Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 1 | Global redistribution of OCT4 binding in PGCs compared with ESCs. a, Cross-section of a human fetal testis (22 weeks) immunostained for OCT4. The panel on the right is an enlargement of the region enclosed within the white broken lines of the left panel. Scale bars, 50 µm; inset, 100 μm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. b, Left panel: heatmap visualization of OCT4 ChIP–seq data, depicting all binding events centred on the peak region within a 5 kb window around the peak. Right panel: distribution and peak heights of OCT4 peaks around the transcription start site (TSS). Peak heights are reported in reads per million. c, Scatterplot comparing OCT4 binding in PGCs and ESCs. Selected genes known to be associated with pluripotency are highlighted in blue, and those highly expressed in the germline are highlighted in red. d, Genome browser representation of ChIP–seq tracks for OCT4 in ESCs (red) and PGCs (yellow) at the OCT4 and PIWIL1 loci. Regions that were bound exclusively by OCT4 in ESCs or PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR (signals per million reads). ChIP–seq were independently repeated twice and similar results were obtained. e, Venn diagram of unique and shared genes bound by OCT4 in ESCs and PGCs. GO analysis results are shown to the right and bottom of the venn diagram. Y-axes represent each category of molecular functions and biological processes. The analysis was performed twice and similar results were obtained based on two independent ChIP–seq data.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: Binding Assay, Immunostaining, ChIP-sequencing

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 2 | Co-occurrence of OCT4 with PAX5 and PRDM1 in PGCs. a, The position weight matrix of an enriched motif found in OCT4 ChIP–seq data from PGCs. The motif resembles the binding motifs for PRDM1 and PAX5. b, Cross-section of a human fetal testis (22 weeks). Upper panel: immunostained for PAX5 (red) and OCT4 (green), and DAPI stained for nuclei (blue). Lower panel: immunostained for PRDM1 (red) and OCT4 (green), and DAPI stained for nuclei (blue). Enlarged panels on the right represent the region enclosed within the white broken lines of the far left panel. White arrows indicate co-localization of PAX5 and OCT4 or PRDM1 and OCT4. Scale bars (orignal images), 100 µm; (expanded images) 50 µm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. c, Venn diagram of unique and shared genes bound by OCT4, PRDM1 and PAX5 in PGCs. The number of genes bound exclusively by each transcription factor or co-bound by multiple transcription factors are labelled. d, Genome browser representation of ChIP–seq tracks for OCT4 (yellow), PAX5 (blue) and PRDM1 (green) at the TBX3 and PIWIL1 loci. Regions that are bound collectively by OCT4, PAX5 and PRDM1 in PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR (signals per million reads). ChIP–seq were independently repeated twice and similar results were obtained. e, GO analysis of co-bound genes. The analysis was performed twice and similar results were obtained. f, GST pull-down assay performed using OCT4 and PAX5 recombinant proteins. Pull-down was repeated three times and similar results were obtained. Unprocessed scans of western blots are shown in Supplementary Fig. 8.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, Staining, Immunostaining, Pull Down Assay, Recombinant, Western Blot

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 5 | PAX5 acts upstream of OCT4. a, Genome browser representation of ChIP–seq tracks at the OCT4 locus. Enhancer regions are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR. ChIP–seq was independently repeated twice and similar results were obtained. b, OCT4 expression in hESCs, control cells and cells overexpressing PAX5 during in vitro differentiation. Data represent the mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. c, Mouse testis xenografts immunostained for GFP and c-KIT. Scale bar, 50 µm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. DAPI stained for nuclei. d, Flow cytometry analysis for GFP and c-KIT of mouse testis xenografts. e, RT-qPCR analysis of OCT4 expression in hPGCs formed in mouse seminiferous tubules by cells overexpressing PAX5, PAX5 knockout cells or control hESCs. Data represent the mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. f, Reporter construct used for measuring OCT4 enhancer activity is shown. The genomic fragment bound by PAX5 and OCT4 (red) was inserted upstream of a luciferase gene driven by a minimal promoter. The y axis represents the fold enrichment of luciferase activity. Data represent the mean ± s.d. of n = 3 independent replicates. Source data for b, e and f are provided in Supplementary Table 2.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Expressing, Control, In Vitro, Two Tailed Test, Immunostaining, Staining, Flow Cytometry, Quantitative RT-PCR, Knock-Out, Construct, Activity Assay, Luciferase

