Structured Review

Millipore nanog
Bisulfite sequencing at promoter regions of <t>OCT4,</t> <t>NANOG,</t> and XIST. To verify whether epigenetic reprogramming occurred by expression of exogenous genes, DNA methylation patterns at promoter regions of pig OCT4, NANOG, and XIST were evaluated by bisulfite sequencing. (A) OCT core promoter: OCT4 core promoter regions were highly methylated. (B) NANOG promoter: Promoter regions of NANOG were methylated to levels similar to somatic cell control. (C) XIST promoter: X chromosome reactivation did not occur in naïve-like piPSCs. Each circle indicates individual CpG dinucleotides. White and dark circles represent unmethylated and methylated CpGs, respectively. Each row represents one individual clone of amplified PCR products.
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Images

1) Product Images from "Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig"

Article Title: Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

Journal: PLoS ONE

doi: 10.1371/journal.pone.0158046

Bisulfite sequencing at promoter regions of OCT4, NANOG, and XIST. To verify whether epigenetic reprogramming occurred by expression of exogenous genes, DNA methylation patterns at promoter regions of pig OCT4, NANOG, and XIST were evaluated by bisulfite sequencing. (A) OCT core promoter: OCT4 core promoter regions were highly methylated. (B) NANOG promoter: Promoter regions of NANOG were methylated to levels similar to somatic cell control. (C) XIST promoter: X chromosome reactivation did not occur in naïve-like piPSCs. Each circle indicates individual CpG dinucleotides. White and dark circles represent unmethylated and methylated CpGs, respectively. Each row represents one individual clone of amplified PCR products.
Figure Legend Snippet: Bisulfite sequencing at promoter regions of OCT4, NANOG, and XIST. To verify whether epigenetic reprogramming occurred by expression of exogenous genes, DNA methylation patterns at promoter regions of pig OCT4, NANOG, and XIST were evaluated by bisulfite sequencing. (A) OCT core promoter: OCT4 core promoter regions were highly methylated. (B) NANOG promoter: Promoter regions of NANOG were methylated to levels similar to somatic cell control. (C) XIST promoter: X chromosome reactivation did not occur in naïve-like piPSCs. Each circle indicates individual CpG dinucleotides. White and dark circles represent unmethylated and methylated CpGs, respectively. Each row represents one individual clone of amplified PCR products.

Techniques Used: Methylation Sequencing, Expressing, DNA Methylation Assay, Methylation, Amplification, Polymerase Chain Reaction

Expression of pluripotent markers in piPSCs. Expression of endogenous and exogenous pluripotent genes was determined by immunostaining and qPCR. (A) OCT4, SOX2, SSEA1, and SSEA4 were expressed in naïve-like piPSCs cultured with LIF. (B) OCT4, SOX2, and SSEA4 were expressed in primed-like piPSCs cultured with bFGF (C) When treated with 2i, SSEA4 was still expressed in naïve-like piPSCs. (D) Expression of NANOG was not detected under any culture conditions as determined by flow cytometric analysis. (E) Exogenous transgenes were highly expressed when treated with doxycycline in piPSCs, while transgenes were not expressed in the absence of doxycycline. Scale bar = 50 μm.
Figure Legend Snippet: Expression of pluripotent markers in piPSCs. Expression of endogenous and exogenous pluripotent genes was determined by immunostaining and qPCR. (A) OCT4, SOX2, SSEA1, and SSEA4 were expressed in naïve-like piPSCs cultured with LIF. (B) OCT4, SOX2, and SSEA4 were expressed in primed-like piPSCs cultured with bFGF (C) When treated with 2i, SSEA4 was still expressed in naïve-like piPSCs. (D) Expression of NANOG was not detected under any culture conditions as determined by flow cytometric analysis. (E) Exogenous transgenes were highly expressed when treated with doxycycline in piPSCs, while transgenes were not expressed in the absence of doxycycline. Scale bar = 50 μm.

Techniques Used: Expressing, Immunostaining, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry

2) Product Images from "Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation"

Article Title: Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00499-18

Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.
Figure Legend Snippet: Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.

Techniques Used: In Vitro, Cell Culture, Immunocytochemistry, Expressing, Marker, Staining, Polymerase Chain Reaction, Sequencing, Negative Control

3) Product Images from "Generation of Transgenic Rats through Induced Pluripotent Stem Cells *"

Article Title: Generation of Transgenic Rats through Induced Pluripotent Stem Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.492900

Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.
Figure Legend Snippet: Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Negative Control, Staining, Positive Control, Polymerase Chain Reaction, Marker, Western Blot, Immunofluorescence, Genomic Sequencing, Methylation

4) 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

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

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

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

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

5) Product Images from "Ablation of Dido3 compromises lineage commitment of stem cells in vitro and during early embryonic development"

Article Title: Ablation of Dido3 compromises lineage commitment of stem cells in vitro and during early embryonic development

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2011.62

Dido3 is necessary for ES cell differentiation in vitro . Wt/Wt and ΔCT/ΔCT ES cells were cultured with LIF to impede differentiation (ES), or were aggregated into EB and maintained without LIF for the indicated time in untreated bacterial plates to induce differentiation. ( a ) AP staining was used as an indicator of undifferentiated and pluripotent stem cells. Photomicrographs show representative results for ES cells and EB at × 20 and × 10 magnification, respectively. ( b ) Western blot analysis was used to assay expression of proteins considered pluripotency markers (Oct4, Nanog, Sox2) in ES cells and EB; β -actin was used as a loading control
Figure Legend Snippet: Dido3 is necessary for ES cell differentiation in vitro . Wt/Wt and ΔCT/ΔCT ES cells were cultured with LIF to impede differentiation (ES), or were aggregated into EB and maintained without LIF for the indicated time in untreated bacterial plates to induce differentiation. ( a ) AP staining was used as an indicator of undifferentiated and pluripotent stem cells. Photomicrographs show representative results for ES cells and EB at × 20 and × 10 magnification, respectively. ( b ) Western blot analysis was used to assay expression of proteins considered pluripotency markers (Oct4, Nanog, Sox2) in ES cells and EB; β -actin was used as a loading control

Techniques Used: Cell Differentiation, In Vitro, Cell Culture, Staining, Western Blot, Expressing

6) Product Images from "Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization"

Article Title: Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0652-9

Reprogramming of human fibroblasts into induced pluripotent stem cells. a , b Fibroblast cells immunolabeled with antibodies against FSP-1 ( a , green) or vimentin ( b , red). DAPI (blue) was used to counterstain the nuclei. c – e iPSCs immunolabeled with antibodies against NANOG ( c ), SOX2 ( d ), and OCT4 ( e ). Expression of these proteins indicates the identity of iPSCs derived from fibroblast reprogramming. f Normal karyotype of an iPSC line determined by G-banding. g Results from PluriTest assay show that our iPSCs have a pluripotent signature (high pluripotency score and low novelty score), and cluster with well-characterized bona-fide iPSC and ESC lines (red background hint; iPSC line 2 is indicated by a yellow arrowhead in the magnified panel) and not with partially differentiated pluripotent cells or somatic tissue (blue background hint). Scale bars: a , b , 50 μm; c – e , 20 μm. FSP-1 fibroblast-specific protein-1
Figure Legend Snippet: Reprogramming of human fibroblasts into induced pluripotent stem cells. a , b Fibroblast cells immunolabeled with antibodies against FSP-1 ( a , green) or vimentin ( b , red). DAPI (blue) was used to counterstain the nuclei. c – e iPSCs immunolabeled with antibodies against NANOG ( c ), SOX2 ( d ), and OCT4 ( e ). Expression of these proteins indicates the identity of iPSCs derived from fibroblast reprogramming. f Normal karyotype of an iPSC line determined by G-banding. g Results from PluriTest assay show that our iPSCs have a pluripotent signature (high pluripotency score and low novelty score), and cluster with well-characterized bona-fide iPSC and ESC lines (red background hint; iPSC line 2 is indicated by a yellow arrowhead in the magnified panel) and not with partially differentiated pluripotent cells or somatic tissue (blue background hint). Scale bars: a , b , 50 μm; c – e , 20 μm. FSP-1 fibroblast-specific protein-1

Techniques Used: Immunolabeling, Expressing, Derivative Assay

7) Product Images from "STON2 negatively modulates stem-like properties in ovarian cancer cells via DNMT1/MUC1 pathway"

Article Title: STON2 negatively modulates stem-like properties in ovarian cancer cells via DNMT1/MUC1 pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0977-y

MUC1 participates in the STON2-mediated modulation of stem-like properties in ovarian cancer cells. a , b 3AO and Caov3 cells were transfected with a MUC1-specific shRNA for 48 h and cultured in spheroid culture conditions for 7 days. Representative images and the relative expression levels of CSC-related markers NANOG and c-MYC were detected by immunoblot analysis ( a ), and the CD44 + CD24 − population was detected by FCM ( b ). c , d 3AO and Caov3 cells were transfected with MUC1 overexpressing plasmid for 48 h, and cultured in spheroid culture conditions for 7 days. Representative images and the relative expression levels of CSC-related markers NANOG and c-MYC were detected by immunoblot analysis ( c ), and the CD44 + CD24 − population was detected using FCM ( d ). e 3AO and Caov3 cells were transfected with a control vector, STON2 overexpression plasmid, or STON2 overexpression plasmid plus MUC1 overexpression plasmid for 48 h and cultured in spheroid culture conditions for 7 days. Sphere-formation (sphere > 50 μm) was assessed. Scale bars, 50 μm. f 3AO cells were transfected with a MUC1-specific shRNA, or MUC1 overexpressing plasmid for 48 h, and cultured in spheroid culture conditions for 7 days. EMT-related markers (E-cadherin, N-cadherin, and fibronectin) were detected using immunoblot analysis. Data represents the mean ± S.E. of three independent experiments. The level of significance is indicated by * P
Figure Legend Snippet: MUC1 participates in the STON2-mediated modulation of stem-like properties in ovarian cancer cells. a , b 3AO and Caov3 cells were transfected with a MUC1-specific shRNA for 48 h and cultured in spheroid culture conditions for 7 days. Representative images and the relative expression levels of CSC-related markers NANOG and c-MYC were detected by immunoblot analysis ( a ), and the CD44 + CD24 − population was detected by FCM ( b ). c , d 3AO and Caov3 cells were transfected with MUC1 overexpressing plasmid for 48 h, and cultured in spheroid culture conditions for 7 days. Representative images and the relative expression levels of CSC-related markers NANOG and c-MYC were detected by immunoblot analysis ( c ), and the CD44 + CD24 − population was detected using FCM ( d ). e 3AO and Caov3 cells were transfected with a control vector, STON2 overexpression plasmid, or STON2 overexpression plasmid plus MUC1 overexpression plasmid for 48 h and cultured in spheroid culture conditions for 7 days. Sphere-formation (sphere > 50 μm) was assessed. Scale bars, 50 μm. f 3AO cells were transfected with a MUC1-specific shRNA, or MUC1 overexpressing plasmid for 48 h, and cultured in spheroid culture conditions for 7 days. EMT-related markers (E-cadherin, N-cadherin, and fibronectin) were detected using immunoblot analysis. Data represents the mean ± S.E. of three independent experiments. The level of significance is indicated by * P

Techniques Used: Transfection, shRNA, Cell Culture, Expressing, Plasmid Preparation, Over Expression

STON2 knockdown promotes stem-like properties of ovarian cancer cells. a 3AO and Caov3 cells were transfected with a STON2-specific shRNA for 48 h, and cultured in spheroid culture conditions for 7 days. The expressions of STON2 and the CSC-related markers NANOG and c-MYC were detected by immunoblot analysis. b Quantitation and representative images of sphere-formation (sphere > 50 μm), Scale bars, 50 μm. c The effect of STON2 knockdown on the CD44 + CD24 − phenotype was analyzed using FCM. d Immunoblot analysis of EMT-related markers (E-cadherin, N-cadherin, and fibronectin). e qPCR analysis of EMT-related markers (E-cadherin, N-cadherin, and fibronectin). f , g 3AO cells were transfected with shNC or shSTON2 for 48 h, cultured in spheroid culture conditions for 7 days, and then injected into NOD/SCID mice ( n = 5 in each group). All mice were sacrificed at week 4 and the tumor incidence evaluated (left panel). Subcutaneous tumors are shown (right panel) ( f ).Tumor diameters of xenografts from mice injected with 1 × 10 5 cells were measured at a regular intervals of 1 week for up to 4 weeks and the tumor volume was calculated ( g ). Data represents mean ± S.E. of three independent experiments. The level of significance is indicated by * P
Figure Legend Snippet: STON2 knockdown promotes stem-like properties of ovarian cancer cells. a 3AO and Caov3 cells were transfected with a STON2-specific shRNA for 48 h, and cultured in spheroid culture conditions for 7 days. The expressions of STON2 and the CSC-related markers NANOG and c-MYC were detected by immunoblot analysis. b Quantitation and representative images of sphere-formation (sphere > 50 μm), Scale bars, 50 μm. c The effect of STON2 knockdown on the CD44 + CD24 − phenotype was analyzed using FCM. d Immunoblot analysis of EMT-related markers (E-cadherin, N-cadherin, and fibronectin). e qPCR analysis of EMT-related markers (E-cadherin, N-cadherin, and fibronectin). f , g 3AO cells were transfected with shNC or shSTON2 for 48 h, cultured in spheroid culture conditions for 7 days, and then injected into NOD/SCID mice ( n = 5 in each group). All mice were sacrificed at week 4 and the tumor incidence evaluated (left panel). Subcutaneous tumors are shown (right panel) ( f ).Tumor diameters of xenografts from mice injected with 1 × 10 5 cells were measured at a regular intervals of 1 week for up to 4 weeks and the tumor volume was calculated ( g ). Data represents mean ± S.E. of three independent experiments. The level of significance is indicated by * P

Techniques Used: Transfection, shRNA, Cell Culture, Quantitation Assay, Real-time Polymerase Chain Reaction, Injection, Mouse Assay

STON2 overexpression inhibits stem-like properties of ovarian cancer cells . a 3AO and Caov3 cells were transfected with STON2 overexpressing plasmid for 48 h and cultured in spheroid culture conditions for 7 days. Expression of STON2 and the CSC-related markers NANOG and c-MYC were determined by immunoblot analysis. b Quantitation and representative images of sphere-formation (sphere > 50 μm), Scale bars, 50 μm. c Effect of STON2 overexpression on the CD44 + CD24 − phenotype was analyzed using FCM. d qPCR analysis of EMT-related markers (E-cadherin, N-cadherin, and fibronectin). Data represents the mean ± S.E. of three independent experiments. The level of significance is indicated by * P
Figure Legend Snippet: STON2 overexpression inhibits stem-like properties of ovarian cancer cells . a 3AO and Caov3 cells were transfected with STON2 overexpressing plasmid for 48 h and cultured in spheroid culture conditions for 7 days. Expression of STON2 and the CSC-related markers NANOG and c-MYC were determined by immunoblot analysis. b Quantitation and representative images of sphere-formation (sphere > 50 μm), Scale bars, 50 μm. c Effect of STON2 overexpression on the CD44 + CD24 − phenotype was analyzed using FCM. d qPCR analysis of EMT-related markers (E-cadherin, N-cadherin, and fibronectin). Data represents the mean ± S.E. of three independent experiments. The level of significance is indicated by * P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Expressing, Quantitation Assay, Real-time Polymerase Chain Reaction

8) Product Images from "NOTCH1 inhibition in vivo results in mammary tumor regression and reduced mammary tumorsphere-forming activity in vitro"

Article Title: NOTCH1 inhibition in vivo results in mammary tumor regression and reduced mammary tumorsphere-forming activity in vitro

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3321

NOTCH1 regulates Nanog expression in mouse mammary tumor cells . (A) Nanog expression is suppressed on NOTCH1 inhibition. RNA was harvested from tumor cell lines left untreated or treated with doxycycline (2 μg/ml) for 24 hours and c-Myc, Hes1, Deltex1, Hey1 , and Nanog expression levels determined with quantitative real-time PCR. Expression is normalized to the untreated control. (B) Doxycycline treatment reduces Nanog protein levels. The mouse mammary tumor lines (8542, 8526) were left untreated or treated with doxycycline (2 μg/ml) for the time periods indicated. Lysates were analyzed for ICN1 and Nanog expression with immunoblotting, and relative band intensities were quantified. (C , D) Nanog is expressed in the nuclei of NOTCH1-transformed mouse mammary tumor cell lines and primary tumorspheres. The mouse mammary tumor cell line 8542 was grown on coverslips, and primary tumorspheres were dissociated and centrifuged onto slides, followed by fixation in 4% paraformaldehyde. The cells were then permeabilized with Triton X-100 and immunostained by using antibodies against cytokeratin 8/18 (red) or Nanog (green). Cells were photographed at 400 × magnification. Scale bars, 100 μm. (E) NOTCH1 regulates NANOG expression in human breast cancer cells. The human basal-like breast cancer cell line MDA-MB-231 was left untreated or treated with the γ-secretase inhibitor Compound E (10 μ M ) for the time periods indicated, and intracellular NOTCH1 and NANOG expression levels examined with immunoblotting.
Figure Legend Snippet: NOTCH1 regulates Nanog expression in mouse mammary tumor cells . (A) Nanog expression is suppressed on NOTCH1 inhibition. RNA was harvested from tumor cell lines left untreated or treated with doxycycline (2 μg/ml) for 24 hours and c-Myc, Hes1, Deltex1, Hey1 , and Nanog expression levels determined with quantitative real-time PCR. Expression is normalized to the untreated control. (B) Doxycycline treatment reduces Nanog protein levels. The mouse mammary tumor lines (8542, 8526) were left untreated or treated with doxycycline (2 μg/ml) for the time periods indicated. Lysates were analyzed for ICN1 and Nanog expression with immunoblotting, and relative band intensities were quantified. (C , D) Nanog is expressed in the nuclei of NOTCH1-transformed mouse mammary tumor cell lines and primary tumorspheres. The mouse mammary tumor cell line 8542 was grown on coverslips, and primary tumorspheres were dissociated and centrifuged onto slides, followed by fixation in 4% paraformaldehyde. The cells were then permeabilized with Triton X-100 and immunostained by using antibodies against cytokeratin 8/18 (red) or Nanog (green). Cells were photographed at 400 × magnification. Scale bars, 100 μm. (E) NOTCH1 regulates NANOG expression in human breast cancer cells. The human basal-like breast cancer cell line MDA-MB-231 was left untreated or treated with the γ-secretase inhibitor Compound E (10 μ M ) for the time periods indicated, and intracellular NOTCH1 and NANOG expression levels examined with immunoblotting.

