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mouse pou5f1 oct4 (Addgene inc)

Name: mouse pou5f1 oct4
Brand: Addgene inc
Rating: 92 (4 reviews)

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Addgene inc mouse pou5f1 oct4
Mouse Pou5f1 Oct4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Characterization of iPSCs on 0 d and during neuronal differentiation on 7 d. A Scheme of modeling NSC differentiation: reference iPSC lines (WISCi004-B and WAi001-B), iPSC lines from one late-onset AD patient (MLUI007-J/H and MLUi008-B/F), and one iPSC line from one matched healthy control (MLUi009-A). B Phase contrast image for iPSCs and NSCs on 0 d and 7 d showing a homogenous differentiation into NSCs (scale bar left 300 μm; scale bar right 100 μm). White arrows indicate the rosette-like formation of NSCs representing a premature stage of neural rosettes. Data are shown for one iPSC line (clone) per donor. C Comparison of the three iPSC lines regarding their expression of pluripotency markers on 0 d and their expression of NSC markers on 7 d. We could prove the presence of pluripotency markers LIN28A, OCT4, NANOG, SOX2 in iPSCs and NSC markers SOX1, SOX2, NES, PAX6, MSI1 in NSCs. RT-PCR analysis by gel electrophoresis visualized as bar charts (one amplicon each from MLUi008-B, MLUi009-A, WISCi004-B was pooled for analysis; n = 3, mean ± SEM). D IF analysis on 0 d showed nuclear localization of OCT4 and cellular distribution of the epithelial marker ECAD in iPSCs (scale bar: 100 μm). E Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d; N = 3 differentiations, mean ± SEM

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Characterization of iPSCs on 0 d and during neuronal differentiation on 7 d. A Scheme of modeling NSC differentiation: reference iPSC lines (WISCi004-B and WAi001-B), iPSC lines from one late-onset AD patient (MLUI007-J/H and MLUi008-B/F), and one iPSC line from one matched healthy control (MLUi009-A). B Phase contrast image for iPSCs and NSCs on 0 d and 7 d showing a homogenous differentiation into NSCs (scale bar left 300 μm; scale bar right 100 μm). White arrows indicate the rosette-like formation of NSCs representing a premature stage of neural rosettes. Data are shown for one iPSC line (clone) per donor. C Comparison of the three iPSC lines regarding their expression of pluripotency markers on 0 d and their expression of NSC markers on 7 d. We could prove the presence of pluripotency markers LIN28A, OCT4, NANOG, SOX2 in iPSCs and NSC markers SOX1, SOX2, NES, PAX6, MSI1 in NSCs. RT-PCR analysis by gel electrophoresis visualized as bar charts (one amplicon each from MLUi008-B, MLUi009-A, WISCi004-B was pooled for analysis; n = 3, mean ± SEM). D IF analysis on 0 d showed nuclear localization of OCT4 and cellular distribution of the epithelial marker ECAD in iPSCs (scale bar: 100 μm). E Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d; N = 3 differentiations, mean ± SEM

Article Snippet: Mouse IgG anti-human OCT4 (POU5F1) , IF: 1:100 , Santa Cruz , Sc-5279.

Techniques: Control, Comparison, Expressing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Marker, Multiplex Assay, Quantitative RT-PCR

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of primers for transcript analysis

Article Snippet: Mouse IgG anti-human OCT4 (POU5F1) , IF: 1:100 , Santa Cruz , Sc-5279.

Techniques:

Single-cell RNA-sequencing roadmap of SSC reprogramming. (A) Schematic for the reprogramming of SSC. Days 16–20, Oct4 -EGFP positive (OG(+)) colonies appeared. SSCM, spermatogonial stem cell culture medium. (B) Bright field (top) and fluorescence images (bottom) of Oct4 -EGFP SSCs (days 0 and 10), Oct4 -EGFP positive (OG(+)) colonies in the early stage of reprogramming (day 19), and gPSC-1 (p1) in the late stage of reprogramming. Scale bar, 200 μm. (C) Uniform manifold approximation and projection (UMAP) plot showing the transcriptome data of reprogramming cells, gPSCs, and ESCs. Reprogramming cells are colored by the day of reprogramming. (D) UMAP plot showing the transcriptome data of reprogramming cells from day 0 to day 27, cells are colored by clusters identified from graphed-based unsupervised clustering. (E) UMAP plot showing the RNA velocity vector field of the early stage of SSC reprogramming. (F) PAGA analysis showing the trajectories of the early stage of SSC reprogramming, each circle represents an individual cluster, the size of circles indicates the number of cells in clusters. (G) Cell trajectory of the successful branch of SSC reprogramming, inferred by Moonocle2. Arrows indicate the developmental order of these cells. (H) Enriched GO terms and P values of differential expressed genes of RPG1/2/3/4 clusters. (I) Violin plots showing the expression level of KEGG pathway ‘Glycolysis/Gluconeogenesis’ (mmu00010) and representative glycolysis-related genes. Dunn’s non-parametric test was performed for pairwise multiple comparison of activity between adjacent clusters, P values were adjusted by Bonferroni method, **** P < 0.0001. (J) Extracellular lactic acid level plotted over time during SSC reprogramming. Unpaired two-tailed Student’s s -test, * P < 0.05. (K) Bar plot showing the number of OG(+) colonies on day 23 of SSC reprogramming treated with 2-DG at different concentration. Unpaired two-tailed Student’s t -test, * P < 0.05. (L) Violin plots showing the expression levels of DNA methylation related genes. (M) Schematic showing the stage division of gPSC-, CiPSC-, and iPSC-reprogramming. (N) Bubble plots showing GO terms and their representative genes upregulated in stage 1 (left) and stage 2 (right) of three reprogramming processes, corresponding to (M). GO terms and representative genes are indicated by colors. See also and .

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: Single-cell RNA-sequencing roadmap of SSC reprogramming. (A) Schematic for the reprogramming of SSC. Days 16–20, Oct4 -EGFP positive (OG(+)) colonies appeared. SSCM, spermatogonial stem cell culture medium. (B) Bright field (top) and fluorescence images (bottom) of Oct4 -EGFP SSCs (days 0 and 10), Oct4 -EGFP positive (OG(+)) colonies in the early stage of reprogramming (day 19), and gPSC-1 (p1) in the late stage of reprogramming. Scale bar, 200 μm. (C) Uniform manifold approximation and projection (UMAP) plot showing the transcriptome data of reprogramming cells, gPSCs, and ESCs. Reprogramming cells are colored by the day of reprogramming. (D) UMAP plot showing the transcriptome data of reprogramming cells from day 0 to day 27, cells are colored by clusters identified from graphed-based unsupervised clustering. (E) UMAP plot showing the RNA velocity vector field of the early stage of SSC reprogramming. (F) PAGA analysis showing the trajectories of the early stage of SSC reprogramming, each circle represents an individual cluster, the size of circles indicates the number of cells in clusters. (G) Cell trajectory of the successful branch of SSC reprogramming, inferred by Moonocle2. Arrows indicate the developmental order of these cells. (H) Enriched GO terms and P values of differential expressed genes of RPG1/2/3/4 clusters. (I) Violin plots showing the expression level of KEGG pathway ‘Glycolysis/Gluconeogenesis’ (mmu00010) and representative glycolysis-related genes. Dunn’s non-parametric test was performed for pairwise multiple comparison of activity between adjacent clusters, P values were adjusted by Bonferroni method, **** P < 0.0001. (J) Extracellular lactic acid level plotted over time during SSC reprogramming. Unpaired two-tailed Student’s s -test, * P < 0.05. (K) Bar plot showing the number of OG(+) colonies on day 23 of SSC reprogramming treated with 2-DG at different concentration. Unpaired two-tailed Student’s t -test, * P < 0.05. (L) Violin plots showing the expression levels of DNA methylation related genes. (M) Schematic showing the stage division of gPSC-, CiPSC-, and iPSC-reprogramming. (N) Bubble plots showing GO terms and their representative genes upregulated in stage 1 (left) and stage 2 (right) of three reprogramming processes, corresponding to (M). GO terms and representative genes are indicated by colors. See also and .

Article Snippet: The primary antibodies used in this study were listed: Goat anti-GFRa1 (R&D, AF714), Mouse anti-ZBTB16 (Santa, sc-28319), Rabbit anti-DDX4 (Abcam, ab13840), Mouse anti-POU5F1 (OCT4) (Abcam, ab19857), Mouse anti-POU5F1 (OCT4) (Santa, sc-5279), Mouse anti-SSEA1(Sigma-Aldrich, MAB4301), Rabbit anti-NANOG (Abcam, ab70482), Rabbit anti-RHOX5 (Abcam, ab31922), Rabbit anti-PRDM14 (Cell Signaling Technology, 83527), Goat anti-OTX2 (R&D, AF1979), Rabbit anti-DPPA3 (Abcam, ab19878), Rabbit anti-L1TD1(Biorbyt, orb35537), Mouse anti-TFAP2C (Santa, sc-12762), Rabbit anti-DNMT3B (proteintech, 26971-1-AP), Rabbit anti-DNMT1 (abclonal, A16729), Mouse anti-UHRF1 (Santa, sc-373750), and Mouse anti-ACTIN (proteintech, CL594-66009).