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 6 | PAX5 and OCT4 act upstream of PRDM1. a, Genome browser representation of ChIP–seq tracks at the PRDM1 locus. Enhancer regions bound by OCT4 and PAX5 in PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR. ChIP–seq was independently repeated twice and similar results were obtained. b, PRDM1 expression in control cells, cells overexpressing PAX5 and PAX5 knockout cells during in vitro differentiation. Data are represented as mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. c, RT-qPCR analysis of PRDM1 expression in hPGCs formed in the mouse seminiferous tubules by PAX5 overexpressing, PAX5 knockout and control hESCs. Data are represented as mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. d, Reporter construct used for measuring PRDM1 enhancer activity is shown. The genomic fragment bound by PAX5 and OCT4 (red) was inserted upstream of a luciferase gene driven by a minimal promoter. The y axis represents the fold enrichment of luciferase activity. Data represent the mean ± s.d. of n = 3 independent replicates. e, RT-qPCR analysis of the expression of genes associated with germline programming. Data represent the mean ± S.D. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. f, Model for gene regulation in pluripotency and germline programmes. In pluripotent stem cells, OCT4, together with other transcription factors (TFs) and cofactors, binds to its own enhancer to activate and maintain its high expression. While differentiating towards germline cells, PAX5 replaces OCT4 and binds to the enhancer of OCT4 to maintain a moderate expression of OCT4. Meanwhile, PAX5 and OCT4 bind to the enhancer of PRDM1 and activate its expression to initiate the germline programme. Source data for b, c, d and e are provided in Supplementary Table 2.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Expressing, Control, Knock-Out, In Vitro, Two Tailed Test, Quantitative RT-PCR, Construct, Activity Assay, Luciferase

Journal: Nature Communications

Article Title: Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

doi: 10.1038/s41467-021-23320-8

Figure Lengend Snippet: a FACS analysis of the percentages of OCT4-Tdtomato+ H1-EPS cells with indicated factor treatment after 2 passages. n = 2 biologically independent samples. None, LCDM medium. b Analysis of cell numbers of feeder-free cultured hEPS cells under the treatments of vitamin C, catalase, and their combination. VC, vitamin C. n = 3 biologically independent samples. ES1-EPS cells were used. c Analysis of cell numbers of feeder-free cultured hEPS cells in different basal culturing media. n = 4 biologically independent samples. ES1-EPS cells were used. d – f Analysis of the effects of different matrix proteins on feeder-free cultured hEPS cells. The percentages of adherent hEPS cells at 1.5 h after seeding was shown in d . Survival of dissociated hEPS cells 24 h after seeding was shown in e . Proliferation of hEPS cells at 72 h after seeding was shown in f . For e , f , index represents the cell number at a specific time point divided by the number of seeding cells. For d – f , n = 3 biologically independent samples. ES1-EPS cells were used. g Representative morphologies of xeno-free hEPS cells with different genetic backgrounds. Similar images were obtained in at least 5 independent experiments. Error bars, mean ± SD. All differences between means with P < 0.01 are indicated. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Statistical significance was analyzed using one-way ANOVA with Tukey multiple comparison test in b – f . Scale bar, 100 μm. Experiments in a – g were all independently repeated at least three times with similar results.

Article Snippet: To evaluate human OCT4 DE transcriptional regulation in primed hPSCs and hEPS cells cultured in different conditions, OCT4 -DE luciferase plasmid (Addgene, 52414) was transfected into the cells by nucleofection (4D-NucleofectorTM System, Lonza).

Techniques: Cell Culture, