 3 ± SD. E. Histograms showing relative expression of the pluripotency markers Nanog and the early differentiation markers Fgf5 in different ESCs cultured for 48 h in N2B27 medium. F. Confocal analysis of ES cells stained with Tuj-1-Alexa 488 and counterstained with the far-red nuclear stain DRAQ5. Wild type, Apc NN and Apc NN expressing Tcf3 (Tcf3 OE) ESCs were used in −4/+4 neural differentiation assay and analyzed by immunofluorescence after 13 days of culture. G. Flow cytometric analysis showing expression of the neural progenitor marker Nestin in Apc NN ESCs stably expressing Tcf3 (Tcf3 OE) and their control cells (parental Apc NN clone and Apc NN transfected with the corresponding empty vector) or wild type ESCs. Cells were analyzed by the −4/+4 neural differentiation assay and stained with specific antibody against Nestin and Tuj1 after 13 days of culture. Wild type (WT) ESCs are shown as control to indicate the Tuj1 positive population which is absent in other genotypes (0.1% in average in Tcf3 OE clones). Numbers in the graph represent the percent of Nestin-positive cells. For wild type ESCs the Nestin-positive populations before and after excluding the mature neurons are shown. See also Figure S4 for defining different FACS gates.
    Figure Legend Snippet: Characterization of Tcf3 over expressing ESCs. A–B. qRT-PCR (A) and western blot (B) analysis of Tcf3 in Apc NN ESCs stably expressing Tcf3. Wild type and Tcf3 −/− ESCs were used for comparison. Actb was used as an internal control. C. Histogram showing reduction of β-catenin/Tcf reporter activity in Apc NN cells stably expressing Tcf3 (Tcf3 OE) compared to parental Apc NN cells and cells expressing the corresponding empty vector. Luciferase signal from TOP or FOP reporter constructs were measured and TOP/FOP ratios are shown in the graph. Bars represent n = 3 ± SD. D. Histogram showing the percent of alkaline phosphatase (AP) positive colonies formed by plating 500 FACS-sorted cells in N2B27 medium after 7 days. N2B27 medium was supplemented with different combinations of LIF, PD and CHIRON. Two independent Apc NN ESC clones (parental clone and transfected with empty vector) and three independent Apc NN ESC clones expressing Tcf3 (Tcf3 OE) were used. Bars represent n = 3 ± SD. E. Histograms showing relative expression of the pluripotency markers Nanog and the early differentiation markers Fgf5 in different ESCs cultured for 48 h in N2B27 medium. F. Confocal analysis of ES cells stained with Tuj-1-Alexa 488 and counterstained with the far-red nuclear stain DRAQ5. Wild type, Apc NN and Apc NN expressing Tcf3 (Tcf3 OE) ESCs were used in −4/+4 neural differentiation assay and analyzed by immunofluorescence after 13 days of culture. G. Flow cytometric analysis showing expression of the neural progenitor marker Nestin in Apc NN ESCs stably expressing Tcf3 (Tcf3 OE) and their control cells (parental Apc NN clone and Apc NN transfected with the corresponding empty vector) or wild type ESCs. Cells were analyzed by the −4/+4 neural differentiation assay and stained with specific antibody against Nestin and Tuj1 after 13 days of culture. Wild type (WT) ESCs are shown as control to indicate the Tuj1 positive population which is absent in other genotypes (0.1% in average in Tcf3 OE clones). Numbers in the graph represent the percent of Nestin-positive cells. For wild type ESCs the Nestin-positive populations before and after excluding the mature neurons are shown. See also Figure S4 for defining different FACS gates.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Stable Transfection, Activity Assay, Plasmid Preparation, Luciferase, Construct, FACS, Clone Assay, Transfection, Cell Culture, Staining, Differentiation Assay, Immunofluorescence, Flow Cytometry, Marker

    Related Articles

    Cell Culture:

    Article Title: A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages
    Article Snippet: NSCs were maintained in NSC medium (DMEM/F-12 (Gibco), 1× NEAA, 0.1 mM 2-mercaptoethanol, 1× penicillin/streptomycin, 1:100 B27 supplement (Invitrogen), 1:200 N2 supplement (PAA), 4.5 μM HEPES (PAA), 0.03 M glucose (Sigma-Aldrich) and 120 μg ml−1 BSA (Invitrogen)) supplemented with 10 ng ml−1 of Egf (Peprotech) and 20 ng ml−1 of Fgf2 (home-made). .. Epiblast stem cells were cultured in N2B27 medium (DMEM/F12 and Neurobasal (both Gibco) in a 1:1 ratio, 1× penicillin/streptomycin, 0.1 mM 2-mercaptoethanol, 2 mM l -glutamine, 1:200 N2 (StemCells) and 1:100 B27 supplement) supplemented with 12 ng ml−1 Fgf2 and 20 ng ml−1 Activin A (home-made). .. 4OHT was used at a concentration of 500 nM and geneticin (G418) at 400 μg ml−1 .

    Article Title: Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction
    Article Snippet: .. In the present study, primary cells were cultured in DMEM/F12 containing 10% NBS (10%-F12) and patented SMCM; similar morphological features of EGJ cells were observed in a previous study for VSMCs in vitro ( ). ..

    In Vitro:

    Article Title: Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction
    Article Snippet: .. In the present study, primary cells were cultured in DMEM/F12 containing 10% NBS (10%-F12) and patented SMCM; similar morphological features of EGJ cells were observed in a previous study for VSMCs in vitro ( ). ..

    Mutagenesis:

    Article Title: Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni
    Article Snippet: .. For growth in liquid defined minimal medium, 108 CFUs of the C . jejuni transposon mutant library were added to 4 ml of DMEM (GIBCO; catalogue number 11965) supplemented with 20 mM Asp, Gln, or Ser. .. The culture tubes were placed on a rotating wheel in 10% CO2 atmosphere for 48 hours, and the bacterial cells were collected by centrifugation.

    other:

    Article Title: Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency
    Article Snippet: Anchorage-independent growth was determined by suspending VPA_AFS cells and NTERA2 cells (5 × 103 cells) in DMEM containing 10% FBS and 0.3% low-melting agarose (GIBCO BRL).

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  • 96
    Thermo Fisher membrane permeant dye oregon green 488 bapta 1 am
    Calcium imaging of parallel fiber bands. A , Schematic illustrating the load site, labeled fibers, location of stimulus electrodes S1 and S2, and field of view in B–F . B , Background fluorescence of fibers labeled with Oregon <t>Green</t> 488 <t>BAPTA-1</t> AM. C , D , Fluorescence evoked by 100 pulses at 100 Hz for electrode S1 ( C ) or electrode S2 ( D ). E , F , Evoked changes in fluorescence for S1 ( E ) and S2 ( F ) in which B has been subtracted away from C and D , respectively. Traces are single trial examples.
    Membrane Permeant Dye Oregon Green 488 Bapta 1 Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher control sirna
    AR knockdown suppresses the androgen-induced GNMT gene. (A) LNCaP cells were cultured in RPMI containing 10% DSS in the absence of antibiotics for 24 h. Cells were then transfected with siRNAs targeting AR or control. Twenty-four hours after transfection, cells were treated with 1 nM R1881 with an equal volume of ethanol being added to the vehicle control. RNA was prepared after a further 48 h and TaqMan RT-PCR for AR and GNMT was performed. Data have been normalised to GAPDH levels and AR or GNMT expression levels in the <t>siRNA</t> control treated with R1881 was set to one. Results are shown as mean values of three independent experiments performed in triplicates with error bars representing s.e.m . (B) LNCaP cells were transfected with siRNAs as for A. Whole cell lysates were prepared and immunoblotted for AR, GNMT (arrowed) and β-actin. Molecular weight markers are expressed in kDa. Suppression of the androgen-regulated genes PSA (C) and NDRG1 (D) following silencing of AR is also shown.
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    Thermo Fisher fzd7
    Knockdown of both <t>Fzd7</t> and CDC42 in healthy pericytes shows impaired tube formation and cell polarity. A: Pericytes (Pc) were transfected with nontargeting siRNA control (siCtrl), siFzd7, sicdc42, and siFzd7/cdc42 for 48 hours. Before seeded on Matrigel,
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    Thermo Fisher pyrene maleimide
    Schematic representation of the <t>pyrene-4-maleimide</t> synthesis and structure. A. Pyrene maleimide structure. B. Pyrene-4-maleimide synthesis route and structure. Ph 3 P: triphenylphosphine; DIAD: diisopropyl azodicarboxylate.
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    Image Search Results


    Calcium imaging of parallel fiber bands. A , Schematic illustrating the load site, labeled fibers, location of stimulus electrodes S1 and S2, and field of view in B–F . B , Background fluorescence of fibers labeled with Oregon Green 488 BAPTA-1 AM. C , D , Fluorescence evoked by 100 pulses at 100 Hz for electrode S1 ( C ) or electrode S2 ( D ). E , F , Evoked changes in fluorescence for S1 ( E ) and S2 ( F ) in which B has been subtracted away from C and D , respectively. Traces are single trial examples.

    Journal: The Journal of Neuroscience

    Article Title: Prolonged Synaptic Currents and Glutamate Spillover at the Parallel Fiber to Stellate Cell Synapse

    doi: 10.1523/JNEUROSCI.20-12-04423.2000

    Figure Lengend Snippet: Calcium imaging of parallel fiber bands. A , Schematic illustrating the load site, labeled fibers, location of stimulus electrodes S1 and S2, and field of view in B–F . B , Background fluorescence of fibers labeled with Oregon Green 488 BAPTA-1 AM. C , D , Fluorescence evoked by 100 pulses at 100 Hz for electrode S1 ( C ) or electrode S2 ( D ). E , F , Evoked changes in fluorescence for S1 ( E ) and S2 ( F ) in which B has been subtracted away from C and D , respectively. Traces are single trial examples.

    Article Snippet: For the experiment shown in Figure , parallel fibers were labeled using local application of the membrane-permeant dye Oregon Green 488 BAPTA-1 AM (Molecular Probes, Eugene, OR) in solution, as described previously ( ; ).

    Techniques: Imaging, Labeling, Fluorescence

    AR knockdown suppresses the androgen-induced GNMT gene. (A) LNCaP cells were cultured in RPMI containing 10% DSS in the absence of antibiotics for 24 h. Cells were then transfected with siRNAs targeting AR or control. Twenty-four hours after transfection, cells were treated with 1 nM R1881 with an equal volume of ethanol being added to the vehicle control. RNA was prepared after a further 48 h and TaqMan RT-PCR for AR and GNMT was performed. Data have been normalised to GAPDH levels and AR or GNMT expression levels in the siRNA control treated with R1881 was set to one. Results are shown as mean values of three independent experiments performed in triplicates with error bars representing s.e.m . (B) LNCaP cells were transfected with siRNAs as for A. Whole cell lysates were prepared and immunoblotted for AR, GNMT (arrowed) and β-actin. Molecular weight markers are expressed in kDa. Suppression of the androgen-regulated genes PSA (C) and NDRG1 (D) following silencing of AR is also shown.

    Journal: Journal of Molecular Endocrinology

    Article Title: Characterisation of the androgen regulation of glycine N-methyltransferase in prostate cancer cells

    doi: 10.1530/JME-13-0169

    Figure Lengend Snippet: AR knockdown suppresses the androgen-induced GNMT gene. (A) LNCaP cells were cultured in RPMI containing 10% DSS in the absence of antibiotics for 24 h. Cells were then transfected with siRNAs targeting AR or control. Twenty-four hours after transfection, cells were treated with 1 nM R1881 with an equal volume of ethanol being added to the vehicle control. RNA was prepared after a further 48 h and TaqMan RT-PCR for AR and GNMT was performed. Data have been normalised to GAPDH levels and AR or GNMT expression levels in the siRNA control treated with R1881 was set to one. Results are shown as mean values of three independent experiments performed in triplicates with error bars representing s.e.m . (B) LNCaP cells were transfected with siRNAs as for A. Whole cell lysates were prepared and immunoblotted for AR, GNMT (arrowed) and β-actin. Molecular weight markers are expressed in kDa. Suppression of the androgen-regulated genes PSA (C) and NDRG1 (D) following silencing of AR is also shown.

    Article Snippet: siRNA transfections Cells were transfected with control siRNA (Negative control N.2, Ambion, Applied Biosystems) or siRNA specific to AR (s1539, Ambion, Applied Biosystems) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocols.