Techniques: RNA Sequencing Assay, Stem Cell Culture, Fluorescence, Plasmid Preparation, Expressing, Activity Assay, Two Tailed Test, Concentration Assay, DNA Methylation Assay

A robust SSC reprogramming system induced by small-molecule compounds. (A) Bar plots showing the number of OG(+) colonies on day 19 treated with individual chemical at 10 µmol/L or 50 μg/mL during SSC reprogramming. Unpaired two-tailed Student’s s -test was performed between DMSO and each chemical group, * P < 0.05. ns, not significant. (B) Bar plots showing the numbers of OG(+) colonies on day 19 treated with individual chemical at different concentration. Unpaired two-tailed Student’s t -test, * P < 0.05. (C) Schematic of SSC reprogramming with the original (top) and 5C (bottom) treatments. (D) AP staining (top) and fluorescence images (bottom) showing the reprogramming cells on day 19 in DMSO and 5C groups. Scale bar, 50 μm. AP staining, alkaline phosphatase staining. (E) Bar plot showing the number of OG(+) colonies at different time points in DMSO and 5C groups. (F) Line chart showing the extracellular lactic acid level plotted over time in DMSO and 5C groups. Unpaired two-tailed Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. (G) Bright field and fluorescence images (top) of gPSCs (p1) from DMSO and 5C groups, respectively. Bar plot (bottom) showing the numbers of gPSC colonies (p1) from these 2 groups. Unpaired two-tailed Student’s t -test; **** P < 0.0001. (H) Bar plot showing the numbers of OG(+) colonies on day 19 in the indicated groups, the original condition (control), DMSO, 5C and 5C withdrawing different chemicals. Unpaired two-tailed Student’s t -test, * P < 0.05, ** P < 0.01, **** P < 0.0001, ns, not significant. (I) Immunofluorescence of OCT4, NANOG and SSEA1 (red) in 5C-gPSC-1. Scale bar, 20 μm. (J) H&E staining of paraffin sections of teratoma derived from subcutaneous injection of 5C-gPSC-1 into nude mice. Scale bar, 100 μm. (K) Bright field and fluorescence images showing E12.5 embryo of KM mouse and chimaera embryo produced by the diploid blastocyst injection of CAG-EGFP 5C-gPSC-2, 2 out of 6 embryos were chimaeras after blastocyst injection. Scale bar, 2 mm. (L) Bright filed and fluorescence images of the E12.5 genital ridges derived from diploid blastocyst injection of 5C-gPSC-1, 7 out of 10 pairs of genital ridges were GFP positive after blastocyst injection. Scale bar, 500 μm. (M) Chimaeras produced from B6D2F1 (black coat color) 5C-gPSC-1. (N) Summary of chimeric assays of diploid blastocyst injection using 5C-gPSC-1.

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: A robust SSC reprogramming system induced by small-molecule compounds. (A) Bar plots showing the number of OG(+) colonies on day 19 treated with individual chemical at 10 µmol/L or 50 μg/mL during SSC reprogramming. Unpaired two-tailed Student’s s -test was performed between DMSO and each chemical group, * P < 0.05. ns, not significant. (B) Bar plots showing the numbers of OG(+) colonies on day 19 treated with individual chemical at different concentration. Unpaired two-tailed Student’s t -test, * P < 0.05. (C) Schematic of SSC reprogramming with the original (top) and 5C (bottom) treatments. (D) AP staining (top) and fluorescence images (bottom) showing the reprogramming cells on day 19 in DMSO and 5C groups. Scale bar, 50 μm. AP staining, alkaline phosphatase staining. (E) Bar plot showing the number of OG(+) colonies at different time points in DMSO and 5C groups. (F) Line chart showing the extracellular lactic acid level plotted over time in DMSO and 5C groups. Unpaired two-tailed Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. (G) Bright field and fluorescence images (top) of gPSCs (p1) from DMSO and 5C groups, respectively. Bar plot (bottom) showing the numbers of gPSC colonies (p1) from these 2 groups. Unpaired two-tailed Student’s t -test; **** P < 0.0001. (H) Bar plot showing the numbers of OG(+) colonies on day 19 in the indicated groups, the original condition (control), DMSO, 5C and 5C withdrawing different chemicals. Unpaired two-tailed Student’s t -test, * P < 0.05, ** P < 0.01, **** P < 0.0001, ns, not significant. (I) Immunofluorescence of OCT4, NANOG and SSEA1 (red) in 5C-gPSC-1. Scale bar, 20 μm. (J) H&E staining of paraffin sections of teratoma derived from subcutaneous injection of 5C-gPSC-1 into nude mice. Scale bar, 100 μm. (K) Bright field and fluorescence images showing E12.5 embryo of KM mouse and chimaera embryo produced by the diploid blastocyst injection of CAG-EGFP 5C-gPSC-2, 2 out of 6 embryos were chimaeras after blastocyst injection. Scale bar, 2 mm. (L) Bright filed and fluorescence images of the E12.5 genital ridges derived from diploid blastocyst injection of 5C-gPSC-1, 7 out of 10 pairs of genital ridges were GFP positive after blastocyst injection. Scale bar, 500 μm. (M) Chimaeras produced from B6D2F1 (black coat color) 5C-gPSC-1. (N) Summary of chimeric assays of diploid blastocyst injection using 5C-gPSC-1.

Techniques: Two Tailed Test, Concentration Assay, Staining, Fluorescence, Immunofluorescence, Derivative Assay, Injection, Produced

5C-gPSCs had true pluripotency identified by tetraploid complementation assay. (A) 5C-gPSC 4N-comp pups and their corresponding placentae. Note: * Pups survived beyond 4 days postnatal; # Pup died within 4 days postnatal. (B) Simple sequence length polymorphism (SSLP) analysis for lineage identification covers markers from different chromosomes, and the 4N-comp mice showed a polymorphic pattern similar with that from the F1-5 ESC, 5C-gPSC-S4, or gPSC-S2 cells originating from B6D2F1 mice, and different from the ICR, C57, or DBA mice. M denotes molecular mass marker. (C) Genotyping analysis of Oct4 -EGFP from F1-5 ESC, 5C-gPSC-S4, or gPSC-S2 4N-comp mice. (D) Bright field (left), fluorescence (middle) and merged (right) images of E3.5 Oct4 -EGFP blastocysts flushed from an ICR female mouse mated with an 5C-gPSC-S4 4N-comp mouse. Scale bar, 100 μm. (E) An 11-week-old 5C-gPSC-S4 4N-comp mouse with a uniformly black coat and its F 1 progeny from its mating to an ICR female mouse. (F) Bar plot showing the developmental rate of full-term 4N-comp pups derived from PSCs, numbers of animals obtained per total number of transferred embryos are shown. Groups are indicated by colors. See for details. (G) Dot plot showing the body weight of 4N-comp mice born by Caesarean section in this study on day 0 postnatal. Groups are indicated by colors. Unpaired two-tailed Student’s t -test, * P < 0.05, ns, not significant. (H) Survival curves of 4N-comp mice obtained in this study. n, total numbers of 4N-comp mice derived from each group. Groups are indicated by colors. (I) Growth curves of 4N-comp mice obtained in this study at the indicated weeks postnatal. Groups are indicated by colors. (J) Bar plot showing the DNA methylation level of the whole genome (left) and total ICRs (right) in ESCs (p10), 5C-gPSCs (p25), gPSCs (p25), respectively. Unpaired two-tailed Student’s t -test, P values were shown above the lines, ** P < 0.01, ns, not significant. (K) Heatmaps showing the mean methylation levels of the paternal ICRs in SSCs (p20), gPSCs (p25), 5C-gPSCs (p25), ESCs (p10). The color key from dark blue to red indicates low to high levels, respectively. (L) Line plot shows the mean methylation levels of Dlk1-Dio3 region in SSCs (p20), 5C-gPSCs (p25), ESCs (p10), gPSCs (p25), respectively. Grey shadows indicate gene regions, the red shadow indicates IG- Gtl2 ICR region. (M) Box plots show the mean DNA methylation levels across gene bodies (from transcription start site (TSS) to transcription end site (TES)) and the 50-bp flanking regions of each miRNA encoded locus in Dlk1-Dio3 region in ESCs, gPSCs and 5C-gPSCs. One dot represents an miRNA encoded locus, the same loci in distinct PSCs are connected by grey lines. Paired two-tailed Student’s t -test was performed between ESCs and other PSCs. (N) Heatmaps showing the mean methylation levels of the maternal ICRs in SSCs (p20), gPSCs (p25), 5C-gPSCs (p25), ESCs (p10). The color key from dark blue to red indicates low to high levels, respectively. (O) qPCR analysis of relative expression of Igf2r , Kcnq1ot1 , Peg3 , and Snrpn normalized to the geometric mean of Rps2 and Gapdh in ESCs, gPSCs, and 5C-gPSCs. Unpaired two-tailed Student’s t -test, ** P < 0.01, *** P < 0.001, ns, not significant. See also .