Techniques Used: Expressing, Inhibition, Real-time Polymerase Chain Reaction, Transformation Assay, Multiple Displacement Amplification

9) 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

Prolonged in vitro cell culture of E14 mouse embryonic stem cells (mESCs) correlates with low Wnt/β-catenin activity. ( a ) Schematic representation of Young (YP) and Old passage (OP) E14 mESCs. ( b ) Representative bright field images of YP- and OP-mESCs. Round-shaped and flat colonies are indicated by white and yellow arrow, respectively. ( c ) Quantitative real-time PCR showing the expression profiles of Axin2 , Lef1 , Tcf1 , Sp5 in YP- and OP- mESCs. The transcriptional levels are normalized to Gapdh as reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and means of n = 3 independent experiments ± SE. ( d , e ) Representative immunofluorescence ( d ) and confocal microphotographs ( e ) of β-catenin. Nuclear demarcation is indicated by white circles (right panel). ( f ) Western blot analysis showing total and nuclear β-catenin protein in YP- and OP-mESCs and its quantification (n = 1) relative to total β-catenin in YP-mESCs. For quantification, densitometric analysis was performed with ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. ( g ) Representative immunofluorescence images showing OCT4 (green), NANOG (red) and their merge in YP- and OP-E14 mESCs. ( h , i ) Representative western blot analysis of OCT4 and NANOG in YP- and OP-mESCs ( h ) and its quantification represented as fold change over the protein amount in YP-mESCs and means of n = 3 independent experiments ± SE ( i ). ( f , h , i ) Full scan blots are available in Supplementary Fig. 6 . ( j – m ) FACS-plot showing the percentage of E-cadherin +( j ) and SSEA1 +cells ( l ) in YP- and OP- mESCs and its quantification ( k , m ) as means of 3 technical replicates ± SE (NS: non stained). ( n , o ) Representative cell cycle FACS profile analyzed with Flowjo software ( n ) and its quantification ( o ) represented as percentage of total cells and means of n = 3 independent experiments ± SE. Scale bar is 200 ( b , d , g ) and 10 μm ( e ). ( d , e left panel, and g ) Nuclei were stained with DAPI. ( f , h ) β-tubulin and H3 were used as loading controls. ( c , i , k , m , o ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p-value
Figure Legend Snippet: Prolonged in vitro cell culture of E14 mouse embryonic stem cells (mESCs) correlates with low Wnt/β-catenin activity. ( a ) Schematic representation of Young (YP) and Old passage (OP) E14 mESCs. ( b ) Representative bright field images of YP- and OP-mESCs. Round-shaped and flat colonies are indicated by white and yellow arrow, respectively. ( c ) Quantitative real-time PCR showing the expression profiles of Axin2 , Lef1 , Tcf1 , Sp5 in YP- and OP- mESCs. The transcriptional levels are normalized to Gapdh as reference gene. Data are represented as fold change (2 −ΔΔCt ) relative to the YP-E14 mESCs and means of n = 3 independent experiments ± SE. ( d , e ) Representative immunofluorescence ( d ) and confocal microphotographs ( e ) of β-catenin. Nuclear demarcation is indicated by white circles (right panel). ( f ) Western blot analysis showing total and nuclear β-catenin protein in YP- and OP-mESCs and its quantification (n = 1) relative to total β-catenin in YP-mESCs. For quantification, densitometric analysis was performed with ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. ( g ) Representative immunofluorescence images showing OCT4 (green), NANOG (red) and their merge in YP- and OP-E14 mESCs. ( h , i ) Representative western blot analysis of OCT4 and NANOG in YP- and OP-mESCs ( h ) and its quantification represented as fold change over the protein amount in YP-mESCs and means of n = 3 independent experiments ± SE ( i ). ( f , h , i ) Full scan blots are available in Supplementary Fig. 6 . ( j – m ) FACS-plot showing the percentage of E-cadherin +( j ) and SSEA1 +cells ( l ) in YP- and OP- mESCs and its quantification ( k , m ) as means of 3 technical replicates ± SE (NS: non stained). ( n , o ) Representative cell cycle FACS profile analyzed with Flowjo software ( n ) and its quantification ( o ) represented as percentage of total cells and means of n = 3 independent experiments ± SE. Scale bar is 200 ( b , d , g ) and 10 μm ( e ). ( d , e left panel, and g ) Nuclei were stained with DAPI. ( f , h ) β-tubulin and H3 were used as loading controls. ( c , i , k , m , o ) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p-value

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

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

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

10) Product Images from "Mouse Meningiocytes Express Sox2 and Yield High Efficiency of Chimeras after Nuclear Reprogramming with Exogenous Factors *Mouse Meningiocytes Express Sox2 and Yield High Efficiency of Chimeras after Nuclear Reprogramming with Exogenous Factors * S⃞"

Article Title: Mouse Meningiocytes Express Sox2 and Yield High Efficiency of Chimeras after Nuclear Reprogramming with Exogenous Factors *Mouse Meningiocytes Express Sox2 and Yield High Efficiency of Chimeras after Nuclear Reprogramming with Exogenous Factors * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M806788200

Generation of iPS cells from mouse meningeal cells. A , DNA microarray comparison between meningeal cells and the indicated cell types. A scatter-plot representation of the expression values for all probe sets is shown. Positions of Oct4, Sox2, and Nanog are marked with arrows . The parallel lines indicate 2-, 3-, 5-, and 10-fold changes in gene expression (up or down). B , microscope immunofluorescence for Sox2 ( green ) of meningeal cells and MEF. 4′,6-Diamidino-2-phenylindole ( DAPI ) staining is shown in blue. Bars indicate the magnification. C , scheme showing the extraction of meningeal membranes from newborn mice and subsequent transduction of meningeal cells using a mixture of retroviruses. D , cells derived from three selected iPS clones formed ES-like colonies and showed positive for AP staining. E , semiquantitative RT-PCR demonstrated integration of the corresponding transgenes in the genome of the three meningeal iPS clones. Sets of primers that amplify both the vector and the transduced factor were used. F , Southern blot also demonstrated integration of the Klf4 transgene in the genome of the three iPS clones. R1 mouse ES cells were used as a control.
Figure Legend Snippet: Generation of iPS cells from mouse meningeal cells. A , DNA microarray comparison between meningeal cells and the indicated cell types. A scatter-plot representation of the expression values for all probe sets is shown. Positions of Oct4, Sox2, and Nanog are marked with arrows . The parallel lines indicate 2-, 3-, 5-, and 10-fold changes in gene expression (up or down). B , microscope immunofluorescence for Sox2 ( green ) of meningeal cells and MEF. 4′,6-Diamidino-2-phenylindole ( DAPI ) staining is shown in blue. Bars indicate the magnification. C , scheme showing the extraction of meningeal membranes from newborn mice and subsequent transduction of meningeal cells using a mixture of retroviruses. D , cells derived from three selected iPS clones formed ES-like colonies and showed positive for AP staining. E , semiquantitative RT-PCR demonstrated integration of the corresponding transgenes in the genome of the three meningeal iPS clones. Sets of primers that amplify both the vector and the transduced factor were used. F , Southern blot also demonstrated integration of the Klf4 transgene in the genome of the three iPS clones. R1 mouse ES cells were used as a control.

Techniques Used: Microarray, Expressing, Microscopy, Immunofluorescence, Staining, Mouse Assay, Transduction, Derivative Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Southern Blot

11) Product Images from "Isolation and characterization of mesenchymal stem cells from human fetus heart"

Article Title: Isolation and characterization of mesenchymal stem cells from human fetus heart

Journal: PLoS ONE

doi: 10.1371/journal.pone.0192244

Expression of pluripotency and embryonic markers and cardiovascular genes by hfC-MSCs and hBM-MSCs. Representative photomicrographs (40X, 20μm) of human fetal cardiac mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (A: OCT-4; B: hoechst dye), Nanog (C: Nanog; D: hoechst dye), SOX-2 (E: SOX-2; F: hoechst dye) and representative photomicrographs of (40X, 20μm) of human bone marrow mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (G: OCT-4; H: hoechst dye), Nanog (I: Nanog; J: hoechst dye), SOX-2 (K: SOX-2; L: hoechst dye).
Figure Legend Snippet: Expression of pluripotency and embryonic markers and cardiovascular genes by hfC-MSCs and hBM-MSCs. Representative photomicrographs (40X, 20μm) of human fetal cardiac mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (A: OCT-4; B: hoechst dye), Nanog (C: Nanog; D: hoechst dye), SOX-2 (E: SOX-2; F: hoechst dye) and representative photomicrographs of (40X, 20μm) of human bone marrow mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (G: OCT-4; H: hoechst dye), Nanog (I: Nanog; J: hoechst dye), SOX-2 (K: SOX-2; L: hoechst dye).

Techniques Used: Expressing

12) Product Images from "The Antihelminthic Niclosamide Inhibits Cancer Stemness, Extracellular Matrix Remodeling, and Metastasis through Dysregulation of the Nuclear β-catenin/c-Myc axis in OSCC"

Article Title: The Antihelminthic Niclosamide Inhibits Cancer Stemness, Extracellular Matrix Remodeling, and Metastasis through Dysregulation of the Nuclear β-catenin/c-Myc axis in OSCC

Journal: Scientific Reports

doi: 10.1038/s41598-018-30692-3

Downregulated β-Catenin expression is critical for inhibition of the cancer stemness, migration, invasion and colony-formation of ALDH+ oral cancer cells. ( A ) Reduced expression levels of β-catenin, Snail, c-Myc, Cyclin D1, Sox2, Oct4 and Nanog proteins in tumorspheres derived from ALDH+ SCC4 cells transfected with β-catenin-specific siRNA, compared with control siRNA-transfected group, using western blot assays. ( B ) Images showing that the number of invaded and migrated ALDH+ SCC4 cells was significantly decreased in the β-catenin-specific siRNA group, compared to the control siRNA or wild type cell group; Original magnification, ×200 (left). Graphical representation of the significant inhibitory effect of β-catenin siRNA transfection on cell migration and invasion (right). ( C ) The number of tumorspheres formed by cells transfected with β-catenin siRNA was significantly less than by the control siRNA or wild type cell group. ( D ) The number of colonies formed by cells transfected with β-catenin siRNA was significantly less than by the control siRNA or wild type cell group. Data represents experiments performed in triplicates and expressed as mean ± SD. *p
Figure Legend Snippet: Downregulated β-Catenin expression is critical for inhibition of the cancer stemness, migration, invasion and colony-formation of ALDH+ oral cancer cells. ( A ) Reduced expression levels of β-catenin, Snail, c-Myc, Cyclin D1, Sox2, Oct4 and Nanog proteins in tumorspheres derived from ALDH+ SCC4 cells transfected with β-catenin-specific siRNA, compared with control siRNA-transfected group, using western blot assays. ( B ) Images showing that the number of invaded and migrated ALDH+ SCC4 cells was significantly decreased in the β-catenin-specific siRNA group, compared to the control siRNA or wild type cell group; Original magnification, ×200 (left). Graphical representation of the significant inhibitory effect of β-catenin siRNA transfection on cell migration and invasion (right). ( C ) The number of tumorspheres formed by cells transfected with β-catenin siRNA was significantly less than by the control siRNA or wild type cell group. ( D ) The number of colonies formed by cells transfected with β-catenin siRNA was significantly less than by the control siRNA or wild type cell group. Data represents experiments performed in triplicates and expressed as mean ± SD. *p

Techniques Used: Expressing, Inhibition, Migration, Derivative Assay, Transfection, Western Blot

13) Product Images from "RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73"

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

Journal: Nature Communications

doi: 10.1038/s41467-017-02786-5

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

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

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

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

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

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

14) Product Images from "Suppression of epithelial–mesenchymal transition and apoptotic pathways by miR-294/302 family synergistically blocks let-7-induced silencing of self-renewal in embryonic stem cells"

Article Title: Suppression of epithelial–mesenchymal transition and apoptotic pathways by miR-294/302 family synergistically blocks let-7-induced silencing of self-renewal in embryonic stem cells

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.205

Combined suppression of the EMT and apoptotic pathways synergistically promotes ESC self-renewal. ( a ) AP staining of let-7c-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with dimethyl sulfoxide (DMSO), RepSox, or RepSox plus CHIR (R C). ( b ) qRT-PCR analysis of pluripotency markers in let-7c-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO, RepSox, or R C. β -Actin gene was used as a control. For each gene, data were normalized to mock-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO. Shown are mean±S.D., n =3. ( c ) Evaluation of synergy between suppression of the EMT pathway and suppression of the apoptotic pathway in promoting the expression of Oct4, Sox2, and Nanog. In all cases, the measured value was at least twofold greater than the value expected from additive effects. ( d ) Western blotting analysis of OCT4, SOX2, and NANOG. Representative gels are shown. Data were first normalized to actin and then to mock-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO. Shown are mean±range. n =2. ( e ) Colony-formation assays. Data were normalized to mock-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO. Shown are mean±S.D., n =3. ( f ) Graphic model of the opposing functions of miR-294/302 and let-7 in regulating ESC self-renewal through antagonistically regulating the EMT and apoptotic pathways
Figure Legend Snippet: Combined suppression of the EMT and apoptotic pathways synergistically promotes ESC self-renewal. ( a ) AP staining of let-7c-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with dimethyl sulfoxide (DMSO), RepSox, or RepSox plus CHIR (R C). ( b ) qRT-PCR analysis of pluripotency markers in let-7c-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO, RepSox, or R C. β -Actin gene was used as a control. For each gene, data were normalized to mock-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO. Shown are mean±S.D., n =3. ( c ) Evaluation of synergy between suppression of the EMT pathway and suppression of the apoptotic pathway in promoting the expression of Oct4, Sox2, and Nanog. In all cases, the measured value was at least twofold greater than the value expected from additive effects. ( d ) Western blotting analysis of OCT4, SOX2, and NANOG. Representative gels are shown. Data were first normalized to actin and then to mock-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO. Shown are mean±range. n =2. ( e ) Colony-formation assays. Data were normalized to mock-transfected Bak− / − , Bax-/flox , Dgcr8−/− ESCs treated with DMSO. Shown are mean±S.D., n =3. ( f ) Graphic model of the opposing functions of miR-294/302 and let-7 in regulating ESC self-renewal through antagonistically regulating the EMT and apoptotic pathways

Techniques Used: Staining, Transfection, Quantitative RT-PCR, Expressing, Western Blot

15) Product Images from "Characterization of immortalized mesenchymal stem cells derived from foetal porcine pancreas"

Article Title: Characterization of immortalized mesenchymal stem cells derived from foetal porcine pancreas

Journal: Cell Proliferation

doi: 10.1111/j.1365-2184.2010.00714.x

Pluripotent ESC markers were expressed in iPMSCs. Immunocytochemistry analysis showed that iPMSCs were positive for pluripotent ESC markers (Oct4, Sox2, Nanog, C‐Myc and Klf4) and PCNA, Bar = 20 μm; RT‐PCR analysis showed that iPMSCs were positive for Oct4, Sox2, Nanog, C‐Myc, Klf4 and PCNA.
Figure Legend Snippet: Pluripotent ESC markers were expressed in iPMSCs. Immunocytochemistry analysis showed that iPMSCs were positive for pluripotent ESC markers (Oct4, Sox2, Nanog, C‐Myc and Klf4) and PCNA, Bar = 20 μm; RT‐PCR analysis showed that iPMSCs were positive for Oct4, Sox2, Nanog, C‐Myc, Klf4 and PCNA.

Techniques Used: Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction

16) Product Images from "Generation of Transgenic Rats through Induced Pluripotent Stem Cells *"

Article Title: Generation of Transgenic Rats through Induced Pluripotent Stem Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.492900

Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.
Figure Legend Snippet: Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Negative Control, Staining, Positive Control, Polymerase Chain Reaction, Marker, Western Blot, Immunofluorescence, Genomic Sequencing, Methylation

17) Product Images from "A Systemic Evaluation of Cardiac Differentiation from mRNA Reprogrammed Human Induced Pluripotent Stem Cells"

Article Title: A Systemic Evaluation of Cardiac Differentiation from mRNA Reprogrammed Human Induced Pluripotent Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0103485

Characterization of transgene free iPSC. A, Micrographs showing morphological changes during mRNA reprograming on day 6 (i), 8 (ii), 15 (iii) and 20 (iv) with live Tra-1-60 (v) staining (day 20). vi, image shows hiPSCs positively stained with alkaline phosphatase. B, Immunostaining of the undifferentiated hiPSC colonies with Oct-4, Sox2, Nanog, SSEA-4, Tra-1-60 and Tra-1-81 antibodies followed by counterstaining with DAPI. Scale bar –200 µm. C, Semi-quantitative gene expression levels of pluripotency associated genes in two hiPSC clones (lane 2 and 3) in comparison with hESC line (lane 1, positive control) with GAPDH as internal loading control. D, A typical normal karyogram of hiPSC clone 1 and Hematoxylin and eosin (H E) staining of teratoma sections of clone 1 showing the presence of ectoderm (neural rosettes), mesoderm (cartilage) and endoderm (secretory tubule). Scale bar –200 µm.
Figure Legend Snippet: Characterization of transgene free iPSC. A, Micrographs showing morphological changes during mRNA reprograming on day 6 (i), 8 (ii), 15 (iii) and 20 (iv) with live Tra-1-60 (v) staining (day 20). vi, image shows hiPSCs positively stained with alkaline phosphatase. B, Immunostaining of the undifferentiated hiPSC colonies with Oct-4, Sox2, Nanog, SSEA-4, Tra-1-60 and Tra-1-81 antibodies followed by counterstaining with DAPI. Scale bar –200 µm. C, Semi-quantitative gene expression levels of pluripotency associated genes in two hiPSC clones (lane 2 and 3) in comparison with hESC line (lane 1, positive control) with GAPDH as internal loading control. D, A typical normal karyogram of hiPSC clone 1 and Hematoxylin and eosin (H E) staining of teratoma sections of clone 1 showing the presence of ectoderm (neural rosettes), mesoderm (cartilage) and endoderm (secretory tubule). Scale bar –200 µm.

Techniques Used: Staining, Immunostaining, Expressing, Clone Assay, Positive Control

18) Product Images from "Human Very Small Embryonic-Like Stem Cells Are Present in Normal Peripheral Blood of Young, Middle-Aged, and Aged Subjects"

Article Title: Human Very Small Embryonic-Like Stem Cells Are Present in Normal Peripheral Blood of Young, Middle-Aged, and Aged Subjects

Journal: Stem Cells International

doi: 10.1155/2016/7651645

Immunofluorescence analysis of PB-derived VSELs. A typical triple-staining with 4′,6-diamidino-2-phenylindole (DAPI) (blue: nuclei), fluorescein isothiocyanate- (FITC-) CD45 (green fluorescence), and tetramethylrhodamine-5-isothiocyanate- (TRITC-) SSEA-4 − , TRA-1-81, OCT-4, or NANOG (red) shows (a) VSELs: small CD45 − cells NANOG + and leukocytes: greater CD45 + NANOG − cells and (b) VSELs: CD45 − cells which express OCT-4 in nuclei or TRA-1-81 and SSEA-4 on the surface.
Figure Legend Snippet: Immunofluorescence analysis of PB-derived VSELs. A typical triple-staining with 4′,6-diamidino-2-phenylindole (DAPI) (blue: nuclei), fluorescein isothiocyanate- (FITC-) CD45 (green fluorescence), and tetramethylrhodamine-5-isothiocyanate- (TRITC-) SSEA-4 − , TRA-1-81, OCT-4, or NANOG (red) shows (a) VSELs: small CD45 − cells NANOG + and leukocytes: greater CD45 + NANOG − cells and (b) VSELs: CD45 − cells which express OCT-4 in nuclei or TRA-1-81 and SSEA-4 on the surface.

Techniques Used: Immunofluorescence, Derivative Assay, Staining, Fluorescence

Relative expressions of PSC markers, Oct-4, Nanog, and Sox2, were measured by RT-qPCR and compared using equal amounts of mRNA isolated from CNT PB of young, middle, and older healthy volunteers. The relative expression of each PSC marker is calculated according to a positive control PCR (RNA isolated from H9 and HUES 3 hESC lines). Data represent the mean ± standard deviation for each age group. The difference in mRNA expression of these markers between the three groups of age was not statistically significant ( P > 0.05, Kruskal-Wallis test).
Figure Legend Snippet: Relative expressions of PSC markers, Oct-4, Nanog, and Sox2, were measured by RT-qPCR and compared using equal amounts of mRNA isolated from CNT PB of young, middle, and older healthy volunteers. The relative expression of each PSC marker is calculated according to a positive control PCR (RNA isolated from H9 and HUES 3 hESC lines). Data represent the mean ± standard deviation for each age group. The difference in mRNA expression of these markers between the three groups of age was not statistically significant ( P > 0.05, Kruskal-Wallis test).

Techniques Used: Quantitative RT-PCR, Isolation, Expressing, Marker, Positive Control, Polymerase Chain Reaction, Standard Deviation

19) Product Images from "Transcriptional regulation of Sox2 by the retinoblastoma family of pocket proteins"

Article Title: Transcriptional regulation of Sox2 by the retinoblastoma family of pocket proteins

Journal: Oncotarget

doi:

Binding of the Rb family of pocket proteins to the Sox2 - SRR2 enhancer and effect of their absence on histone marks a, Chromatin immunoprecipitation (ChIP) assay using antibodies against p107, p130, and pRb followed by semi-quantitative PCR using primers amplifying the Sox2-SRR2 . Primers amplifying β-actin promoter and IgG were used as negative controls. b, ChIP of repressive H3K27me3 and active H3K4me3 histone marks in the Sox2-SRR2 enhancer of wt, Rb -null, and p130 -null MEFs, using two different sets of primers amplifying the Sox2-SRR2 enhancer (upper and middle panels). Control ChIP assay using primers amplifying Nanog promoter is shown at the bottom panel. All data correspond to the average ± s.d. of qPCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p
Figure Legend Snippet: Binding of the Rb family of pocket proteins to the Sox2 - SRR2 enhancer and effect of their absence on histone marks a, Chromatin immunoprecipitation (ChIP) assay using antibodies against p107, p130, and pRb followed by semi-quantitative PCR using primers amplifying the Sox2-SRR2 . Primers amplifying β-actin promoter and IgG were used as negative controls. b, ChIP of repressive H3K27me3 and active H3K4me3 histone marks in the Sox2-SRR2 enhancer of wt, Rb -null, and p130 -null MEFs, using two different sets of primers amplifying the Sox2-SRR2 enhancer (upper and middle panels). Control ChIP assay using primers amplifying Nanog promoter is shown at the bottom panel. All data correspond to the average ± s.d. of qPCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test

Cells lacking Rb or p130 express higher levels of Sox2 a, Sox2 (left) or Nanog (right) mRNA levels in wt, Rb -null, p107 -null, and p130 -null primary MEFs as assessed by TaqMan expression analysis. Absolute values are referenced to the levels obtained with primary wt cells. b, Sox2 mRNA levels in wt, RB -null, p107 -null, and p130 -null immortalized fibroblasts assessed as in (a). Absolute values are referenced to the levels obtained with wt cells. c, Sox2 mRNA (upper panel) and protein expression by Western blot (lower panel) in cell extracts from wt and SV40 large-T antigen (LT) immortalized fibroblasts. Absolute values are referenced to the levels obtained with wt cells. d, Graph showing the analysis of GFP expression by flow cytometry from EOS pluripotency reporter plasmid introduced in reprogrammable primary (i4F-MEFs, black line) or immortalized (i4F-MEFs-shp53, green line) cells (upper panel). The settings were previously adjusted to consider i4F-MEFs without EOS plasmid as GFP negative. Sox2 mRNA levels expressed by wt or immortalized wt-shp53 MEFs, i4F or immortalized i4F-shp53 MEFs, and ESCs, measured by qRT-PCR (lower panel). Values are referenced to the levels obtained using wt primary MEFs and in log 10 scale. All data correspond to the average ± s.d. of qRT-PCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p
Figure Legend Snippet: Cells lacking Rb or p130 express higher levels of Sox2 a, Sox2 (left) or Nanog (right) mRNA levels in wt, Rb -null, p107 -null, and p130 -null primary MEFs as assessed by TaqMan expression analysis. Absolute values are referenced to the levels obtained with primary wt cells. b, Sox2 mRNA levels in wt, RB -null, p107 -null, and p130 -null immortalized fibroblasts assessed as in (a). Absolute values are referenced to the levels obtained with wt cells. c, Sox2 mRNA (upper panel) and protein expression by Western blot (lower panel) in cell extracts from wt and SV40 large-T antigen (LT) immortalized fibroblasts. Absolute values are referenced to the levels obtained with wt cells. d, Graph showing the analysis of GFP expression by flow cytometry from EOS pluripotency reporter plasmid introduced in reprogrammable primary (i4F-MEFs, black line) or immortalized (i4F-MEFs-shp53, green line) cells (upper panel). The settings were previously adjusted to consider i4F-MEFs without EOS plasmid as GFP negative. Sox2 mRNA levels expressed by wt or immortalized wt-shp53 MEFs, i4F or immortalized i4F-shp53 MEFs, and ESCs, measured by qRT-PCR (lower panel). Values are referenced to the levels obtained using wt primary MEFs and in log 10 scale. All data correspond to the average ± s.d. of qRT-PCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p

Techniques Used: Expressing, Western Blot, Flow Cytometry, Cytometry, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Absence of Rb allows two-factor (Oct4 and Klf4) reprogramming a, Representative pictures of iPSC colonies expressing GFP from the EOS pluripotency reporter plasmid (top panels), and stained for alkaline phosphatase (AP, bottom panels). Shown are iPSC colonies obtained in wt MEFs after three-factor expression (Oct4, Klf4, and Sox2; 3F-OKS; left panel), and Rb -null MEFs after three-factor (3F-OKS; middle panel) or two-factor (Oct4, Klf4; 2F-OK; right panel) expression. b, Pluripotency factor ( Oct4 , Sox2 , Klf4 , and Nanog ) mRNA expression by qRT-PCR in iPSCs obtained from wt primary MEFs reprogrammed by 3F-OKS, or Rb -null with 3F-OKS or 2F-OK. Null expression from MEFs is shown as negative control, and expression in ESCs as positive control. c, Western blot analysis of the expression of pluripotency factors (Oct4, Sox2, and Nanog) in the same set of cells as in (b). d, Representative pictures of embryoid bodies (EBs) obtained after in vitro spontaneous differentiation of iPSCs generated from wt primary MEFs reprogrammed by 3F-OKS, or Rb -null with 3F-OKS or 2F-OK. e, Differentiation factor ( Nkx2.5 , Dlx3 , and Gata4 ) mRNA expression by qRT-PCR in EBs obtained from iPSCs generated from Rb -null primary MEFs reprogrammed by 2F-OK. Values are referred to the expression obtained for the corresponding iPSCs. All data correspond to the average ± s.d. of qRT-PCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p
Figure Legend Snippet: Absence of Rb allows two-factor (Oct4 and Klf4) reprogramming a, Representative pictures of iPSC colonies expressing GFP from the EOS pluripotency reporter plasmid (top panels), and stained for alkaline phosphatase (AP, bottom panels). Shown are iPSC colonies obtained in wt MEFs after three-factor expression (Oct4, Klf4, and Sox2; 3F-OKS; left panel), and Rb -null MEFs after three-factor (3F-OKS; middle panel) or two-factor (Oct4, Klf4; 2F-OK; right panel) expression. b, Pluripotency factor ( Oct4 , Sox2 , Klf4 , and Nanog ) mRNA expression by qRT-PCR in iPSCs obtained from wt primary MEFs reprogrammed by 3F-OKS, or Rb -null with 3F-OKS or 2F-OK. Null expression from MEFs is shown as negative control, and expression in ESCs as positive control. c, Western blot analysis of the expression of pluripotency factors (Oct4, Sox2, and Nanog) in the same set of cells as in (b). d, Representative pictures of embryoid bodies (EBs) obtained after in vitro spontaneous differentiation of iPSCs generated from wt primary MEFs reprogrammed by 3F-OKS, or Rb -null with 3F-OKS or 2F-OK. e, Differentiation factor ( Nkx2.5 , Dlx3 , and Gata4 ) mRNA expression by qRT-PCR in EBs obtained from iPSCs generated from Rb -null primary MEFs reprogrammed by 2F-OK. Values are referred to the expression obtained for the corresponding iPSCs. All data correspond to the average ± s.d. of qRT-PCR data. Statistical significance was assessed by the two-tailed Student's t-test: *** p

Techniques Used: Expressing, Plasmid Preparation, Staining, Quantitative RT-PCR, Negative Control, Positive Control, Western Blot, In Vitro, Generated, Two Tailed Test

20) Product Images from "Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters"

Article Title: Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084457

HeLa/TSA cells were enriched of cancer stem-like cells. A , HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog mRNA levels. The columns represent the average of three separate experiments; error bars, SD. B , The quantitative analysis of Sox2, Oct4, Nanog, Mdr-1 and UbB protein levels by western blotting. C , HeLa/TSA mammosphere cells were analyzed by immunofluorescence for Sox2, Oct4 and Nanog. The representative photographs were taken using a fluorescence microscope (original magnification, ×200). The nuclei were presented as detected using DAPI (4′, 6-Diamidino-2-phenylindole) staining. D , The analysis of the side-population cell fraction in HeLa and HeLa/TSA cell cultures. Left panel: HeLa; right panel: HeLa/TSA. E, The analysis of CD44 and CD24 expression of HeLa and HeLa/TSA cells. Left panel: HeLa; right panel: HeLa/TSA.
Figure Legend Snippet: HeLa/TSA cells were enriched of cancer stem-like cells. A , HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog mRNA levels. The columns represent the average of three separate experiments; error bars, SD. B , The quantitative analysis of Sox2, Oct4, Nanog, Mdr-1 and UbB protein levels by western blotting. C , HeLa/TSA mammosphere cells were analyzed by immunofluorescence for Sox2, Oct4 and Nanog. The representative photographs were taken using a fluorescence microscope (original magnification, ×200). The nuclei were presented as detected using DAPI (4′, 6-Diamidino-2-phenylindole) staining. D , The analysis of the side-population cell fraction in HeLa and HeLa/TSA cell cultures. Left panel: HeLa; right panel: HeLa/TSA. E, The analysis of CD44 and CD24 expression of HeLa and HeLa/TSA cells. Left panel: HeLa; right panel: HeLa/TSA.

Techniques Used: Western Blot, Immunofluorescence, Fluorescence, Microscopy, Staining, Expressing

UbB siRNA could down-regulate the SP and spheroids formation. A , HeLa cells were transfected with 10 nM UbB-targeting siRNA, or stealth RNAi control, for 72 hours. Real-time PCR analysis of relative UbB mRNA expression was proceeded. GAPDH was used as a reference control. B , The quantitative analysis of the UbB protein level by western blotting. Cells were treated as described in A. Beta-actin was used as a reference control. C , HeLa and HeLa/TSA mammosphere cells were infected with LV-UbBsi, which was cloned with UbBsi2, for 72 hours and subjected to analysis by western blotting for UbB protein level. D , LV-UbBsi-infected mammosphere cells were analyzed for the UbB, UbC, UbA52 and UbA80 mRNA levels. GAPDH was used as a reference control. The columns represent the average of three separate experiments; error bars, SD. E , LV-UbBsi-infected HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog mRNA levels. GAPDH was used as a reference control. The columns represent the average of three separate experiments; error bars, SD. F . LV-UbBsi-infected HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog protein levels. Beta-actin was used as a reference control. G , The analysis of the side-population cell fraction in LV-UbBsi-infected HeLa and HeLa/TSA cell cultures. H , The spheroid formation assays were performed on LV-UbBsi-infected cells.
Figure Legend Snippet: UbB siRNA could down-regulate the SP and spheroids formation. A , HeLa cells were transfected with 10 nM UbB-targeting siRNA, or stealth RNAi control, for 72 hours. Real-time PCR analysis of relative UbB mRNA expression was proceeded. GAPDH was used as a reference control. B , The quantitative analysis of the UbB protein level by western blotting. Cells were treated as described in A. Beta-actin was used as a reference control. C , HeLa and HeLa/TSA mammosphere cells were infected with LV-UbBsi, which was cloned with UbBsi2, for 72 hours and subjected to analysis by western blotting for UbB protein level. D , LV-UbBsi-infected mammosphere cells were analyzed for the UbB, UbC, UbA52 and UbA80 mRNA levels. GAPDH was used as a reference control. The columns represent the average of three separate experiments; error bars, SD. E , LV-UbBsi-infected HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog mRNA levels. GAPDH was used as a reference control. The columns represent the average of three separate experiments; error bars, SD. F . LV-UbBsi-infected HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog protein levels. Beta-actin was used as a reference control. G , The analysis of the side-population cell fraction in LV-UbBsi-infected HeLa and HeLa/TSA cell cultures. H , The spheroid formation assays were performed on LV-UbBsi-infected cells.

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Infection, Clone Assay

21) Product Images from "Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells *"

Article Title: Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.526442

Pin1 overexpression inhibits senescence, increases differentiation, and decreases cell death of CPCs. A , Cyclin B but not Cyclin D is decreased by Pin1 overexpression as seen by immunoblots and densitometric analyses in EGFP or Pin1 overexpressing CPCs ( n = 6). B , p53 and Rb expression is decreased in Pin1 overexpressing CPCs as seen by immunoblots and densitometric analyses ( n = 5). C , EGFP or Pin1 overexpressing CPCs were cultured until passage 22 and treated with SA-β-gal assay. EGFP expressing CPCs had more senescent cells ( blue cells indicated by arrows ) as seen in inverted microscopic images ( left ) and counts ( right ), whereas Pin1 overexpression significantly decreased SA-β-gal + cells ( n = 3). D , EGFP or Pin1 overexpressing CPCs were cultured in the presence or absence of serum and treated with propidium iodide. The percentage of dead cells were determined as cells positive for propidium iodide as measured by flow cytometry ( n = 3). E , immunoblots and densitometric analyses showing expression of α-SMA in EGFP or Pin1 overexpressing CPCs after treatment with dexamethasone for 6 days ( n = 4). F, quantitative RT-PCR analyses showing expression of cardiac troponin T ( cTNT ) in EGFP or Pin1 overexpressing CPCs after treatment with dexamethasone for 6 days relative to a 3-day Dex treatment ( n = 4). G , immunoblots and densitometric analyses showing expression of c-kit, c-Myc, Oct4, KLF4, and Nanog in EGFP or Pin1 overexpressing CPCs ( n = 4). H , CPC lines stably expressing EGFP or EGFP and Pin1 ( Pin1 ) were single cell sorted based on GFP expression and cultured for 8 days. Microscopic images ( left ) and counts ( right ) showing no differences in size or number of GFP + clones that grow out over 8 days ( left ). *, p
Figure Legend Snippet: Pin1 overexpression inhibits senescence, increases differentiation, and decreases cell death of CPCs. A , Cyclin B but not Cyclin D is decreased by Pin1 overexpression as seen by immunoblots and densitometric analyses in EGFP or Pin1 overexpressing CPCs ( n = 6). B , p53 and Rb expression is decreased in Pin1 overexpressing CPCs as seen by immunoblots and densitometric analyses ( n = 5). C , EGFP or Pin1 overexpressing CPCs were cultured until passage 22 and treated with SA-β-gal assay. EGFP expressing CPCs had more senescent cells ( blue cells indicated by arrows ) as seen in inverted microscopic images ( left ) and counts ( right ), whereas Pin1 overexpression significantly decreased SA-β-gal + cells ( n = 3). D , EGFP or Pin1 overexpressing CPCs were cultured in the presence or absence of serum and treated with propidium iodide. The percentage of dead cells were determined as cells positive for propidium iodide as measured by flow cytometry ( n = 3). E , immunoblots and densitometric analyses showing expression of α-SMA in EGFP or Pin1 overexpressing CPCs after treatment with dexamethasone for 6 days ( n = 4). F, quantitative RT-PCR analyses showing expression of cardiac troponin T ( cTNT ) in EGFP or Pin1 overexpressing CPCs after treatment with dexamethasone for 6 days relative to a 3-day Dex treatment ( n = 4). G , immunoblots and densitometric analyses showing expression of c-kit, c-Myc, Oct4, KLF4, and Nanog in EGFP or Pin1 overexpressing CPCs ( n = 4). H , CPC lines stably expressing EGFP or EGFP and Pin1 ( Pin1 ) were single cell sorted based on GFP expression and cultured for 8 days. Microscopic images ( left ) and counts ( right ) showing no differences in size or number of GFP + clones that grow out over 8 days ( left ). *, p

Techniques Used: Over Expression, Western Blot, Expressing, Cell Culture, β-Gal Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Stable Transfection, Clone Assay

22) Product Images from "Reprogramming of murine and human somatic cells using a single polycistronic vector"

Article Title: Reprogramming of murine and human somatic cells using a single polycistronic vector

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0811426106

4F2A iPS cells express pluripotency markers. ( A ) Immunostaining of Oct4 protein indicates high titer infections can be achieved with the 4F2A. MEFs were cultured in DOX media for 2 days after transduction with 4F2A + rtTA. ( B ) Morphology changes in Nanog-GFP
Figure Legend Snippet: 4F2A iPS cells express pluripotency markers. ( A ) Immunostaining of Oct4 protein indicates high titer infections can be achieved with the 4F2A. MEFs were cultured in DOX media for 2 days after transduction with 4F2A + rtTA. ( B ) Morphology changes in Nanog-GFP

Techniques Used: Immunostaining, Cell Culture, Transduction

23) Product Images from "Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity"

Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr1253

Proposed model for Tet1 -mediated epigenetic and transcriptional regulation of mESC self-renewal and pluripotency. Red arrows denote regulatory interactions inferred from data generated for this study. Tet1 , regulated by Oct4 ( 28 ), regulates DNA methylation (5mC) by converting 5mC to 5hmC ( 16 , 28 ). Tet1 regulates LIF/Stat3 signaling by facilitating Stat3 binding by an yet to be determined mechanism, and regulates the transcriptional regulatory module comprising Nanog, Esrrb, Tcl1, Tbx3, Klf2/4, Prdm14 and Lefty1/2. Tet1's regulation of Tet2 confers tight regulation of 5mC to 5hmC conversion. Tet1's negative regulation of de novo DNA methyltransferase Dnmt3b may provide an additional layer of Tet1-mediated regulation of 5mC. Silent and active promoters on the chromatin are denoted by broad red and green arrows, respectively.
Figure Legend Snippet: Proposed model for Tet1 -mediated epigenetic and transcriptional regulation of mESC self-renewal and pluripotency. Red arrows denote regulatory interactions inferred from data generated for this study. Tet1 , regulated by Oct4 ( 28 ), regulates DNA methylation (5mC) by converting 5mC to 5hmC ( 16 , 28 ). Tet1 regulates LIF/Stat3 signaling by facilitating Stat3 binding by an yet to be determined mechanism, and regulates the transcriptional regulatory module comprising Nanog, Esrrb, Tcl1, Tbx3, Klf2/4, Prdm14 and Lefty1/2. Tet1's regulation of Tet2 confers tight regulation of 5mC to 5hmC conversion. Tet1's negative regulation of de novo DNA methyltransferase Dnmt3b may provide an additional layer of Tet1-mediated regulation of 5mC. Silent and active promoters on the chromatin are denoted by broad red and green arrows, respectively.

Techniques Used: Generated, DNA Methylation Assay, Binding Assay

Tet1 negatively regulates de novo DNA methyltransferase Dnmt3b . ( A ) ChIP assay of select Stat3 target regions using an antibody against H3K9me3 in control and Tet1 KD mESCs (48 h). The y -axis represents enrichment over input normalized to a positive control region ( Myod1 ) for H3K9me3. Error bars represent SEM of three experiments. ( B ) Relative mRNA levels of DNA methyltransferases Dnmt1 , Dnmt3a and Dnmt3b in control and Tet1 KD mESCs 96 h after transfection. The mRNA levels in control cells are set as 1. Data are normalized to Actin. Error bars represent SEM of three experiments. ( C ) Western blot analysis showing protein levels of Nanog, Dnmt3a and Dnmt3b in control and Tet1 KD mESCs 96 h after transfection. Ran is used as a loading control. ( D ) Genome browser shot showing a region containing the Dnmt3b gene and results from Tet1 and Sin3a ChIP-Seq experiments by various groups (GSM numbers denote GEO accession). The red open rectangle highlights Tet1 occupancy at the promoter region of Dnmt3b , where a CpG island is present (green-filled rectangle). ( E ) Relative 5hmC levels at Dnmt3b locus in control and Tet1 KD (96 h) mESCs. Error bars represent SEM of three experiments.
Figure Legend Snippet: Tet1 negatively regulates de novo DNA methyltransferase Dnmt3b . ( A ) ChIP assay of select Stat3 target regions using an antibody against H3K9me3 in control and Tet1 KD mESCs (48 h). The y -axis represents enrichment over input normalized to a positive control region ( Myod1 ) for H3K9me3. Error bars represent SEM of three experiments. ( B ) Relative mRNA levels of DNA methyltransferases Dnmt1 , Dnmt3a and Dnmt3b in control and Tet1 KD mESCs 96 h after transfection. The mRNA levels in control cells are set as 1. Data are normalized to Actin. Error bars represent SEM of three experiments. ( C ) Western blot analysis showing protein levels of Nanog, Dnmt3a and Dnmt3b in control and Tet1 KD mESCs 96 h after transfection. Ran is used as a loading control. ( D ) Genome browser shot showing a region containing the Dnmt3b gene and results from Tet1 and Sin3a ChIP-Seq experiments by various groups (GSM numbers denote GEO accession). The red open rectangle highlights Tet1 occupancy at the promoter region of Dnmt3b , where a CpG island is present (green-filled rectangle). ( E ) Relative 5hmC levels at Dnmt3b locus in control and Tet1 KD (96 h) mESCs. Error bars represent SEM of three experiments.

Techniques Used: Chromatin Immunoprecipitation, Positive Control, Transfection, Western Blot

24) Product Images from "Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells *"

Article Title: Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.077156

Dies1 suppression maintains ESCs in undifferentiated state in the absence of LIF. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. This marker of ESC undifferentiated state was clearly observed in more than 50% of the colonies of Dies1 KD cells, although only about 10% of the NS shRNA transfected colonies were AP-positive. The experiments were done in triplicate. *, p ≤ 0.001. B , Dies1 KD ESCs grown 13 days in neural differentiation medium still expressed Oct3/4 and Nanog, although NS-transfected cells are completely negative for these master genes of stemness. Scale bars , 250 μm. C , mRNA levels of Oct3/4 and Nanog were measured in cells cultured as in A . The two mRNAs were strongly reduced in NS shRNA-transfected cells ( black bars ), although they were clearly detectable at 7 and 13 days after the induction of differentiation in Dies1 KD cells ( gray bars ). D , expression of a Dies1 cDNA resistant to the shRNA used to silence endogenous Dies1 rescued the phenotype of Dies1 KD ESCs. In cells expressing recombinant Dies1 ( gray bars ), Oct3/4 mRNA was expressed at the same levels of controls ( black bars ). E , as in D , Oct3/4 protein levels are rescued by Dies1 overexpression.
Figure Legend Snippet: Dies1 suppression maintains ESCs in undifferentiated state in the absence of LIF. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. This marker of ESC undifferentiated state was clearly observed in more than 50% of the colonies of Dies1 KD cells, although only about 10% of the NS shRNA transfected colonies were AP-positive. The experiments were done in triplicate. *, p ≤ 0.001. B , Dies1 KD ESCs grown 13 days in neural differentiation medium still expressed Oct3/4 and Nanog, although NS-transfected cells are completely negative for these master genes of stemness. Scale bars , 250 μm. C , mRNA levels of Oct3/4 and Nanog were measured in cells cultured as in A . The two mRNAs were strongly reduced in NS shRNA-transfected cells ( black bars ), although they were clearly detectable at 7 and 13 days after the induction of differentiation in Dies1 KD cells ( gray bars ). D , expression of a Dies1 cDNA resistant to the shRNA used to silence endogenous Dies1 rescued the phenotype of Dies1 KD ESCs. In cells expressing recombinant Dies1 ( gray bars ), Oct3/4 mRNA was expressed at the same levels of controls ( black bars ). E , as in D , Oct3/4 protein levels are rescued by Dies1 overexpression.

Techniques Used: Stable Transfection, Transfection, Staining, Marker, shRNA, Cell Culture, Expressing, Recombinant, Over Expression

Inhibition of Nodal/Activin signaling pathway masks the effects of Dies1 silencing in ESCs. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. Cells were treated with SB-431542 or with DMSO as a control. % of colonies AP-positive was calculated in triplicate experiments. *, p ≤ 0.001. B , Western blot analysis of Oct3/4 and Nanog in the cells treated as described in A .
Figure Legend Snippet: Inhibition of Nodal/Activin signaling pathway masks the effects of Dies1 silencing in ESCs. A , ESCs stably transfected with NS or Dies1 silencing shRNAs were grown for 6 days in the absence of LIF and then stained for AP. Cells were treated with SB-431542 or with DMSO as a control. % of colonies AP-positive was calculated in triplicate experiments. *, p ≤ 0.001. B , Western blot analysis of Oct3/4 and Nanog in the cells treated as described in A .

Techniques Used: Inhibition, Stable Transfection, Transfection, Staining, Western Blot

25) Product Images from "Generation of fertile offspring from Kitw/Kitwv mice through differentiation of gene corrected nuclear transfer embryonic stem cells"

Article Title: Generation of fertile offspring from Kitw/Kitwv mice through differentiation of gene corrected nuclear transfer embryonic stem cells

Journal: Cell Research

doi: 10.1038/cr.2015.74

). (A) Immunostaining of the W-1R EpiLCs (d0, d1, and d2) with anti-Oct4 (left) and anti-Nanog (middle). Nuclei were stained with Hoechst 33342 (blue; right). Scale bar = 50 μm. (B) Gene expression profiles during the EpiLC induction (triple repeats). The value for ntESCs (W-1R, P28) was set as 0. The average value is shown in the log 2 scale. (C) FACS sorting of SSEA1 and integrin β3 double-positive cells on day 2, 4, and 6 aggregates differentiated from W-1 (upper) and W-1R (lower) cells. (D) Gene expression profiles of SSEA1 and integrin β3 double-positive cells on day 6 after PGCLC induction. The average value is plotted on the log 2 scale with SDs. (E) Western blot analyses of H3K9me2 and H3K27me3 in W-1R ntESCs, W-1R d2 EpiLCs, and W-1R d6 PGCLCs (SSEA1 and integrin β3 double-positive cells). (F) Bisulfite sequencing of DMRs of Igf2r , Snrpn , H19 and Kcnq1ot1 in wild-type tail tip tissues, W-1R ntESCs and W-1R day-6 PGCLCs (SSEA1 and integrin β3 double-positive cells). White and black circles indicate unmethylated and methylated CpGs, respectively.
Figure Legend Snippet: ). (A) Immunostaining of the W-1R EpiLCs (d0, d1, and d2) with anti-Oct4 (left) and anti-Nanog (middle). Nuclei were stained with Hoechst 33342 (blue; right). Scale bar = 50 μm. (B) Gene expression profiles during the EpiLC induction (triple repeats). The value for ntESCs (W-1R, P28) was set as 0. The average value is shown in the log 2 scale. (C) FACS sorting of SSEA1 and integrin β3 double-positive cells on day 2, 4, and 6 aggregates differentiated from W-1 (upper) and W-1R (lower) cells. (D) Gene expression profiles of SSEA1 and integrin β3 double-positive cells on day 6 after PGCLC induction. The average value is plotted on the log 2 scale with SDs. (E) Western blot analyses of H3K9me2 and H3K27me3 in W-1R ntESCs, W-1R d2 EpiLCs, and W-1R d6 PGCLCs (SSEA1 and integrin β3 double-positive cells). (F) Bisulfite sequencing of DMRs of Igf2r , Snrpn , H19 and Kcnq1ot1 in wild-type tail tip tissues, W-1R ntESCs and W-1R day-6 PGCLCs (SSEA1 and integrin β3 double-positive cells). White and black circles indicate unmethylated and methylated CpGs, respectively.

Techniques Used: Immunostaining, Staining, Expressing, FACS, Western Blot, Methylation Sequencing, Methylation

The pluripotency of Kit w /Kit wv ). (A) Scheme for derivation of ntESCs from SCNT embryos. (B) Image of TTFs obtained from the Kit w /Kit wv adult mouse. Scale bar = 100 μm. (C) The TTF nuclear-transferred blastocysts. Scale bar = 100 μm. (D) Morphology of W-1 embryonic stem cell line. Scale bar = 100 μm. (E) Karyotype of the W-1 embryonic stem cell line. (F) Immunostaining of W-1 ESCs (Oct4, Nanog, and SSEA1). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 50 μm. (G) W-1 ESCs produced “all-ES” mouse by tetraploid complementation. (H) DNA sequencing of the W and WV point mutation loci in the W-1 “all-ES” mouse.
Figure Legend Snippet: The pluripotency of Kit w /Kit wv ). (A) Scheme for derivation of ntESCs from SCNT embryos. (B) Image of TTFs obtained from the Kit w /Kit wv adult mouse. Scale bar = 100 μm. (C) The TTF nuclear-transferred blastocysts. Scale bar = 100 μm. (D) Morphology of W-1 embryonic stem cell line. Scale bar = 100 μm. (E) Karyotype of the W-1 embryonic stem cell line. (F) Immunostaining of W-1 ESCs (Oct4, Nanog, and SSEA1). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 50 μm. (G) W-1 ESCs produced “all-ES” mouse by tetraploid complementation. (H) DNA sequencing of the W and WV point mutation loci in the W-1 “all-ES” mouse.