    Techniques: Cell Culture, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight

    Knockdown of both Fzd7 and CDC42 in healthy pericytes shows impaired tube formation and cell polarity. A: Pericytes (Pc) were transfected with nontargeting siRNA control (siCtrl), siFzd7, sicdc42, and siFzd7/cdc42 for 48 hours. Before seeded on Matrigel,

    Journal: The American Journal of Pathology

    Article Title: Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis

    doi: 10.1016/j.ajpath.2014.09.013

    Figure Lengend Snippet: Knockdown of both Fzd7 and CDC42 in healthy pericytes shows impaired tube formation and cell polarity. A: Pericytes (Pc) were transfected with nontargeting siRNA control (siCtrl), siFzd7, sicdc42, and siFzd7/cdc42 for 48 hours. Before seeded on Matrigel,

    Article Snippet: To achieve gene knockdown, 2 μmol/L siRNA of Fzd7 (catalog number M-003671-02-0005; Thermo Fisher Scientific Inc., Rockford, IL) and/or 2 μmol/L siRNA of cdc42 (catalog number M-005057-01-0005; Thermo Fisher Scientific Inc.) or nontargeting siRNA control (catalog number D-001810-10-05; Thermo Fisher Scientific Inc.) was transfected into healthy pericytes.

    Techniques: Transfection

    Model representing that Wnt/planar cell polarity signaling contributes to the association between pericytes and developing blood vessels via the Wnt5a-Fzd7-cdc42 axis. The up-regulation of cdc42 on Wnt5a/Fzd7 signaling results in increased motility and

    Journal: The American Journal of Pathology

    Article Title: Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis

    doi: 10.1016/j.ajpath.2014.09.013

    Figure Lengend Snippet: Model representing that Wnt/planar cell polarity signaling contributes to the association between pericytes and developing blood vessels via the Wnt5a-Fzd7-cdc42 axis. The up-regulation of cdc42 on Wnt5a/Fzd7 signaling results in increased motility and

    Article Snippet: To achieve gene knockdown, 2 μmol/L siRNA of Fzd7 (catalog number M-003671-02-0005; Thermo Fisher Scientific Inc., Rockford, IL) and/or 2 μmol/L siRNA of cdc42 (catalog number M-005057-01-0005; Thermo Fisher Scientific Inc.) or nontargeting siRNA control (catalog number D-001810-10-05; Thermo Fisher Scientific Inc.) was transfected into healthy pericytes.

    Techniques:

    Transfection of Fzd7 and cdc42 expression constructs in PAH pericytes improve their ability to associate with vascular tubes. A: PAH pericytes were transfected with GFP-tagged Fzd7 and cdc42 plasmids (green) and seeded with PMVECs (red) in Matrigel-coated

    Journal: The American Journal of Pathology

    Article Title: Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis

    doi: 10.1016/j.ajpath.2014.09.013

    Figure Lengend Snippet: Transfection of Fzd7 and cdc42 expression constructs in PAH pericytes improve their ability to associate with vascular tubes. A: PAH pericytes were transfected with GFP-tagged Fzd7 and cdc42 plasmids (green) and seeded with PMVECs (red) in Matrigel-coated

    Article Snippet: To achieve gene knockdown, 2 μmol/L siRNA of Fzd7 (catalog number M-003671-02-0005; Thermo Fisher Scientific Inc., Rockford, IL) and/or 2 μmol/L siRNA of cdc42 (catalog number M-005057-01-0005; Thermo Fisher Scientific Inc.) or nontargeting siRNA control (catalog number D-001810-10-05; Thermo Fisher Scientific Inc.) was transfected into healthy pericytes.

    Techniques: Transfection, Expressing, Construct

    Reduction of Fzd7 and cdc42 prevents pericyte-induced microvessel formation in SCID mice. Representative images of hematoxylin and eosin staining ( A–D ) and immunofluorescence ( E–H ) show the appearance of PMVECs alone ( A ), PMVECs plus healthy

    Journal: The American Journal of Pathology

    Article Title: Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis

    doi: 10.1016/j.ajpath.2014.09.013

    Figure Lengend Snippet: Reduction of Fzd7 and cdc42 prevents pericyte-induced microvessel formation in SCID mice. Representative images of hematoxylin and eosin staining ( A–D ) and immunofluorescence ( E–H ) show the appearance of PMVECs alone ( A ), PMVECs plus healthy

    Article Snippet: To achieve gene knockdown, 2 μmol/L siRNA of Fzd7 (catalog number M-003671-02-0005; Thermo Fisher Scientific Inc., Rockford, IL) and/or 2 μmol/L siRNA of cdc42 (catalog number M-005057-01-0005; Thermo Fisher Scientific Inc.) or nontargeting siRNA control (catalog number D-001810-10-05; Thermo Fisher Scientific Inc.) was transfected into healthy pericytes.