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: 5C-gPSCs had true pluripotency identified by tetraploid complementation assay. (A) 5C-gPSC 4N-comp pups and their corresponding placentae. Note: * Pups survived beyond 4 days postnatal; # Pup died within 4 days postnatal. (B) Simple sequence length polymorphism (SSLP) analysis for lineage identification covers markers from different chromosomes, and the 4N-comp mice showed a polymorphic pattern similar with that from the F1-5 ESC, 5C-gPSC-S4, or gPSC-S2 cells originating from B6D2F1 mice, and different from the ICR, C57, or DBA mice. M denotes molecular mass marker. (C) Genotyping analysis of Oct4 -EGFP from F1-5 ESC, 5C-gPSC-S4, or gPSC-S2 4N-comp mice. (D) Bright field (left), fluorescence (middle) and merged (right) images of E3.5 Oct4 -EGFP blastocysts flushed from an ICR female mouse mated with an 5C-gPSC-S4 4N-comp mouse. Scale bar, 100 μm. (E) An 11-week-old 5C-gPSC-S4 4N-comp mouse with a uniformly black coat and its F 1 progeny from its mating to an ICR female mouse. (F) Bar plot showing the developmental rate of full-term 4N-comp pups derived from PSCs, numbers of animals obtained per total number of transferred embryos are shown. Groups are indicated by colors. See for details. (G) Dot plot showing the body weight of 4N-comp mice born by Caesarean section in this study on day 0 postnatal. Groups are indicated by colors. Unpaired two-tailed Student’s t -test, * P < 0.05, ns, not significant. (H) Survival curves of 4N-comp mice obtained in this study. n, total numbers of 4N-comp mice derived from each group. Groups are indicated by colors. (I) Growth curves of 4N-comp mice obtained in this study at the indicated weeks postnatal. Groups are indicated by colors. (J) Bar plot showing the DNA methylation level of the whole genome (left) and total ICRs (right) in ESCs (p10), 5C-gPSCs (p25), gPSCs (p25), respectively. Unpaired two-tailed Student’s t -test, P values were shown above the lines, ** P < 0.01, ns, not significant. (K) Heatmaps showing the mean methylation levels of the paternal ICRs in SSCs (p20), gPSCs (p25), 5C-gPSCs (p25), ESCs (p10). The color key from dark blue to red indicates low to high levels, respectively. (L) Line plot shows the mean methylation levels of Dlk1-Dio3 region in SSCs (p20), 5C-gPSCs (p25), ESCs (p10), gPSCs (p25), respectively. Grey shadows indicate gene regions, the red shadow indicates IG- Gtl2 ICR region. (M) Box plots show the mean DNA methylation levels across gene bodies (from transcription start site (TSS) to transcription end site (TES)) and the 50-bp flanking regions of each miRNA encoded locus in Dlk1-Dio3 region in ESCs, gPSCs and 5C-gPSCs. One dot represents an miRNA encoded locus, the same loci in distinct PSCs are connected by grey lines. Paired two-tailed Student’s t -test was performed between ESCs and other PSCs. (N) Heatmaps showing the mean methylation levels of the maternal ICRs in SSCs (p20), gPSCs (p25), 5C-gPSCs (p25), ESCs (p10). The color key from dark blue to red indicates low to high levels, respectively. (O) qPCR analysis of relative expression of Igf2r , Kcnq1ot1 , Peg3 , and Snrpn normalized to the geometric mean of Rps2 and Gapdh in ESCs, gPSCs, and 5C-gPSCs. Unpaired two-tailed Student’s t -test, ** P < 0.01, *** P < 0.001, ns, not significant. See also .

Techniques: Sequencing, Marker, Fluorescence, Derivative Assay, Two Tailed Test, DNA Methylation Assay, Methylation, Expressing

A reversed developmental trajectory of germ cell in SSC reprogramming. (A) UMAP plot showing the integration of single-cell transcriptome data from successful branch of SSC reprogramming (DMSO- and 5C groups), mouse in vivo germ cells and gonadal somatic cells from E6.5 to PND5.5. Grey dots indicate in vivo germ cells or germline somatic cells, colored dots or triangles with black outline indicate reprogramming cells or ESCs. (B) Scmap projection of the successful branch of SSC reprogramming (left, 5C group; right, DMSO group) and mouse in vivo germ cells. The color key from bright yellow to dark blue indicates low to high ratio of projected cells. SPG, spermatogonia; in vivo GC dev., in vivo germ cell development. (C) Heatmap showing the expression of DEGs of in vivo germ cell development and their expression dynamics in SSC reprogramming, GO terms enriched in both processes were highlighted. (D) Violin plots showing the expression levels of canonical marker genes of in vivo germ cells in the successful branch of SSC reprogramming. (E) Immunofluorescence of Oct4 -EGFP (green) co-stained with RHOX5 (top, red) and L1TD1 (bottom, red), respectively, in domed colonies during SSC reprogramming. Scale bar, 50 μm. (F) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (pink) and TFAP2C (red) in domed colonies during SSC reprogramming. Scale bar, 50 μm. (G) Immunofluorescence of Oct4 -EGFP (green) co-stained with OTX2 (red) in domed colonies during SSC reprogramming. Scale bar, 50 μm. (H) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (pink) and OTX2 (red) in domed colonies at different time points during SSC reprogramming. Note, * The diameters of detected colonies. Scale bar, 50 μm. (I) Boxplot showing the number of PRDM14(+)OTX2(–), PRDM14(+)OTX2(+), PRDM14(–)OTX2(+) colonies (left) and their percentage (right) in OG(+) colonies at different time points during SSC reprogramming, corresponding to . See also .

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: A reversed developmental trajectory of germ cell in SSC reprogramming. (A) UMAP plot showing the integration of single-cell transcriptome data from successful branch of SSC reprogramming (DMSO- and 5C groups), mouse in vivo germ cells and gonadal somatic cells from E6.5 to PND5.5. Grey dots indicate in vivo germ cells or germline somatic cells, colored dots or triangles with black outline indicate reprogramming cells or ESCs. (B) Scmap projection of the successful branch of SSC reprogramming (left, 5C group; right, DMSO group) and mouse in vivo germ cells. The color key from bright yellow to dark blue indicates low to high ratio of projected cells. SPG, spermatogonia; in vivo GC dev., in vivo germ cell development. (C) Heatmap showing the expression of DEGs of in vivo germ cell development and their expression dynamics in SSC reprogramming, GO terms enriched in both processes were highlighted. (D) Violin plots showing the expression levels of canonical marker genes of in vivo germ cells in the successful branch of SSC reprogramming. (E) Immunofluorescence of Oct4 -EGFP (green) co-stained with RHOX5 (top, red) and L1TD1 (bottom, red), respectively, in domed colonies during SSC reprogramming. Scale bar, 50 μm. (F) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (pink) and TFAP2C (red) in domed colonies during SSC reprogramming. Scale bar, 50 μm. (G) Immunofluorescence of Oct4 -EGFP (green) co-stained with OTX2 (red) in domed colonies during SSC reprogramming. Scale bar, 50 μm. (H) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (pink) and OTX2 (red) in domed colonies at different time points during SSC reprogramming. Note, * The diameters of detected colonies. Scale bar, 50 μm. (I) Boxplot showing the number of PRDM14(+)OTX2(–), PRDM14(+)OTX2(+), PRDM14(–)OTX2(+) colonies (left) and their percentage (right) in OG(+) colonies at different time points during SSC reprogramming, corresponding to . See also .

Techniques: In Vivo, Expressing, Marker, Immunofluorescence, Staining

Identification of key regulators in SSC reprogramming. (A) Volcano plots showing differentially expressed genes (DEGs, | log (fold change) | ≥ 1, adjusted P < 0.01) in RPG1/2/3/4. Red and blue dots indicate upregulated and downregulated genes, respectively. Transcription factors are highlighted in bold. (B) Heatmap showing the regulon activities from SCENIC analysis in the successful branch of SSC reprogramming and representative enriched GO terms of indicated regulons. The color key from blue to red indicates low to high regulon activity. (C) Heatmaps showing the expression pattern of Rhox5 related genes, target genes of Prdm14 and Otx2 , respectively. The color key from blue to red indicates low to high expression level. (D) Schematic of the gene editing by CRISPR-CasRx in SSC reprogramming. (E) Fluorescence images (left) and boxplot (right) showing the number of OG(+) colonies reprogrammed from empty vector (sgNC)–, vectors targeting Rhox5 (sg Rhox5 1# and sg Rhox5 2#)-transfected SSCs in 5C condition on day 19. Scale bar, 50 μm. Unpaired two-tailed Student’s t -test, ** P < 0.01, *** P < 0.001. NC, negative control. (F) (i) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (top, pink), TFAP2C (top, red), and OTX2 (bottom, red) in domed colonies reprogrammed from sgNC- and sg Rhox5 2#-transfected SSCs, respectively. Scale bar, 50 μm. (ii) Dot plots showing the intensity of PRDM14, TFAP2C, and OTX2 corresponding to (i), Unpaired two-tailed Student’s t -test, **** P < 0.0001. See also .

Journal: Protein & Cell

Article Title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

doi: 10.1093/procel/pwac044

Figure Lengend Snippet: Identification of key regulators in SSC reprogramming. (A) Volcano plots showing differentially expressed genes (DEGs, | log (fold change) | ≥ 1, adjusted P < 0.01) in RPG1/2/3/4. Red and blue dots indicate upregulated and downregulated genes, respectively. Transcription factors are highlighted in bold. (B) Heatmap showing the regulon activities from SCENIC analysis in the successful branch of SSC reprogramming and representative enriched GO terms of indicated regulons. The color key from blue to red indicates low to high regulon activity. (C) Heatmaps showing the expression pattern of Rhox5 related genes, target genes of Prdm14 and Otx2 , respectively. The color key from blue to red indicates low to high expression level. (D) Schematic of the gene editing by CRISPR-CasRx in SSC reprogramming. (E) Fluorescence images (left) and boxplot (right) showing the number of OG(+) colonies reprogrammed from empty vector (sgNC)–, vectors targeting Rhox5 (sg Rhox5 1# and sg Rhox5 2#)-transfected SSCs in 5C condition on day 19. Scale bar, 50 μm. Unpaired two-tailed Student’s t -test, ** P < 0.01, *** P < 0.001. NC, negative control. (F) (i) Immunofluorescence of Oct4 -EGFP (green) co-stained with PRDM14 (top, pink), TFAP2C (top, red), and OTX2 (bottom, red) in domed colonies reprogrammed from sgNC- and sg Rhox5 2#-transfected SSCs, respectively. Scale bar, 50 μm. (ii) Dot plots showing the intensity of PRDM14, TFAP2C, and OTX2 corresponding to (i), Unpaired two-tailed Student’s t -test, **** P < 0.0001. See also .

Techniques: Activity Assay, Expressing, CRISPR, Fluorescence, Plasmid Preparation, Transfection, Two Tailed Test, Negative Control, Immunofluorescence, Staining

Dual immunofluorescence assay of undifferentiated pluripotent stem cell lines, H9, HES4 and iPSC stained with OCT4 or SG1 antibodies. Phase contrast image of stem cell colonies (Phase) and same colony stained with Hoechst dye (Hoechst; blue), OCT4 antibody (OCT4; green), and SG1 antibody (SG1; red).

Journal: PLoS ONE

Article Title: A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

doi: 10.1371/journal.pone.0020663

Figure Lengend Snippet: Dual immunofluorescence assay of undifferentiated pluripotent stem cell lines, H9, HES4 and iPSC stained with OCT4 or SG1 antibodies. Phase contrast image of stem cell colonies (Phase) and same colony stained with Hoechst dye (Hoechst; blue), OCT4 antibody (OCT4; green), and SG1 antibody (SG1; red).

Article Snippet: Primary antibodies used included rabbit polyclonal α-OCT4 (1∶100, Cell Signaling, catalog # 2750), goat polyclonal α-OCT4 (1∶100, Santa Cruz, catalog # SC-8628), mouse monoclonal α-OCT4 (POU5F1; 1∶100, Sigma catalog # P0082), mouse monoclonal α-TRA-1-60 (1∶500, Cell Signaling, catalog # 4746), rabbit polyclonal α-DNMT3B (H-230, 1∶100, Santa Cruz, catalog # 20704), rabbit polyclonal α-DNMT3B (CS, 1∶100, Cell Signaling, catalog # 2161) and rabbit polyclonal SG1 (1∶100).

Techniques: Immunofluorescence, Staining

Western blot analysis using DNMT3B exon 10 peptide antibody (SG1), OCT4 and GAPDH (control) antibodies to detect proteins expressed in pluripotent stem (PSCs) and spontaneously differentiated cells (SDCs; 14–15 days).

Journal: PLoS ONE

Article Title: A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

doi: 10.1371/journal.pone.0020663

Figure Lengend Snippet: Western blot analysis using DNMT3B exon 10 peptide antibody (SG1), OCT4 and GAPDH (control) antibodies to detect proteins expressed in pluripotent stem (PSCs) and spontaneously differentiated cells (SDCs; 14–15 days).

Techniques: Western Blot

Mixed populations of pluripotent and early-stage spontaneously differentiated cells (4–5 days minus zbFGF) obtained from (A) BG01 hESC line or (B) foreskin-1 iPSC line were stained with SG1 antibody and compared to cells stained with α-OCT4 polyclonal and two commercially available α-DNMT3B polyclonal antibodies, one from Cell Signaling (CS) and one from Santa Cruz (H-230). Phase contrast image of stem cell colonies (Phase) and same cells stained with Hoechst dye (Hoecsht; blue), α-OCT4 antibody (OCT4; green) and one of three different α-DNMT3B antibodies (DNMT3B; red) as indicated on the right. The α-DNMT3B antibodies used included the custom peptide antibody, SG1 (top), or one of two commercial antibodies, CS (middle) or H-230 (bottom). Compact colonies of pluripotent stem cells are indicated by large arrows, while dispersed spontaneously differentiated cells are indicated by small arrows in the phase contrast images.

Journal: PLoS ONE

Article Title: A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

doi: 10.1371/journal.pone.0020663

Figure Lengend Snippet: Mixed populations of pluripotent and early-stage spontaneously differentiated cells (4–5 days minus zbFGF) obtained from (A) BG01 hESC line or (B) foreskin-1 iPSC line were stained with SG1 antibody and compared to cells stained with α-OCT4 polyclonal and two commercially available α-DNMT3B polyclonal antibodies, one from Cell Signaling (CS) and one from Santa Cruz (H-230). Phase contrast image of stem cell colonies (Phase) and same cells stained with Hoechst dye (Hoecsht; blue), α-OCT4 antibody (OCT4; green) and one of three different α-DNMT3B antibodies (DNMT3B; red) as indicated on the right. The α-DNMT3B antibodies used included the custom peptide antibody, SG1 (top), or one of two commercial antibodies, CS (middle) or H-230 (bottom). Compact colonies of pluripotent stem cells are indicated by large arrows, while dispersed spontaneously differentiated cells are indicated by small arrows in the phase contrast images.

Techniques: Staining

RNA extracted from H9 cells induced to differentiate by removal of zbFGF from the media for the indicated number of days (0, 3, 6, 9, 12 or 15) was subjected to qRT-PCR analysis. Relative expression levels of OCT4 transcripts (black bars) in comparison to DNMT3B exon 10 containing transcripts (white bars) are plotted as a function of the number of days of spontaneous differentiation. Duplicate qRT-PCR experiments were performed for each sample; the mean of the two experiments is plotted with SEM indicated by the error bars.

Journal: PLoS ONE

Article Title: A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

doi: 10.1371/journal.pone.0020663

Figure Lengend Snippet: RNA extracted from H9 cells induced to differentiate by removal of zbFGF from the media for the indicated number of days (0, 3, 6, 9, 12 or 15) was subjected to qRT-PCR analysis. Relative expression levels of OCT4 transcripts (black bars) in comparison to DNMT3B exon 10 containing transcripts (white bars) are plotted as a function of the number of days of spontaneous differentiation. Duplicate qRT-PCR experiments were performed for each sample; the mean of the two experiments is plotted with SEM indicated by the error bars.

Techniques: Quantitative RT-PCR, Expressing

Mixed populations of pluripotent and spontaneously differentiated cells (4–5 days minus zbFGF) obtained from the H9 hESC line were analyzed by dual immunofluorescence staining using SG1 rabbit polyclonal antibody and mouse monoclonal antibodies to OCT4 (A) or TRA-1-60 (B). A. Brightfield images of stem cell colonies (Brightfield) and same cells stained with Hoechst dye (Hoecsht; blue), α-OCT4 antibody (OCT4; green) and α-DNMT3B exon 10 encoded peptide (SG1; red) are shown. OCT4 and SG1 staining patterns are overlaid (Merge) and the area outlined by the white box in the Merge panel is shown in the Magnification panel to facilitate visualization of precise staining patterns in individual cells. The large arrow in the Magnification panel indicates a cell exhibiting high level expression of both OCT4 and SG1 (in this case, the cell is undergoing mitosis and the SG1 antibody ‘paints’ the chromatids of the dividing cell), the small arrow identifies a cell exhibiting high OCT4 but low SG1 staining, while the large arrowhead indicates a cell still expressing high levels of OCT4 that is not stained by the SG1 antibody. B. Similar analysis as in A (above) using a monoclonal antibody that detects the stem cell marker TRA-1-60 (green). As opposed to the intracellular proteins (above), the TRA-1-60 antibody detects the expected TRA-1-60 expression on the cell surface. While high-level TRA-1-60 expression is detected on almost all cells (both PSCs and SDCs), SG1 staining is more tightly restricted to PSCs or those cells in very early stages of spontaneous differentiation. All images are shown at 100× magnification with the exception of the two “Magnification” panels, which were increased in size by about 12 fold in order to allow visualization of individual cells.

Journal: PLoS ONE

Article Title: A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

doi: 10.1371/journal.pone.0020663

Figure Lengend Snippet: Mixed populations of pluripotent and spontaneously differentiated cells (4–5 days minus zbFGF) obtained from the H9 hESC line were analyzed by dual immunofluorescence staining using SG1 rabbit polyclonal antibody and mouse monoclonal antibodies to OCT4 (A) or TRA-1-60 (B). A. Brightfield images of stem cell colonies (Brightfield) and same cells stained with Hoechst dye (Hoecsht; blue), α-OCT4 antibody (OCT4; green) and α-DNMT3B exon 10 encoded peptide (SG1; red) are shown. OCT4 and SG1 staining patterns are overlaid (Merge) and the area outlined by the white box in the Merge panel is shown in the Magnification panel to facilitate visualization of precise staining patterns in individual cells. The large arrow in the Magnification panel indicates a cell exhibiting high level expression of both OCT4 and SG1 (in this case, the cell is undergoing mitosis and the SG1 antibody ‘paints’ the chromatids of the dividing cell), the small arrow identifies a cell exhibiting high OCT4 but low SG1 staining, while the large arrowhead indicates a cell still expressing high levels of OCT4 that is not stained by the SG1 antibody. B. Similar analysis as in A (above) using a monoclonal antibody that detects the stem cell marker TRA-1-60 (green). As opposed to the intracellular proteins (above), the TRA-1-60 antibody detects the expected TRA-1-60 expression on the cell surface. While high-level TRA-1-60 expression is detected on almost all cells (both PSCs and SDCs), SG1 staining is more tightly restricted to PSCs or those cells in very early stages of spontaneous differentiation. All images are shown at 100× magnification with the exception of the two “Magnification” panels, which were increased in size by about 12 fold in order to allow visualization of individual cells.

Techniques: Immunofluorescence, Staining, Expressing, Marker

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