Techniques Used: Immunostaining, Staining, Produced, DNA Sequencing, Mutagenesis

26) Product Images from "KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription"

Article Title: KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.91

KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1
Figure Legend Snippet: KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1

Techniques Used: Chromatin Immunoprecipitation

27) Product Images from "Chronic Heart Failure Is Associated With Transforming Growth Factor Beta-Dependent Yield and Functional Decline in Atrial Explant-Derived c-Kit+ Cells"

Article Title: Chronic Heart Failure Is Associated With Transforming Growth Factor Beta-Dependent Yield and Functional Decline in Atrial Explant-Derived c-Kit+ Cells

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.113.000317

TGF‐β inhibition suppressed EMT and upregulated pluripotency gene expression in c‐Kit+ cells. TGF‐β signaling was inhibited by suppression of TGF‐β receptor type 1 (SB) or by suppression of Smad2/3 phosphorylation (SIS) for 7 days. A, SB and SIS treatments increased amounts of round phase‐bright cells compared with control. Scale bar=100 μm. B, Western blot analysis of c‐Kit+ cells treated with SB and SIS showed reductions of pSmad2/3 levels. C, qRT‐PCR analysis of EMT‐ and pluripotency‐related gene expression in SB‐ and SIS‐treated c‐Kit+ cells from sham and CHF explants. Fold changes were calculated as a ratio of the expression in the SB‐ or SIS‐treated group to the expression in the control group. n=5 per condition. *Fold changes > 2. D, Western blot analysis of Nanog in SB‐ and SIS‐treated c‐Kit+ cells. β‐Actin was used as a loading control. Representative blots are shown. E, Densitometry analysis of Nanog. n=3 per condition. * P
Figure Legend Snippet: TGF‐β inhibition suppressed EMT and upregulated pluripotency gene expression in c‐Kit+ cells. TGF‐β signaling was inhibited by suppression of TGF‐β receptor type 1 (SB) or by suppression of Smad2/3 phosphorylation (SIS) for 7 days. A, SB and SIS treatments increased amounts of round phase‐bright cells compared with control. Scale bar=100 μm. B, Western blot analysis of c‐Kit+ cells treated with SB and SIS showed reductions of pSmad2/3 levels. C, qRT‐PCR analysis of EMT‐ and pluripotency‐related gene expression in SB‐ and SIS‐treated c‐Kit+ cells from sham and CHF explants. Fold changes were calculated as a ratio of the expression in the SB‐ or SIS‐treated group to the expression in the control group. n=5 per condition. *Fold changes > 2. D, Western blot analysis of Nanog in SB‐ and SIS‐treated c‐Kit+ cells. β‐Actin was used as a loading control. Representative blots are shown. E, Densitometry analysis of Nanog. n=3 per condition. * P

Techniques Used: Inhibition, Expressing, Western Blot, Quantitative RT-PCR

28) Product Images from "Nanog safeguards early embryogenesis against global activation of maternal β-catenin activity by interfering with TCF factors"

Article Title: Nanog safeguards early embryogenesis against global activation of maternal β-catenin activity by interfering with TCF factors

Journal: bioRxiv

doi: 10.1101/821702

Wnt/β-catenin activity is hyper-activated in nanog mutant. ( a ) Translation of Nanog protein totally disappeared in MZ nanog . Nanog protein can be detected as early as 64-cell stage and vanished at 75% epiboly stage in WT embryos, while no Nanog protein was detected in MZ nanog . ( b ) Immunolocalization of Nanog on cryosections of WT and MZ nanog at sphere stage. Nanog is localized in cell nucleus of WT and disappeared in MZ nanog . ( c ) Injection of low dose of β-catenin mRNA (200 pg) induced slightly up-regulation of boz and chd in WT embryos, but highly increasing in MZ nanog embryos, indicating that Wnt activity is over-activated because of nanog LOF. Injection or co-injection was performed at one-cell stage. boz was detected at 4 hpf, chd was detected at 4.5 hpf. ( d ) The percentage of embryos counted in WISH of (c) . ( e ) RT-qPCR showed the expression of boz is significantly up-regulated in MZ nanog . Embryos were collected at 4 hpf. ( f ) TOPflash activity assay showed that Wnt/β-catenin signaling activity was significantly up-regulated in MZ nanog embryos, and could be restored by overexpression of Wnt antagonists, gsk3b or ck1a . ( g ) Ectopic expression of chd in MZ nanog was also rescued by gsk3b and ck1a overexpression. chd was detected at 4.5 hpf. ( h ) Overexpression of gsk3b or ck1a was confirmed to rescue the early development defect of MZ nanog . Embryos were injected at one-cell stage and phenotype was observed at 8 hpf. ** means p
Figure Legend Snippet: Wnt/β-catenin activity is hyper-activated in nanog mutant. ( a ) Translation of Nanog protein totally disappeared in MZ nanog . Nanog protein can be detected as early as 64-cell stage and vanished at 75% epiboly stage in WT embryos, while no Nanog protein was detected in MZ nanog . ( b ) Immunolocalization of Nanog on cryosections of WT and MZ nanog at sphere stage. Nanog is localized in cell nucleus of WT and disappeared in MZ nanog . ( c ) Injection of low dose of β-catenin mRNA (200 pg) induced slightly up-regulation of boz and chd in WT embryos, but highly increasing in MZ nanog embryos, indicating that Wnt activity is over-activated because of nanog LOF. Injection or co-injection was performed at one-cell stage. boz was detected at 4 hpf, chd was detected at 4.5 hpf. ( d ) The percentage of embryos counted in WISH of (c) . ( e ) RT-qPCR showed the expression of boz is significantly up-regulated in MZ nanog . Embryos were collected at 4 hpf. ( f ) TOPflash activity assay showed that Wnt/β-catenin signaling activity was significantly up-regulated in MZ nanog embryos, and could be restored by overexpression of Wnt antagonists, gsk3b or ck1a . ( g ) Ectopic expression of chd in MZ nanog was also rescued by gsk3b and ck1a overexpression. chd was detected at 4.5 hpf. ( h ) Overexpression of gsk3b or ck1a was confirmed to rescue the early development defect of MZ nanog . Embryos were injected at one-cell stage and phenotype was observed at 8 hpf. ** means p

Techniques Used: Activity Assay, Mutagenesis, Injection, Quantitative RT-PCR, Expressing, Over Expression

Knockdown of nanog leads to dorsalization and posteriorization. (a) nanog mRNA is maternally transcribed and vanishes at 75% epiboly stage. (b) Two different phenotypes are observed at two doses of nanog MO (0.5 ng/embryo; low dose, LD, and 1.2 ng/embryo, moderate dose, MD) injected embryos, telencephalon defect and dorsalization. Phenotype was observed at 36 hpf. n represents embryo numbers. (c) Western blot detection of Nanog in two doses of nanog MO injected embryos. Nanog translation was blocked in all detected stages in moderate dose of nanog MO injected embryos, and few of Nanog protein can be detected at early stage in low dose of nanog MO injected embryos. (d) Relative Nanog signal intensity in western blots. (e) and (f) The expressions of forebrain marker six3b and telencephalon marker emx1 are absent in low dose of MO injected embryos. krox20 was used as a stage-control marker. Red arrows indicate the expression region of six3b or emx1 . ( g ) and (h) The dorsal marker gene, chd , and maternal β-catenin target, boz , were both up-regulated in moderate dose of nanog MO injected embryos. (i) and ( j ) The expression of dorsal neuroectoderm marker otx2 , is expanded in low dose of nanog MO injected embryos. Red arrow indicates the ventral expansion of otx2 . (k) and (l) The ventral epidermal ectoderm marker foxi1 is eliminated in low dose of nanog MO injected embryos. Red arrow indicates the ventral absence of foxi1 . foxi1 and otx2 were detected at 90% epiboly stage, six3b and emx1 were detected at 2-somite stage. chd was detected at 4.5 hpf, boz was detected at 4 hpf. *** means p
Figure Legend Snippet: Knockdown of nanog leads to dorsalization and posteriorization. (a) nanog mRNA is maternally transcribed and vanishes at 75% epiboly stage. (b) Two different phenotypes are observed at two doses of nanog MO (0.5 ng/embryo; low dose, LD, and 1.2 ng/embryo, moderate dose, MD) injected embryos, telencephalon defect and dorsalization. Phenotype was observed at 36 hpf. n represents embryo numbers. (c) Western blot detection of Nanog in two doses of nanog MO injected embryos. Nanog translation was blocked in all detected stages in moderate dose of nanog MO injected embryos, and few of Nanog protein can be detected at early stage in low dose of nanog MO injected embryos. (d) Relative Nanog signal intensity in western blots. (e) and (f) The expressions of forebrain marker six3b and telencephalon marker emx1 are absent in low dose of MO injected embryos. krox20 was used as a stage-control marker. Red arrows indicate the expression region of six3b or emx1 . ( g ) and (h) The dorsal marker gene, chd , and maternal β-catenin target, boz , were both up-regulated in moderate dose of nanog MO injected embryos. (i) and ( j ) The expression of dorsal neuroectoderm marker otx2 , is expanded in low dose of nanog MO injected embryos. Red arrow indicates the ventral expansion of otx2 . (k) and (l) The ventral epidermal ectoderm marker foxi1 is eliminated in low dose of nanog MO injected embryos. Red arrow indicates the ventral absence of foxi1 . foxi1 and otx2 were detected at 90% epiboly stage, six3b and emx1 were detected at 2-somite stage. chd was detected at 4.5 hpf, boz was detected at 4 hpf. *** means p

Techniques Used: Injection, Western Blot, Marker, Expressing

Nanog interferes with the binding of β-catenin to TCF7 in vitro . ( a ) Nanog interacts with TCF7 through its N-terminal. Different Myc-tagged Nanog were constructed and co-transfection with HA-Tcf7 in HEK293T cells. Deletion of Nanog N’ terminal could not coprecipitate with Tcf7, indicating that Nanog physically interacts with Tcf7 through N’ terminal. ( b ) TCF7 combines with Nanog through its Gro-binding domain. Different Myc-tagged Tcf7 were constructed and co-transfection with HA-Nanog in HEK293T cells. Deletion of Tcf7 Groucho-binding domain (GroBD) could not coprecipitate with Nanog, indicating that Tcf7 binds with Nanog through GroBD. ( c ) Nanog and β-catenin competitively binds with Tcf7. Co-transfection of increasing amount of Nanog decreases the interaction of β-catenin and TCF7 in a dose-dependent manner. More nanog was transfected into Tcf7 and β-catenin co-transfected cells, less β-catenin could be coprecipitated. The molecular weight of HA-Nanog is around 55 KDa and HA-β-catenin is around 100 KDa, so we can easily distinguish the anti-HA bands through protein size. ( d ) Co-transfected of Ctnnbip1 facilities the binding of Nanog with TCF7 and degradation of β-catenin. Two different amounts of HA-ctnnbip1 was co-transfected with Tcf7, Nanog and β-catenin in HEK293T cells, as the Ctnnbip1 expressed, more Nanog was coprecipitated and less β-catenin was pulled down. The molecular weight of HA-ctnnbip1 is around 10 KDa. Note that HA-β-catenin level is reduced when Ctnnbip1 is expressed.
Figure Legend Snippet: Nanog interferes with the binding of β-catenin to TCF7 in vitro . ( a ) Nanog interacts with TCF7 through its N-terminal. Different Myc-tagged Nanog were constructed and co-transfection with HA-Tcf7 in HEK293T cells. Deletion of Nanog N’ terminal could not coprecipitate with Tcf7, indicating that Nanog physically interacts with Tcf7 through N’ terminal. ( b ) TCF7 combines with Nanog through its Gro-binding domain. Different Myc-tagged Tcf7 were constructed and co-transfection with HA-Nanog in HEK293T cells. Deletion of Tcf7 Groucho-binding domain (GroBD) could not coprecipitate with Nanog, indicating that Tcf7 binds with Nanog through GroBD. ( c ) Nanog and β-catenin competitively binds with Tcf7. Co-transfection of increasing amount of Nanog decreases the interaction of β-catenin and TCF7 in a dose-dependent manner. More nanog was transfected into Tcf7 and β-catenin co-transfected cells, less β-catenin could be coprecipitated. The molecular weight of HA-Nanog is around 55 KDa and HA-β-catenin is around 100 KDa, so we can easily distinguish the anti-HA bands through protein size. ( d ) Co-transfected of Ctnnbip1 facilities the binding of Nanog with TCF7 and degradation of β-catenin. Two different amounts of HA-ctnnbip1 was co-transfected with Tcf7, Nanog and β-catenin in HEK293T cells, as the Ctnnbip1 expressed, more Nanog was coprecipitated and less β-catenin was pulled down. The molecular weight of HA-ctnnbip1 is around 10 KDa. Note that HA-β-catenin level is reduced when Ctnnbip1 is expressed.

Techniques Used: Binding Assay, In Vitro, Construct, Cotransfection, Transfection, Molecular Weight

Confrontation of the β-catenin transcriptional activity in nucleus rescues the developmental defect of MZ nanog . ( a ) Overexpression of tcf7-Δ βBD, tcf7-ΔHMG , or ctnnbip1 mRNA rescued the dorsalization phenotype of MZ nanog , while overexpression of tcf7-ΔGroBD did not. Phenotype was observed at 8 hpf. At least 50 embryos were injected and three independent experiments were performed. ( b ) Excessive and ectopic expression of chd in MZ nanog was rescued by overexpression of tcf7-Δ βBD, tcf7-ΔHMG , or ctnnbip1 . WISH analysis of chd was detected at 4.5 hpf. ( c ) Detection of TOPflash activity in rescued MZ nanog embryos. ** means p
Figure Legend Snippet: Confrontation of the β-catenin transcriptional activity in nucleus rescues the developmental defect of MZ nanog . ( a ) Overexpression of tcf7-Δ βBD, tcf7-ΔHMG , or ctnnbip1 mRNA rescued the dorsalization phenotype of MZ nanog , while overexpression of tcf7-ΔGroBD did not. Phenotype was observed at 8 hpf. At least 50 embryos were injected and three independent experiments were performed. ( b ) Excessive and ectopic expression of chd in MZ nanog was rescued by overexpression of tcf7-Δ βBD, tcf7-ΔHMG , or ctnnbip1 . WISH analysis of chd was detected at 4.5 hpf. ( c ) Detection of TOPflash activity in rescued MZ nanog embryos. ** means p

Techniques Used: Activity Assay, Over Expression, Injection, Expressing

nanog negatively regulates Wnt/β-catenin signaling. ( a ) The embryos injected with low dose of nanog MO (0.5 ng) exhibit the similar phenotypes-telencephalon defect - with wnt8a (1 pg) overexpression embryos and tcf7l1a (1.6 ng) knockdown embryos. Phenotype was observed at 36 hpf. ( b ) Embryos injected with nanog MO (160 pg), wnt8a mRNA (0.1 pg) or tcf7l1a MO (800 pg) at extremely low dose show no obvious defect, respectively. However, co-injection of nanog MO with wnt8a mRNA or tcf7l1a MO at the same dose resulted in telencephalon truncated. n represent the number of embryos we observed. ( c ) Wnt/β-catenin activity is over-activated in nanog morphants and can be rescued by knockdown of wnt8a . The expression of zygotic Wnt target genes, sp5l and frzb were up-regulated in nanog morphant whereas Wnt antagonist dkk1b was reduced, and this expression defect can be restored by knockdown of wnt8a . so did the telencephalon defect in nanog morphant. gfp mRNA was used as injection control. sp5l was detected at 75% epiboly, frzb and dkk1b were detected at shield stage, six3b and emx1 were detected at 2-somite stage, krox20 was used as stage control. ( d ) The percentage of embryos counted in WISH (c) . ( e ) Analysis of TOPflash activity showed Wnt/β-catenin activity is up-regulated in both of low dose and moderate dose of nanog MO injected embryos. Embryos were collected at 8 hpf. ( e ) Analysis of TOPflash activity showed co-transfection of Nanog with β-catenin in HEK293T cells inhibited the up-regulated Wnt/β-catenin activity induced by β-catenin in a dose-dependent manner. ( f ) Overexpression of nanog in HEK293T cells can also inhibit the up-regulated Wnt/β-catenin activity induced by ΔN-β-catenin (constitutively activated β-catenin). ** means p
Figure Legend Snippet: nanog negatively regulates Wnt/β-catenin signaling. ( a ) The embryos injected with low dose of nanog MO (0.5 ng) exhibit the similar phenotypes-telencephalon defect - with wnt8a (1 pg) overexpression embryos and tcf7l1a (1.6 ng) knockdown embryos. Phenotype was observed at 36 hpf. ( b ) Embryos injected with nanog MO (160 pg), wnt8a mRNA (0.1 pg) or tcf7l1a MO (800 pg) at extremely low dose show no obvious defect, respectively. However, co-injection of nanog MO with wnt8a mRNA or tcf7l1a MO at the same dose resulted in telencephalon truncated. n represent the number of embryos we observed. ( c ) Wnt/β-catenin activity is over-activated in nanog morphants and can be rescued by knockdown of wnt8a . The expression of zygotic Wnt target genes, sp5l and frzb were up-regulated in nanog morphant whereas Wnt antagonist dkk1b was reduced, and this expression defect can be restored by knockdown of wnt8a . so did the telencephalon defect in nanog morphant. gfp mRNA was used as injection control. sp5l was detected at 75% epiboly, frzb and dkk1b were detected at shield stage, six3b and emx1 were detected at 2-somite stage, krox20 was used as stage control. ( d ) The percentage of embryos counted in WISH (c) . ( e ) Analysis of TOPflash activity showed Wnt/β-catenin activity is up-regulated in both of low dose and moderate dose of nanog MO injected embryos. Embryos were collected at 8 hpf. ( e ) Analysis of TOPflash activity showed co-transfection of Nanog with β-catenin in HEK293T cells inhibited the up-regulated Wnt/β-catenin activity induced by β-catenin in a dose-dependent manner. ( f ) Overexpression of nanog in HEK293T cells can also inhibit the up-regulated Wnt/β-catenin activity induced by ΔN-β-catenin (constitutively activated β-catenin). ** means p

Techniques Used: Injection, Over Expression, Activity Assay, Expressing, Cotransfection

N-terminal of Nanog is required for its Wnt/β-catenin repressive activity. ( a ) The expression of mesendoderm marker, mxtx2 , strictly zygotic gene, blf , and microRNA-430 precursor ( mir-430 ) were reduced, even absent in MZ nanog embryos, while miR-430 target, sod1 was significantly increased in MZ nanog . Overexpression of nanog_ FL, nanog _ΔCT, or vp16 - nanog can restore the expression of mxtx2 , blf , sod1 and mir-430 in MZ nanog . mxtx2 , blf and sod1 were detected at shield stage, mir-430 was detected at 4 hpf. ( b ) The percentage of embryos counted in WISH of (a) . ( c ) Overexpression of full length of nanog ( nanog_ FL), C-terminal truncated Nanog ( nanog _ΔCT), and vp16- nanog homeodomain ( vp16 - nanog ) can rescue the developmental defects of MZ nanog . Both of nanog_ FL and nanog _ΔCT rescued embryos showed WT-like phenotype, and could grow up and reproduce offspring, while vp16 - nanog rescued embryos still shows a phenotype of head truncation. Phenotype was observed at 36 hpf. ( d ) The expression of neuroectoderm marker, otx2 , and forebrain marker six3b were absent in MZ nanog embryos, and can be restored by overexpression of nanog_ FL or nanog _ΔCT, but not vp16 - nanog . ( e ) The percentage of embryos counted in WISH of (d) . ( f ) TOPflash activity showed co-transfection of VP16-nanog with β-catenin in HEK293T cells could not inhibit the up-regulated Wnt/β-catenin activity induced by β-catenin.
Figure Legend Snippet: N-terminal of Nanog is required for its Wnt/β-catenin repressive activity. ( a ) The expression of mesendoderm marker, mxtx2 , strictly zygotic gene, blf , and microRNA-430 precursor ( mir-430 ) were reduced, even absent in MZ nanog embryos, while miR-430 target, sod1 was significantly increased in MZ nanog . Overexpression of nanog_ FL, nanog _ΔCT, or vp16 - nanog can restore the expression of mxtx2 , blf , sod1 and mir-430 in MZ nanog . mxtx2 , blf and sod1 were detected at shield stage, mir-430 was detected at 4 hpf. ( b ) The percentage of embryos counted in WISH of (a) . ( c ) Overexpression of full length of nanog ( nanog_ FL), C-terminal truncated Nanog ( nanog _ΔCT), and vp16- nanog homeodomain ( vp16 - nanog ) can rescue the developmental defects of MZ nanog . Both of nanog_ FL and nanog _ΔCT rescued embryos showed WT-like phenotype, and could grow up and reproduce offspring, while vp16 - nanog rescued embryos still shows a phenotype of head truncation. Phenotype was observed at 36 hpf. ( d ) The expression of neuroectoderm marker, otx2 , and forebrain marker six3b were absent in MZ nanog embryos, and can be restored by overexpression of nanog_ FL or nanog _ΔCT, but not vp16 - nanog . ( e ) The percentage of embryos counted in WISH of (d) . ( f ) TOPflash activity showed co-transfection of VP16-nanog with β-catenin in HEK293T cells could not inhibit the up-regulated Wnt/β-catenin activity induced by β-catenin.

Techniques Used: Activity Assay, Expressing, Marker, Over Expression, Cotransfection

Tle3a and Tle3b do not contribute to the repression of maternal β-catenin activity. (a) Detection of nuclear localization of maternal β-catenin at 3.5 hpf by immunostainning. Upper, animal view. Lower, lateral view. Total-β-catenin was co-stained with DAPI. (b) wnt8a1 , wnt8a2 , β-catenin , tle3a , tle3b and nanog are maternally expressed during oogenesis and unfertilized eggs. Results showed cryosection and whole-mount in situ hybridization. (c) Injection of tle3a MO, tle3b MO, or tle3 MOs together, does not lead to early developmental defect. MO was injected at one-cell stage and at least 50 embryos were injected and observed. (d) The CRISPR/Cas9 target of tle3a is located within exon 6, and a 10-bp deletion mutant ( tle3a ihb355 ) was obtained. (e) The CRISPR/Cas9 target of tle3b is located at the splicing site of exon 2 and intron 2, and a 119-bp insertion mutant ( tle3b ihb354 ) was obtained. Target sequence is in bold and PAM sequence is in Green. (f) The maternal and zygotic mutant of tle3a or tle3b , or double mutant of tle3a and tle3b , showed no early developmental defect. (g) The mRNA expression level of tle3a was reduced in MZ tle3a at 3hpf. (h) The mRNA expression level of tle3b was reduced in MZ tle3b at 3hpf. ** means p
Figure Legend Snippet: Tle3a and Tle3b do not contribute to the repression of maternal β-catenin activity. (a) Detection of nuclear localization of maternal β-catenin at 3.5 hpf by immunostainning. Upper, animal view. Lower, lateral view. Total-β-catenin was co-stained with DAPI. (b) wnt8a1 , wnt8a2 , β-catenin , tle3a , tle3b and nanog are maternally expressed during oogenesis and unfertilized eggs. Results showed cryosection and whole-mount in situ hybridization. (c) Injection of tle3a MO, tle3b MO, or tle3 MOs together, does not lead to early developmental defect. MO was injected at one-cell stage and at least 50 embryos were injected and observed. (d) The CRISPR/Cas9 target of tle3a is located within exon 6, and a 10-bp deletion mutant ( tle3a ihb355 ) was obtained. (e) The CRISPR/Cas9 target of tle3b is located at the splicing site of exon 2 and intron 2, and a 119-bp insertion mutant ( tle3b ihb354 ) was obtained. Target sequence is in bold and PAM sequence is in Green. (f) The maternal and zygotic mutant of tle3a or tle3b , or double mutant of tle3a and tle3b , showed no early developmental defect. (g) The mRNA expression level of tle3a was reduced in MZ tle3a at 3hpf. (h) The mRNA expression level of tle3b was reduced in MZ tle3b at 3hpf. ** means p

Techniques Used: Activity Assay, Staining, In Situ Hybridization, Injection, CRISPR, Mutagenesis, Sequencing, Expressing

Nanog does not regulate the transcription of Wnt/β-catenin components and nuclei localization of β-catenin. ( a ) In situ hybridization and ( b, c ) RT-PCR showed the maternal transcription of wnt8a1 , wnt8a2 , and β-catenin2 were not affected in MZ nanog when compared with WT. ( d ) Western blot and ( e ) statistical results show that the level of nuclear β-catenin (anti-active β-catenin) in MZ nanog was not increased. Anti-total β-catenin was used as β-catenin expression control and anti-β-actin was used as internal control. 2 pg of wnt8a mRNA was injected at one-cell stage in WT and used as positive control. Embryos were collected at 4 hpf. Experiments were carried out for triplicates. ( f ) Immunolocalization of β-catenin on whole-mount embryos at 1000-cell stage (3hpf) shows that the nuclear β-catenin in both of WT and MZ nanog was localized in dorsal margin cells and devoid of ventral cells, indicating that Nanog does not regulate the nuclei localization of β-catenin. Signal was observed at animal view. NS, no significant difference. * means p
Figure Legend Snippet: Nanog does not regulate the transcription of Wnt/β-catenin components and nuclei localization of β-catenin. ( a ) In situ hybridization and ( b, c ) RT-PCR showed the maternal transcription of wnt8a1 , wnt8a2 , and β-catenin2 were not affected in MZ nanog when compared with WT. ( d ) Western blot and ( e ) statistical results show that the level of nuclear β-catenin (anti-active β-catenin) in MZ nanog was not increased. Anti-total β-catenin was used as β-catenin expression control and anti-β-actin was used as internal control. 2 pg of wnt8a mRNA was injected at one-cell stage in WT and used as positive control. Embryos were collected at 4 hpf. Experiments were carried out for triplicates. ( f ) Immunolocalization of β-catenin on whole-mount embryos at 1000-cell stage (3hpf) shows that the nuclear β-catenin in both of WT and MZ nanog was localized in dorsal margin cells and devoid of ventral cells, indicating that Nanog does not regulate the nuclei localization of β-catenin. Signal was observed at animal view. NS, no significant difference. * means p

Techniques Used: In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Injection, Positive Control

29) Product Images from "The Role of Chromatin Density in Cell Population Heterogeneity during Stem Cell Differentiation"

Article Title: The Role of Chromatin Density in Cell Population Heterogeneity during Stem Cell Differentiation

Journal: Scientific Reports

doi: 10.1038/s41598-017-13731-3

Relationship between density changes and gene expression variation in core pluripotency genes. ( a ) The densities of OCT4, NANOG, and SOX2 change during stem cell differentiation. ( b – d ) hESC was differentiated to NPC and MES, followed by immunostaining
Figure Legend Snippet: Relationship between density changes and gene expression variation in core pluripotency genes. ( a ) The densities of OCT4, NANOG, and SOX2 change during stem cell differentiation. ( b – d ) hESC was differentiated to NPC and MES, followed by immunostaining

Techniques Used: Expressing, Cell Differentiation, Immunostaining

30) Product Images from "Isolation and characterization of mesenchymal stem cells from human fetus heart"

Article Title: Isolation and characterization of mesenchymal stem cells from human fetus heart

Journal: PLoS ONE

doi: 10.1371/journal.pone.0192244

Expression of pluripotency and embryonic markers and cardiovascular genes by hfC-MSCs and hBM-MSCs. Representative photomicrographs (40X, 20μm) of human fetal cardiac mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (A: OCT-4; B: hoechst dye), Nanog (C: Nanog; D: hoechst dye), SOX-2 (E: SOX-2; F: hoechst dye) and representative photomicrographs of (40X, 20μm) of human bone marrow mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (G: OCT-4; H: hoechst dye), Nanog (I: Nanog; J: hoechst dye), SOX-2 (K: SOX-2; L: hoechst dye).
Figure Legend Snippet: Expression of pluripotency and embryonic markers and cardiovascular genes by hfC-MSCs and hBM-MSCs. Representative photomicrographs (40X, 20μm) of human fetal cardiac mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (A: OCT-4; B: hoechst dye), Nanog (C: Nanog; D: hoechst dye), SOX-2 (E: SOX-2; F: hoechst dye) and representative photomicrographs of (40X, 20μm) of human bone marrow mesenchymal stem cells (hfC-MSCs) showing expression of OCT-4 (G: OCT-4; H: hoechst dye), Nanog (I: Nanog; J: hoechst dye), SOX-2 (K: SOX-2; L: hoechst dye).

Techniques Used: Expressing

31) Product Images from "Opposing microRNA families regulate self-renewal in mouse embryonic stem cells"

Article Title: Opposing microRNA families regulate self-renewal in mouse embryonic stem cells

Journal: Nature

doi: 10.1038/nature08725

The let-7 and ESCC miRNA families have opposing roles in regulating ESC self-renewal. (a) Transfected miRNAs with the seed sequence highlighted. (b) Pou5f1/Oct4 immunofluorescence staining after transfection of let-7c, miR-294 and combinations of let-7c with miR-294, mutant-miR-294, miR-291a-5p, or miR-130b in Dgcr8 -/- (i) and wild-type (ii) ESCs. Representative images, n = 3. (c) qRT-PCR for Pou5f1/Oct4, Sox2, and Nanog normalized to beta-actin after miRNA introduction as in b. n = 3-8. * indicates p
Figure Legend Snippet: The let-7 and ESCC miRNA families have opposing roles in regulating ESC self-renewal. (a) Transfected miRNAs with the seed sequence highlighted. (b) Pou5f1/Oct4 immunofluorescence staining after transfection of let-7c, miR-294 and combinations of let-7c with miR-294, mutant-miR-294, miR-291a-5p, or miR-130b in Dgcr8 -/- (i) and wild-type (ii) ESCs. Representative images, n = 3. (c) qRT-PCR for Pou5f1/Oct4, Sox2, and Nanog normalized to beta-actin after miRNA introduction as in b. n = 3-8. * indicates p

Techniques Used: Transfection, Sequencing, Immunofluorescence, Staining, Mutagenesis, Quantitative RT-PCR

32) 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

Non-human primate rsPSCs a , Phase-contrast images of colony morphologies of rhesus macaque rsESCs (ORMES22 and ORMES23), rhesus macaque rsiPSCs and chimpanzee rsiPSCs. b , Immunofluorescence images of NANOG, SOX2, DNMT3b, TRA-1-60 and TRA-1-80 protein expression in ORMES23 rsESCs. ORMES23 rsESCs were also stained for alkaline phosphatase (AP) activity and OCT4 immunohistochemistry (top right). c , Haematoxylin and eosin staining images of teratomas generated by chimpanzee rsiPSCs show lineage differentiation towards three germ layers. d for details). A, anterior; P, posterior; D, distal. e , Fluorescence images of grafted embryos after in vitro culture. GFP-labelled ORMES23 rsESCs were grafted to posterior, distal and anterior regions of epiblasts of isolated non-intact and non-viable E7.5 mouse embryos and cultured in vitro for 36 h before fixation and visualization by an inverted fluorescence microscope. Arrowhead indicates a cell clump failed to distribute. Dashed line indicates dispersed cells in the posterior region of grafted embryo. Blue, DAPI. Insets show higher-magnification images of GFP-labelled cells. f , Top, quantification of the extent of cell spreading of GFP-labelledORMES23 rsESCs after being grafted to different regions of E7.5 mouse epiblasts. Bottom, incorporation efficiency of grafted GFP-labelled ORMES23 rsESCs in mouse E7.5 epiblasts. Error bars indicate s.d. ( n , indicated on the graph, independent experiments); t -test, * P
Figure Legend Snippet: Non-human primate rsPSCs a , Phase-contrast images of colony morphologies of rhesus macaque rsESCs (ORMES22 and ORMES23), rhesus macaque rsiPSCs and chimpanzee rsiPSCs. b , Immunofluorescence images of NANOG, SOX2, DNMT3b, TRA-1-60 and TRA-1-80 protein expression in ORMES23 rsESCs. ORMES23 rsESCs were also stained for alkaline phosphatase (AP) activity and OCT4 immunohistochemistry (top right). c , Haematoxylin and eosin staining images of teratomas generated by chimpanzee rsiPSCs show lineage differentiation towards three germ layers. d for details). A, anterior; P, posterior; D, distal. e , Fluorescence images of grafted embryos after in vitro culture. GFP-labelled ORMES23 rsESCs were grafted to posterior, distal and anterior regions of epiblasts of isolated non-intact and non-viable E7.5 mouse embryos and cultured in vitro for 36 h before fixation and visualization by an inverted fluorescence microscope. Arrowhead indicates a cell clump failed to distribute. Dashed line indicates dispersed cells in the posterior region of grafted embryo. Blue, DAPI. Insets show higher-magnification images of GFP-labelled cells. f , Top, quantification of the extent of cell spreading of GFP-labelledORMES23 rsESCs after being grafted to different regions of E7.5 mouse epiblasts. Bottom, incorporation efficiency of grafted GFP-labelled ORMES23 rsESCs in mouse E7.5 epiblasts. Error bars indicate s.d. ( n , indicated on the graph, independent experiments); t -test, * P

Techniques Used: Immunofluorescence, Expressing, Staining, Activity Assay, Immunohistochemistry, Generated, Fluorescence, In Vitro, Isolation, Cell Culture, Microscopy

F/R1-based culture supports self-renewal of human ESCs as well as iPSC generation a , Expression of pluripotency markers SOX2, NANOG, TRA-1-60, TRA-1-80 and SSEA-4 in human H1 rsESCs. b , Representative bright-field images showing colonies visualized by alkaline phosphatase (AP) staining after being plated at clonal density (1,000 cells per well) and cultured for 6 days. Y27632 was added at 10 µM. c , Real-time quantitative PCR analysis of expression of pluripotency marker genes ( OCT4 , SOX2 , NANOG and LIN28 ) and lineage marker genes ( T , NKX1-2 and WNT3 ) in H1 ESCs, human-foreskin-fibroblast-derived iPSCs, H1 rsESCs and human-foreskin-fibroblast-derived rs-iPSCs. Error bars indicate s.d. ( n = 3, biological replicates). d , Haematoxylin and eosin staining images of teratomas generated from human H1 rsESCs show lineage differentiation towards three germlayers. e , Karyotype analysis of human H9 rsESCs indicates a normal diploid chromosome content. f , Representative bright-field images showing morphologies of putative iPSC colonies in conventional F/A-based human ESC culture and F/R1-based culture conditions (top). Alkaline phosphatase staining at day 25 post-nucleofection indicates a larger colony size in F/R1-based culture (bottom). g , Efficiency of iPSC generation in conventional F/A-based human ESC culture conditions and F/R1-based culture conditions. Error bars indicate s.d. ( n = 3, independent experiments). h , Quality of human iPSC-like colonies generated in F/A-based and F/R1-based culture conditions. Partial and full alkaline-phosphatase-positive iPSC-like colonies were counted separately using the criteria shown on the right. Error bars indicate s.d. ( n = 3, independent experiments). i , Phase-contrast image showing morphology of human-foreskin-fibroblast-derived rs-iPSCs. j , Graphic representation of H3K4me3 and H3K27me3 ChIP-Seq signals near the transcription start site (TSS) for Polycomb target genes in H1 ESCs and H1 rsESCs. k , Average H3K27me3 signal at Polycomb target genes in H1 rsESCs (purple) and H1 ESCs (green). i , Cell-cycle profiles of H1 ESCs and H1 rsESCs were analysed by flow cytometry. m , Flow cytometry analysis of OCT4, SOX2, NANOG and TRA-1-60 protein expression in H1 ESCs and H1 rsESCs.
Figure Legend Snippet: F/R1-based culture supports self-renewal of human ESCs as well as iPSC generation a , Expression of pluripotency markers SOX2, NANOG, TRA-1-60, TRA-1-80 and SSEA-4 in human H1 rsESCs. b , Representative bright-field images showing colonies visualized by alkaline phosphatase (AP) staining after being plated at clonal density (1,000 cells per well) and cultured for 6 days. Y27632 was added at 10 µM. c , Real-time quantitative PCR analysis of expression of pluripotency marker genes ( OCT4 , SOX2 , NANOG and LIN28 ) and lineage marker genes ( T , NKX1-2 and WNT3 ) in H1 ESCs, human-foreskin-fibroblast-derived iPSCs, H1 rsESCs and human-foreskin-fibroblast-derived rs-iPSCs. Error bars indicate s.d. ( n = 3, biological replicates). d , Haematoxylin and eosin staining images of teratomas generated from human H1 rsESCs show lineage differentiation towards three germlayers. e , Karyotype analysis of human H9 rsESCs indicates a normal diploid chromosome content. f , Representative bright-field images showing morphologies of putative iPSC colonies in conventional F/A-based human ESC culture and F/R1-based culture conditions (top). Alkaline phosphatase staining at day 25 post-nucleofection indicates a larger colony size in F/R1-based culture (bottom). g , Efficiency of iPSC generation in conventional F/A-based human ESC culture conditions and F/R1-based culture conditions. Error bars indicate s.d. ( n = 3, independent experiments). h , Quality of human iPSC-like colonies generated in F/A-based and F/R1-based culture conditions. Partial and full alkaline-phosphatase-positive iPSC-like colonies were counted separately using the criteria shown on the right. Error bars indicate s.d. ( n = 3, independent experiments). i , Phase-contrast image showing morphology of human-foreskin-fibroblast-derived rs-iPSCs. j , Graphic representation of H3K4me3 and H3K27me3 ChIP-Seq signals near the transcription start site (TSS) for Polycomb target genes in H1 ESCs and H1 rsESCs. k , Average H3K27me3 signal at Polycomb target genes in H1 rsESCs (purple) and H1 ESCs (green). i , Cell-cycle profiles of H1 ESCs and H1 rsESCs were analysed by flow cytometry. m , Flow cytometry analysis of OCT4, SOX2, NANOG and TRA-1-60 protein expression in H1 ESCs and H1 rsESCs.

Techniques Used: Expressing, Staining, Cell Culture, Real-time Polymerase Chain Reaction, Marker, Derivative Assay, Generated, Chromatin Immunoprecipitation, Flow Cytometry, Cytometry

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

Highly efficient derivation of rsEpiSCs a , Derivation of rsEpiSCs with different passaging methods: collagenase IV (top) and trypsin (bottom). Shown are phase-contrast images of day 4 epiblast outgrowths and cells at passage 1 (P1) and passage 10 (P10). b , Derivation efficiency is compared between EpiSCs and rsEpiSCs using isolated E5.75 epiblasts from three different mouse strains. c , Derivation of rsEpiSCs using epiblasts isolated from different developmental stages of postimplantation embryos. Typical morphologies of staged embryos at E5.25, 5.75, 6.5, 7.25 and 7.5 are shown in the left panel. Day 2 and day 4 epiblast outgrowths as well as colonies of P1 are shown. d , Real-time quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), naive ( Rex1 and Klf2 ) and primed ( Otx2 and Fgf5 ) PSC marker genes in mouse ESCs and rsEpiSCs derived from different post-implantation developmental stages. Error bars indicate s.d. ( n = 3, biological samples). e , Derivation of rsEpiSCs from E3.5 pre-implantation blastocysts. Zona pellucida was removed with acidic Tyrode’s solution, followed by the removal of trophectoderm by immunosurgery. Isolated inner cell mass was used for the derivation of rsEpiSCs. Arrows and arrowheads point to the intact trophectoderm and destroyed trophectoderm, respectively. f , Schematic representation of dissection of isolated E6.5 epiblasts into four pieces: anterior-proximal (AP), anterior-distal (AD), posterior-proximal (PP) and posterior-distal (PD). g , Derivation of rsEpiSCs from four regions of E6.5 epiblasts. Day 2 and day 4 epiblast outgrowths as well as colonies of passage 5 (P5) are shown. h , Real-time quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), naive ( Rex1 and Klf2 ) and primed ( Otx2 and Fgf5 ) PSC marker genes in rsEpiSCs derived from whole, AP, AD, PP and PD regions of E6.5 epiblasts. Error bars indicate s.d. ( n = 3, biological samples). i , Derivation of rsEpiSCs using other Wnt inhibitors: XAV939 (2.5 µM) and IWP2 (2.5 µM). Day 4 epiblast outgrowths and colonies at passage 10 (P10) were shown. j , Real-time quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), endodermal ( Sox17 and Gsc ), mesodermal ( Eomes and Mixl1 ) and neuroectodermal ( Sox1 and Pax6 ) marker genes in rsEpiSCs derived using different Wnt inhibitors. Error bars indicate s.d. ( d, h, j , n = 3, biological replicates).
Figure Legend Snippet: Highly efficient derivation of rsEpiSCs a , Derivation of rsEpiSCs with different passaging methods: collagenase IV (top) and trypsin (bottom). Shown are phase-contrast images of day 4 epiblast outgrowths and cells at passage 1 (P1) and passage 10 (P10). b , Derivation efficiency is compared between EpiSCs and rsEpiSCs using isolated E5.75 epiblasts from three different mouse strains. c , Derivation of rsEpiSCs using epiblasts isolated from different developmental stages of postimplantation embryos. Typical morphologies of staged embryos at E5.25, 5.75, 6.5, 7.25 and 7.5 are shown in the left panel. Day 2 and day 4 epiblast outgrowths as well as colonies of P1 are shown. d , Real-time quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), naive ( Rex1 and Klf2 ) and primed ( Otx2 and Fgf5 ) PSC marker genes in mouse ESCs and rsEpiSCs derived from different post-implantation developmental stages. Error bars indicate s.d. ( n = 3, biological samples). e , Derivation of rsEpiSCs from E3.5 pre-implantation blastocysts. Zona pellucida was removed with acidic Tyrode’s solution, followed by the removal of trophectoderm by immunosurgery. Isolated inner cell mass was used for the derivation of rsEpiSCs. Arrows and arrowheads point to the intact trophectoderm and destroyed trophectoderm, respectively. f , Schematic representation of dissection of isolated E6.5 epiblasts into four pieces: anterior-proximal (AP), anterior-distal (AD), posterior-proximal (PP) and posterior-distal (PD). g , Derivation of rsEpiSCs from four regions of E6.5 epiblasts. Day 2 and day 4 epiblast outgrowths as well as colonies of passage 5 (P5) are shown. h , Real-time quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), naive ( Rex1 and Klf2 ) and primed ( Otx2 and Fgf5 ) PSC marker genes in rsEpiSCs derived from whole, AP, AD, PP and PD regions of E6.5 epiblasts. Error bars indicate s.d. ( n = 3, biological samples). i , Derivation of rsEpiSCs using other Wnt inhibitors: XAV939 (2.5 µM) and IWP2 (2.5 µM). Day 4 epiblast outgrowths and colonies at passage 10 (P10) were shown. j , Real-time quantitative PCR analysis of expression of pluripotent ( Oct4 , Sox2 and Nanog ), endodermal ( Sox17 and Gsc ), mesodermal ( Eomes and Mixl1 ) and neuroectodermal ( Sox1 and Pax6 ) marker genes in rsEpiSCs derived using different Wnt inhibitors. Error bars indicate s.d. ( d, h, j , n = 3, biological replicates).

Techniques Used: Passaging, Isolation, Real-time Polymerase Chain Reaction, Expressing, Marker, Derivative Assay, Dissection

33) Product Images from "KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription"

Article Title: KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.91

KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1
Figure Legend Snippet: KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1

Techniques Used: Chromatin Immunoprecipitation

34) Product Images from "KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription"

Article Title: KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.91

KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1
Figure Legend Snippet: KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1

Techniques Used: Chromatin Immunoprecipitation

35) Product Images from "Heat shock induces the depletion of Oct4 in mouse blastocysts and stem cells"

Article Title: Heat shock induces the depletion of Oct4 in mouse blastocysts and stem cells

Journal: bioRxiv

doi: 10.1101/264044

Impact of heat shock on Oct4 expression in mouse blastocysts and ESCs. (A) Immunostaining of Oct4, Klf4, Sox2 and Nanog in the ICMs of E3.5 blastocysts. The blastocysts were incubated for 1 h at the indicated temperature, and the nuclei were shown by DAPI staining (blue). (B and C) Western blot analysis of Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 from E14 and JM83A mESCs. Lamin B and GAPDH were used as controls. C: 37 °C; H: 42 °C for 1 h; R: recovery at 37 °C for 6 h following 42 °C treatment (B). Quantification data represent the mean ± SD of three independent experiments. ∗p
Figure Legend Snippet: Impact of heat shock on Oct4 expression in mouse blastocysts and ESCs. (A) Immunostaining of Oct4, Klf4, Sox2 and Nanog in the ICMs of E3.5 blastocysts. The blastocysts were incubated for 1 h at the indicated temperature, and the nuclei were shown by DAPI staining (blue). (B and C) Western blot analysis of Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 from E14 and JM83A mESCs. Lamin B and GAPDH were used as controls. C: 37 °C; H: 42 °C for 1 h; R: recovery at 37 °C for 6 h following 42 °C treatment (B). Quantification data represent the mean ± SD of three independent experiments. ∗p

Techniques Used: Expressing, Immunostaining, Incubation, Staining, Western Blot

36) Product Images from "Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors"

Article Title: Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0167550

Characterization of bovine-induced trophoblastic cells (biTBCs). (A) Alkaline phosphatase activity in biTBCs. (B) CDX2 expression in biTBCs. (C) OCT3/4 expression in biTBCs. (D) NANOG expression in biTBCs. (A) scale bars = 500 μm. (B)-(D) scale bars = 100 μm.
Figure Legend Snippet: Characterization of bovine-induced trophoblastic cells (biTBCs). (A) Alkaline phosphatase activity in biTBCs. (B) CDX2 expression in biTBCs. (C) OCT3/4 expression in biTBCs. (D) NANOG expression in biTBCs. (A) scale bars = 500 μm. (B)-(D) scale bars = 100 μm.

Techniques Used: Activity Assay, Expressing

37) Product Images from "MicroRNA-33b Inhibits Breast Cancer Metastasis by Targeting HMGA2, SALL4 and Twist1"

Article Title: MicroRNA-33b Inhibits Breast Cancer Metastasis by Targeting HMGA2, SALL4 and Twist1

Journal: Scientific Reports

doi: 10.1038/srep09995

miR-33b inhibits breast cancer cell stemness. (A) Representative images of the mammospheres formed by BT-549/ctrl and BT-549/miR-33b cells. Quantification of primary, secondary and tertiary mammospheres formed by BT-549/ctrl and BT-549/miR-33b cells. (B) Representative images of the mammospheres formed by MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. Quantification of primary, secondary and tertiary mammosphere formation formed by MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (C,D) Tumorsphere assays for BT-549/ctrl and BT-549/miR-33b (C) and MDA-MB-231/ctrl and MDA-MB-231/miR-33b (D) cells were performed by limiting dilution with 1,000 cells to one cell per well of a 96-well ultra-low attachment plate. The numbers of mammospheres were scored at the end of 7 days. This experiment was performed three times, with four wells per cell dilution. (E) FACS analysis of the CD44 + /CD24 − stem cell subpopulation in MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (F) qRT-PCR analysis of the mRNA expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 in BT-549/ctrl and BT-549/miR-33b cells as well as in MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (G) Western blot analysis of Bmi-1, Nanog, Oct4, Sox2, HMGA2, SALL4 and Twist1 expression in BT-549/ctrl and BT-549/miR-33b cells as well as in MDA-MB-231/ctrl and MDA-MB-231/ miR-33b cells. The full-length blots were presented in the Supplementary Figure 8 . Scale bars, 100 μm. Data represent the mean ± s.d. *: P
Figure Legend Snippet: miR-33b inhibits breast cancer cell stemness. (A) Representative images of the mammospheres formed by BT-549/ctrl and BT-549/miR-33b cells. Quantification of primary, secondary and tertiary mammospheres formed by BT-549/ctrl and BT-549/miR-33b cells. (B) Representative images of the mammospheres formed by MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. Quantification of primary, secondary and tertiary mammosphere formation formed by MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (C,D) Tumorsphere assays for BT-549/ctrl and BT-549/miR-33b (C) and MDA-MB-231/ctrl and MDA-MB-231/miR-33b (D) cells were performed by limiting dilution with 1,000 cells to one cell per well of a 96-well ultra-low attachment plate. The numbers of mammospheres were scored at the end of 7 days. This experiment was performed three times, with four wells per cell dilution. (E) FACS analysis of the CD44 + /CD24 − stem cell subpopulation in MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (F) qRT-PCR analysis of the mRNA expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 in BT-549/ctrl and BT-549/miR-33b cells as well as in MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (G) Western blot analysis of Bmi-1, Nanog, Oct4, Sox2, HMGA2, SALL4 and Twist1 expression in BT-549/ctrl and BT-549/miR-33b cells as well as in MDA-MB-231/ctrl and MDA-MB-231/ miR-33b cells. The full-length blots were presented in the Supplementary Figure 8 . Scale bars, 100 μm. Data represent the mean ± s.d. *: P

Techniques Used: Multiple Displacement Amplification, FACS, Quantitative RT-PCR, Expressing, Western Blot

Knockdown miR-33b promotes the self-renewal of MCF-10A cells. (A) qRT-PCR analysis of the knockdown efficiency of miR-33b in MCF-10A cells. (B) Representative images of the mammospheres formed by MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (C) Quantification of primary, secondary and tertiary mammospheres formed by MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (D) A limiting dilution assay was performed on the MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b from 1 to 1,000 cells per well. The numbers of mammospheres were scored at the end of 7 days. (E) FACS analysis of the CD44 + /CD24 − stem cell subpopulation in MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (F) qRT-PCR analysis of the mRNA expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 as well as the downstream target genes of miR-33b, HMGA2, SALL4 and Twist1 in MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (G) Western blot analysis of the protein expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 as well as the downstream target genes of miR-33b, HMGA2, SALL4 and Twist1. The full-length blots were presented in the Supplementary Figure 10 . Scale bars, 100 μm. Data represent the mean ± s.d. *: P
Figure Legend Snippet: Knockdown miR-33b promotes the self-renewal of MCF-10A cells. (A) qRT-PCR analysis of the knockdown efficiency of miR-33b in MCF-10A cells. (B) Representative images of the mammospheres formed by MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (C) Quantification of primary, secondary and tertiary mammospheres formed by MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (D) A limiting dilution assay was performed on the MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b from 1 to 1,000 cells per well. The numbers of mammospheres were scored at the end of 7 days. (E) FACS analysis of the CD44 + /CD24 − stem cell subpopulation in MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (F) qRT-PCR analysis of the mRNA expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 as well as the downstream target genes of miR-33b, HMGA2, SALL4 and Twist1 in MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (G) Western blot analysis of the protein expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 as well as the downstream target genes of miR-33b, HMGA2, SALL4 and Twist1. The full-length blots were presented in the Supplementary Figure 10 . Scale bars, 100 μm. Data represent the mean ± s.d. *: P

Techniques Used: Quantitative RT-PCR, Limiting Dilution Assay, FACS, Expressing, Western Blot

38) 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

Tcf3 downregulation in wild-type ES cells impairs but does not fully inhibit neural differentiation. A. Immunohistochemistry analysis was used to evaluate the neural differentiation in teratoma samples derived from Tcf3 −/− or their wild type control (GS1) ESCs. Immunostaining with specific antibodies revealed retention of the pluripotency marker Oct4 and expression of the neural markers GFAP, neurofilaments (2H3) and synaptic vesicles (SV2) in Tcf3 −/− teratomas. Thionin staining was used to evaluate cartilage differentiation. B. RNAs were isolated from different teratoma samples and analyzed by qRT-PCR for differentiation markers. Dot plots show normalized qRT-PCR values for the neural markers Map2 , β- III-Tubulin and GFAP and for the pluripotency markers Oct4 and Nanog among the different teratoma samples. Each dot represents one sample.
Figure Legend Snippet: Tcf3 downregulation in wild-type ES cells impairs but does not fully inhibit neural differentiation. A. Immunohistochemistry analysis was used to evaluate the neural differentiation in teratoma samples derived from Tcf3 −/− or their wild type control (GS1) ESCs. Immunostaining with specific antibodies revealed retention of the pluripotency marker Oct4 and expression of the neural markers GFAP, neurofilaments (2H3) and synaptic vesicles (SV2) in Tcf3 −/− teratomas. Thionin staining was used to evaluate cartilage differentiation. B. RNAs were isolated from different teratoma samples and analyzed by qRT-PCR for differentiation markers. Dot plots show normalized qRT-PCR values for the neural markers Map2 , β- III-Tubulin and GFAP and for the pluripotency markers Oct4 and Nanog among the different teratoma samples. Each dot represents one sample.

Techniques Used: Immunohistochemistry, Derivative Assay, Immunostaining, Marker, Expressing, Staining, Isolation, Quantitative RT-PCR

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

Wnt signaling downregulates Tcf3 expression in mouse ESCs. A. qRT-PCR analysis of Tcf3 in wild type, Apc NN and Apc Min/Min ESCs. Actb was used as an internal control; bars represent n = 2 ± SD. B. Western blot analysis of the core pluripotency markers Oct4, Nanog, Sox2 and Tcf3 on protein lysates isolated from two independent Apc NN clones and wild type control ESCs. Actb and Tubulin were used as an internal control. C–D. qRT-PCR analysis of Tcf3 in wild type ESCs treated for different time intervals with Wnt3a conditioned medium (C), or with the GSK-inhibitor SB-216763 (D). L-medium and DMSO were employed as controls, respectively. Actb was used as an internal control; bars represent n = 2 ± SD. E. Time course western blot analysis of Tcf3 expression in wild type ESCs treated with Wnt3a conditioned medium (Wnt3a CM) or control L-medium (LM). Actb was used as an internal control. F. qRT-PCR analysis of Tcf3 and Wnt target genes Axin2 and Cdx1 in wild type ESC treated for 48 h with different concentrations of GSK-inhibitor SB-216763 or DMSO as control. Actb was used as an internal control; bars represent n = 2 ± SD.
Figure Legend Snippet: Wnt signaling downregulates Tcf3 expression in mouse ESCs. A. qRT-PCR analysis of Tcf3 in wild type, Apc NN and Apc Min/Min ESCs. Actb was used as an internal control; bars represent n = 2 ± SD. B. Western blot analysis of the core pluripotency markers Oct4, Nanog, Sox2 and Tcf3 on protein lysates isolated from two independent Apc NN clones and wild type control ESCs. Actb and Tubulin were used as an internal control. C–D. qRT-PCR analysis of Tcf3 in wild type ESCs treated for different time intervals with Wnt3a conditioned medium (C), or with the GSK-inhibitor SB-216763 (D). L-medium and DMSO were employed as controls, respectively. Actb was used as an internal control; bars represent n = 2 ± SD. E. Time course western blot analysis of Tcf3 expression in wild type ESCs treated with Wnt3a conditioned medium (Wnt3a CM) or control L-medium (LM). Actb was used as an internal control. F. qRT-PCR analysis of Tcf3 and Wnt target genes Axin2 and Cdx1 in wild type ESC treated for 48 h with different concentrations of GSK-inhibitor SB-216763 or DMSO as control. Actb was used as an internal control; bars represent n = 2 ± SD.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Isolation, Clone Assay

39) Product Images from "The cohesin-associated protein Wapal is required for proper Polycomb-mediated gene silencing"

Article Title: The cohesin-associated protein Wapal is required for proper Polycomb-mediated gene silencing

Journal: Epigenetics & Chromatin

doi: 10.1186/s13072-016-0063-7

a ChIP-qPCR was performed at six genomic loci ( HoxD1 , Sox11 , Twist1 , Snai2 , Ppp1r3c , and Lefty1 ) all derepressed after Wapal depletion with an antibody specific to H3K27me3. Nanog , which is well expressed in ESCs, is used as a negative control. Two additional negative controls (Ctrl 1 and Ctrl 2) are included—genomic regions within gene deserts. Genomic coordinates amplified by each primer set are given in Additional file 15 : Table S4. Asterisk indicates a statistically significant difference from input ( p value
Figure Legend Snippet: a ChIP-qPCR was performed at six genomic loci ( HoxD1 , Sox11 , Twist1 , Snai2 , Ppp1r3c , and Lefty1 ) all derepressed after Wapal depletion with an antibody specific to H3K27me3. Nanog , which is well expressed in ESCs, is used as a negative control. Two additional negative controls (Ctrl 1 and Ctrl 2) are included—genomic regions within gene deserts. Genomic coordinates amplified by each primer set are given in Additional file 15 : Table S4. Asterisk indicates a statistically significant difference from input ( p value

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Amplification

a The genomic distribution of Wapal-binding sites. b Overlap of Wapal-binding sites with CTCF, Nanog, or core subunits of the cohesin complex Smc3 and Rad21. Percentages listed indicate the area(s) of overlap of Wapal-binding sites with a specific factor. c ChIP-seq tracks for Adm to demonstrate the binding of Wapal, CTCF, and cohesin components. The y - axis represents the # of ChIP-seq tags recovered for a given genomic bin. The range for each track is displayed to the right of track name. A lower threshold of 2 was set for all tracks to minimize background. In all cases, the y - axis for BirA and Wapal are the same to demonstrate binding specificity
Figure Legend Snippet: a The genomic distribution of Wapal-binding sites. b Overlap of Wapal-binding sites with CTCF, Nanog, or core subunits of the cohesin complex Smc3 and Rad21. Percentages listed indicate the area(s) of overlap of Wapal-binding sites with a specific factor. c ChIP-seq tracks for Adm to demonstrate the binding of Wapal, CTCF, and cohesin components. The y - axis represents the # of ChIP-seq tags recovered for a given genomic bin. The range for each track is displayed to the right of track name. A lower threshold of 2 was set for all tracks to minimize background. In all cases, the y - axis for BirA and Wapal are the same to demonstrate binding specificity

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

a The 100 probes most upregulated ( red ) or downregulated ( green ) with shRNA #1 to Wapal were identified and the Log2 fold change (Log2 FC) calculated for Wapal shRNA #2, Smc3 shRNAs, and from published studies in which the pluripotency-associated TFs Nanog or Oct4 were depleted by siRNAs. Log2 FC is displayed, with the color scheme indicated. b GSEA was used (see “ Methods ” section for details) to identify pathways globally dysregulated by Wapal depletion. Six gene sets from pluripotent cells which all exhibited substantial enrichment in Wapal-depleted samples are shown. Positive normalized enrichment score (NES) indicates that the gene set is enriched in Wapal-depleted samples as compared to samples infected with the empty vector. False discovery rate (FDR
Figure Legend Snippet: a The 100 probes most upregulated ( red ) or downregulated ( green ) with shRNA #1 to Wapal were identified and the Log2 fold change (Log2 FC) calculated for Wapal shRNA #2, Smc3 shRNAs, and from published studies in which the pluripotency-associated TFs Nanog or Oct4 were depleted by siRNAs. Log2 FC is displayed, with the color scheme indicated. b GSEA was used (see “ Methods ” section for details) to identify pathways globally dysregulated by Wapal depletion. Six gene sets from pluripotent cells which all exhibited substantial enrichment in Wapal-depleted samples are shown. Positive normalized enrichment score (NES) indicates that the gene set is enriched in Wapal-depleted samples as compared to samples infected with the empty vector. False discovery rate (FDR

Techniques Used: shRNA, Infection, Plasmid Preparation

40) Product Images from "KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription"

Article Title: KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.91

KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1
Figure Legend Snippet: KDM5B is a Nanog and Oct4 target and critical for ESC self-renewal. ( A ) UCSC genome browser track depicting Oct4 and Nanog ChIP-Seq occupancy in the vicinity of the murine KDM5B genomic locus. ( B ) Confirmation of Oct4 and Nanog occupancy at KDM5B (P1

Techniques Used: Chromatin Immunoprecipitation

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other:

Article Title: Stress-Induced Enzyme Activation Primes Murine Embryonic Stem Cells to Differentiate Toward the First Extraembryonic Lineage
Article Snippet: Anti-rabbit Nanog was from Chemicon/Millipore (AB5731; Billerica, MA) and Rex1 antibodies were from Abcam (AB28141; Cambridge, MA).

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  • 94
    Millipore primary antibodies against nanog
    ChIP analysis reveals <t>Nanog</t> - binding sites regulating p27 KIP1 expression. (A) Schematic representation (not drawn to scale) of the p27 genomic locus (RefSeq: NM_009875) highlighting the location of putative Nanog-binding sites designated ‘D’ (primer pairs designated as D1 and D2) and ‘P’ (primer pairs designated as P1 and P2) in the upstream region of the p27 KIP1 gene ( Chen et al., 2008 ; Marson et al., 2008 ). PCR primer pairs were designed for these sites for ChIP analyses (dumbbell shaped; Table S2 ). (B) ChIP analysis reveals that Nanog protein in ESCs binds within the upstream region of the p27 KIP1 gene. Oct4-GiP MEF and Oct4-GiP ESCs were cultured, harvested and processed for ChIP analysis with beads only, IgG and Nanog antibody. The Oct4-GiP MEF cell line was used as a negative control. Input <t>DNA</t> (10%) was used as a control for ChIP. Beads only and IgG served as negative controls. Putative Nanog-binding regions were amplified by the designed primer pairs (D1, D2, P1 and P2). Primer pairs were also designed randomly in the 3′UTR region of the p27 KIP1 gene to serve as a negative (desert) control (Dc). (C) RT-qPCR analysis on the ChIP samples explained in B using primers pairs ‘P’ (P1) and ‘D’ (D1) to amplify the Nanog-binding p27 KIP1 sites. RT-qPCR was also performed on the Dc primer set but no amplification was observed other than the input samples (data not shown). Two independent biological replicates were performed for ChIP analysis. ** P
    Primary Antibodies Against Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pluripotency markers nanog
    Reprogramming of fibroblasts into pluripotent stem cells. ( a ) Schematic diagram of pluripotent reprogramming. Fibroblasts were transfected with lentiviruses carrying Oct4-Sox2-Klf3-c-Myc (OSKM) transcription factors and were induced into <t>pluripotency.</t> iPSC: induced pluripotent stem cell; lncRNA: long noncoding RNA. ( b ) Typical images of iPSC reprogramming. After OSKM transfection, cells were cultured on MEF feeder cells in mouse stem cell culture. Cell images were taken at different stages of reprogramming. Red arrows: dynamic changes of cell morphology. ( c ) Characterization of iPSCs. After induction, iPSC colonies were stained for alkaline phosphatase, a stem cell marker. At the end of reprogramming, iPSC colonies were expanded on MEF feeder cells. After expansion, stable iPSCs were examined for pluripotency by immunochemical staining of pluripotent biomarkers, including <t>NANOG</t> and SSEA4. iPSCs were used for further testing of teratoma formation in nude mice. ( d ) Schematic diagram of RNA-seq for fibroblasts and iPSCs. Thousands of RNAs were found to be differentially expressed after reprogramming.
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    Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, <t>NANOG,</t> PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.
    Nanog, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ChIP analysis reveals Nanog - binding sites regulating p27 KIP1 expression. (A) Schematic representation (not drawn to scale) of the p27 genomic locus (RefSeq: NM_009875) highlighting the location of putative Nanog-binding sites designated ‘D’ (primer pairs designated as D1 and D2) and ‘P’ (primer pairs designated as P1 and P2) in the upstream region of the p27 KIP1 gene ( Chen et al., 2008 ; Marson et al., 2008 ). PCR primer pairs were designed for these sites for ChIP analyses (dumbbell shaped; Table S2 ). (B) ChIP analysis reveals that Nanog protein in ESCs binds within the upstream region of the p27 KIP1 gene. Oct4-GiP MEF and Oct4-GiP ESCs were cultured, harvested and processed for ChIP analysis with beads only, IgG and Nanog antibody. The Oct4-GiP MEF cell line was used as a negative control. Input DNA (10%) was used as a control for ChIP. Beads only and IgG served as negative controls. Putative Nanog-binding regions were amplified by the designed primer pairs (D1, D2, P1 and P2). Primer pairs were also designed randomly in the 3′UTR region of the p27 KIP1 gene to serve as a negative (desert) control (Dc). (C) RT-qPCR analysis on the ChIP samples explained in B using primers pairs ‘P’ (P1) and ‘D’ (D1) to amplify the Nanog-binding p27 KIP1 sites. RT-qPCR was also performed on the Dc primer set but no amplification was observed other than the input samples (data not shown). Two independent biological replicates were performed for ChIP analysis. ** P

    Journal: Journal of Cell Science

    Article Title: Nanog induces suppression of senescence through downregulation of p27KIP1 expression

    doi: 10.1242/jcs.167932

    Figure Lengend Snippet: ChIP analysis reveals Nanog - binding sites regulating p27 KIP1 expression. (A) Schematic representation (not drawn to scale) of the p27 genomic locus (RefSeq: NM_009875) highlighting the location of putative Nanog-binding sites designated ‘D’ (primer pairs designated as D1 and D2) and ‘P’ (primer pairs designated as P1 and P2) in the upstream region of the p27 KIP1 gene ( Chen et al., 2008 ; Marson et al., 2008 ). PCR primer pairs were designed for these sites for ChIP analyses (dumbbell shaped; Table S2 ). (B) ChIP analysis reveals that Nanog protein in ESCs binds within the upstream region of the p27 KIP1 gene. Oct4-GiP MEF and Oct4-GiP ESCs were cultured, harvested and processed for ChIP analysis with beads only, IgG and Nanog antibody. The Oct4-GiP MEF cell line was used as a negative control. Input DNA (10%) was used as a control for ChIP. Beads only and IgG served as negative controls. Putative Nanog-binding regions were amplified by the designed primer pairs (D1, D2, P1 and P2). Primer pairs were also designed randomly in the 3′UTR region of the p27 KIP1 gene to serve as a negative (desert) control (Dc). (C) RT-qPCR analysis on the ChIP samples explained in B using primers pairs ‘P’ (P1) and ‘D’ (D1) to amplify the Nanog-binding p27 KIP1 sites. RT-qPCR was also performed on the Dc primer set but no amplification was observed other than the input samples (data not shown). Two independent biological replicates were performed for ChIP analysis. ** P

    Article Snippet: After dilution, the protein–DNA complexes were immunoprecipitated overnight at 4°C with rotation using primary antibodies against Nanog (AB5731, Millipore) and rabbit control IgG ChIP grade (ab46540, Abcam).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Expressing, Polymerase Chain Reaction, Cell Culture, Negative Control, Amplification, Quantitative RT-PCR

    Reprogramming of fibroblasts into pluripotent stem cells. ( a ) Schematic diagram of pluripotent reprogramming. Fibroblasts were transfected with lentiviruses carrying Oct4-Sox2-Klf3-c-Myc (OSKM) transcription factors and were induced into pluripotency. iPSC: induced pluripotent stem cell; lncRNA: long noncoding RNA. ( b ) Typical images of iPSC reprogramming. After OSKM transfection, cells were cultured on MEF feeder cells in mouse stem cell culture. Cell images were taken at different stages of reprogramming. Red arrows: dynamic changes of cell morphology. ( c ) Characterization of iPSCs. After induction, iPSC colonies were stained for alkaline phosphatase, a stem cell marker. At the end of reprogramming, iPSC colonies were expanded on MEF feeder cells. After expansion, stable iPSCs were examined for pluripotency by immunochemical staining of pluripotent biomarkers, including NANOG and SSEA4. iPSCs were used for further testing of teratoma formation in nude mice. ( d ) Schematic diagram of RNA-seq for fibroblasts and iPSCs. Thousands of RNAs were found to be differentially expressed after reprogramming.

    Journal: Scientific Data

    Article Title: Combined RNA-seq and RAT-seq mapping of long noncoding RNAs in pluripotent reprogramming

    doi: 10.1038/sdata.2018.255

    Figure Lengend Snippet: Reprogramming of fibroblasts into pluripotent stem cells. ( a ) Schematic diagram of pluripotent reprogramming. Fibroblasts were transfected with lentiviruses carrying Oct4-Sox2-Klf3-c-Myc (OSKM) transcription factors and were induced into pluripotency. iPSC: induced pluripotent stem cell; lncRNA: long noncoding RNA. ( b ) Typical images of iPSC reprogramming. After OSKM transfection, cells were cultured on MEF feeder cells in mouse stem cell culture. Cell images were taken at different stages of reprogramming. Red arrows: dynamic changes of cell morphology. ( c ) Characterization of iPSCs. After induction, iPSC colonies were stained for alkaline phosphatase, a stem cell marker. At the end of reprogramming, iPSC colonies were expanded on MEF feeder cells. After expansion, stable iPSCs were examined for pluripotency by immunochemical staining of pluripotent biomarkers, including NANOG and SSEA4. iPSCs were used for further testing of teratoma formation in nude mice. ( d ) Schematic diagram of RNA-seq for fibroblasts and iPSCs. Thousands of RNAs were found to be differentially expressed after reprogramming.

    Article Snippet: The isolated iPSCs were also examined for the expression of the pluripotency markers NANOG and SSEA1 using Fluorescent Mouse ES/iPS Cell Characterization Kit (SCR077, Millipore, CA), as previously described , .

    Techniques: Transfection, Cell Culture, Stem Cell Culture, Staining, Marker, Mouse Assay, RNA Sequencing Assay

    Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.

    Journal: Molecular and Cellular Biology

    Article Title: Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation

    doi: 10.1128/MCB.00499-18

    Figure Lengend Snippet: Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.

    Article Snippet: Primary antibodies used were OCT4 (1:100; Millipore), NANOG (1:100; Millipore), CUX1 (1:100; Abcam), glial fibrillary acidic protein (GFAP) (1:250; Dako), TUBB3/TUJ1 (1:1,000; Covance), BRN2 (1:100; Abcam), PAX6 (1:1,000; Developmental Studies Hybridoma Bank [DSHB]), anti-phospho-histone H3 (Ser10) (1:200; Cell Signaling Technology) and were applied for 3 to 4 h at room temperature or overnight at 4°C.

    Techniques: In Vitro, Cell Culture, Immunocytochemistry, Expressing, Marker, Staining, Polymerase Chain Reaction, Sequencing, Negative Control

    Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Generation of Transgenic Rats through Induced Pluripotent Stem Cells *

    doi: 10.1074/jbc.M113.492900

    Figure Lengend Snippet: Characterization of riPSCs. A , RT-PCR analysis of the expression of endogenous and transgenic Yamanaka factors. DA5-3 and transgenic REFs were used as positive controls. Normal REFs were used as a negative control. B , karyotype of riPS-1 (passage 18, 2 N = 42). Scale bar represents 10 μm. C , AP staining of riPS cells (riPS-1, P18). Scale bar represents 100 μm. D , RT-PCR analysis the expression of pluripotent markers of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. E , Q-PCR analysis of the expression of pluripotency marker genes Oct4 , Nanog , Sox2 , and Rex1 in riPSCs (riPS-1, P15). DA5-3 was used as a positive control, and REFs as a negative control. Expression values are relative to β-actin gene expression set as 1. Error bars represent the S.D. ( n = 3). F , Western blot detection of Oct4, Nanog, and Sox2 expression of rat iPS cells. DA5-3 was used as positive control and REFs as negative control. G , Oct4, Nanog, Sox2, and SSEA-1 expression in riPSCs (riPS-1, P18) was determined by immunofluorescence. DNA ( blue ) was stained with Hoechst 33342. Scale bars represent 50 μm. H , bisulfite genomic sequencing of the enhancer region ( blue ) and promoter region ( red ) of rat Oct4. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

    Article Snippet: Primary antibodies against the following markers were used: Oct4 (1:100; Santa Cruz Biotechnology), Sox2 (1:100; Santa Cruz Biotechnology), Nanog (1:100; Millipore), SSEA-1 (1:100; Santa Cruz Biotechnology), and Esrrb (1:100; Santa Cruz Biotechnology).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Negative Control, Staining, Positive Control, Polymerase Chain Reaction, Marker, Western Blot, Immunofluorescence, Genomic Sequencing, Methylation