    Techniques: Mouse Assay, Staining, Immunofluorescence

    Schematic representation of the pyrene-4-maleimide synthesis and structure. A. Pyrene maleimide structure. B. Pyrene-4-maleimide synthesis route and structure. Ph 3 P: triphenylphosphine; DIAD: diisopropyl azodicarboxylate.

    Journal: PLoS ONE

    Article Title: A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation

    doi: 10.1371/journal.pone.0026691

    Figure Lengend Snippet: Schematic representation of the pyrene-4-maleimide synthesis and structure. A. Pyrene maleimide structure. B. Pyrene-4-maleimide synthesis route and structure. Ph 3 P: triphenylphosphine; DIAD: diisopropyl azodicarboxylate.

    Article Snippet: Pyrene maleimide (Invitrogen, Carlsbad, CA) and pyrene-4-maleimide stock solutions (25 mM) were prepared in dimetylsulfoxide.

    Techniques:

    Monomer and excimer emission in the presence of organic compounds. A. Pyrene maleimide. B. Pyrene-4-maleimide. C. Pyrene maleimide vs. pyrene-4-maleimide in mercaptoethanol. D. Pyrene maleimide vs. pyrene-4-maleimide in butanethiol. The concentrations of the fluorescent probes and organic compounds were 3 µM and 1 mM, respectively. The labels in panels A and C also apply to panels B and D, respectively. Peaks 1–4 are indicated (see text). Data in panels A–C were normalized to peak 1 intensity in mercaptoethanol. Data in panel D were normalized to peak 4 intensity in butanethiol.

    Journal: PLoS ONE

    Article Title: A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation

    doi: 10.1371/journal.pone.0026691

    Figure Lengend Snippet: Monomer and excimer emission in the presence of organic compounds. A. Pyrene maleimide. B. Pyrene-4-maleimide. C. Pyrene maleimide vs. pyrene-4-maleimide in mercaptoethanol. D. Pyrene maleimide vs. pyrene-4-maleimide in butanethiol. The concentrations of the fluorescent probes and organic compounds were 3 µM and 1 mM, respectively. The labels in panels A and C also apply to panels B and D, respectively. Peaks 1–4 are indicated (see text). Data in panels A–C were normalized to peak 1 intensity in mercaptoethanol. Data in panel D were normalized to peak 4 intensity in butanethiol.

    Article Snippet: Pyrene maleimide (Invitrogen, Carlsbad, CA) and pyrene-4-maleimide stock solutions (25 mM) were prepared in dimetylsulfoxide.

    Techniques:

    Double-stranded DNA models with pyrene maleimides attached via thiol linkers. A. Stick representation view along the DNA long axis. Pyrene-4-maleimide attached to DNA 14-14c and DNA 14-12c is shown in green and red, respectively. Pyrene maleimide attached to DNA 14-14c is shown in blue. The 5′ thiol was present in the 14-bp long strand in all cases. The 3′ thiol was present in the 14-bp or 12-bp long complementary strands. B. Stick representation view perpendicular to that in panel A. Only the pyrenes are shown for clarity. Color coding as in panel A. See Materials and Methods for details.

    Journal: PLoS ONE

    Article Title: A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation

    doi: 10.1371/journal.pone.0026691

    Figure Lengend Snippet: Double-stranded DNA models with pyrene maleimides attached via thiol linkers. A. Stick representation view along the DNA long axis. Pyrene-4-maleimide attached to DNA 14-14c and DNA 14-12c is shown in green and red, respectively. Pyrene maleimide attached to DNA 14-14c is shown in blue. The 5′ thiol was present in the 14-bp long strand in all cases. The 3′ thiol was present in the 14-bp or 12-bp long complementary strands. B. Stick representation view perpendicular to that in panel A. Only the pyrenes are shown for clarity. Color coding as in panel A. See Materials and Methods for details.

    Article Snippet: Pyrene maleimide (Invitrogen, Carlsbad, CA) and pyrene-4-maleimide stock solutions (25 mM) were prepared in dimetylsulfoxide.

    Techniques:

    Emission of pyrene compounds reacted with thiol-modified DNA. A. Emission spectra of single-stranded DNA containing a 5′ end thiol group (DNA 14 ) reacted with pyrene-4-maleimide. Data were normalized to peak 1 intensity from reduced DNA 14 . [DNA 14 ] was 0.5 µM, and [pyrene-4-maleimide] was 2 µM. B. Emission spectra of double-stranded DNA reacted with pyrene maleimide. DNA 14-14c : DNA 14 annealed to fully complementary DNA with a 3′ end thiol group (DNA 14c ). DNA 14-12c : DNA 14 annealed to a 3′ end two-base shorter complementary oligonucleotide with a 3′ end thiol group. DNA 14-10c : DNA 14 annealed to a 3′ end four-base shorter complementary oligonucleotide with a 3′ end thiol group. C. Emission spectra of double-stranded DNA reacted with pyrene-4-maleimide. Insert: schematic representation of the experimental system showing the double-stranded DNAs labeled with pyrene-4-maleimide. The pyrenes are represented by green rhomboids. Data in panels B and C were normalized to peak 1 intensity. The labels in panel B also apply to panel C. D. Excimer/monomer emission ratio. The values were calculated as: excimer/monomer = I peak 4 /I peak 1 , where I is the highest intensity of the peak, and peak 1 and peak 4 correspond to the excimer and monomer emission peaks. Averages ± SEM from experiments such as those shown in panels B and C (n = 3 for pyrene maleimide, and n = 4 for pyrene-4-maleimide). The asterisk denotes P

    Journal: PLoS ONE

    Article Title: A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation

    doi: 10.1371/journal.pone.0026691

    Figure Lengend Snippet: Emission of pyrene compounds reacted with thiol-modified DNA. A. Emission spectra of single-stranded DNA containing a 5′ end thiol group (DNA 14 ) reacted with pyrene-4-maleimide. Data were normalized to peak 1 intensity from reduced DNA 14 . [DNA 14 ] was 0.5 µM, and [pyrene-4-maleimide] was 2 µM. B. Emission spectra of double-stranded DNA reacted with pyrene maleimide. DNA 14-14c : DNA 14 annealed to fully complementary DNA with a 3′ end thiol group (DNA 14c ). DNA 14-12c : DNA 14 annealed to a 3′ end two-base shorter complementary oligonucleotide with a 3′ end thiol group. DNA 14-10c : DNA 14 annealed to a 3′ end four-base shorter complementary oligonucleotide with a 3′ end thiol group. C. Emission spectra of double-stranded DNA reacted with pyrene-4-maleimide. Insert: schematic representation of the experimental system showing the double-stranded DNAs labeled with pyrene-4-maleimide. The pyrenes are represented by green rhomboids. Data in panels B and C were normalized to peak 1 intensity. The labels in panel B also apply to panel C. D. Excimer/monomer emission ratio. The values were calculated as: excimer/monomer = I peak 4 /I peak 1 , where I is the highest intensity of the peak, and peak 1 and peak 4 correspond to the excimer and monomer emission peaks. Averages ± SEM from experiments such as those shown in panels B and C (n = 3 for pyrene maleimide, and n = 4 for pyrene-4-maleimide). The asterisk denotes P

    Article Snippet: Pyrene maleimide (Invitrogen, Carlsbad, CA) and pyrene-4-maleimide stock solutions (25 mM) were prepared in dimetylsulfoxide.

    Techniques: Modification, Labeling

    Long lifetime of pyrene-4-maleimide excimer emission. DNA 14 : reduced single-stranded DNA 14 adduct. DNA 14-14c : double-stranded DNA 14-14c adduct. IRF: instrument response function. The red lines are fits of the data to multi-exponential functions, with the two weighted residuals (R i ) vs. time plots corresponding to the double-stranded (top) and single-stranded (bottom) data fits.

    Journal: PLoS ONE

    Article Title: A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation

    doi: 10.1371/journal.pone.0026691

    Figure Lengend Snippet: Long lifetime of pyrene-4-maleimide excimer emission. DNA 14 : reduced single-stranded DNA 14 adduct. DNA 14-14c : double-stranded DNA 14-14c adduct. IRF: instrument response function. The red lines are fits of the data to multi-exponential functions, with the two weighted residuals (R i ) vs. time plots corresponding to the double-stranded (top) and single-stranded (bottom) data fits.

    Article Snippet: Pyrene maleimide (Invitrogen, Carlsbad, CA) and pyrene-4-maleimide stock solutions (25 mM) were prepared in dimetylsulfoxide.

    Techniques: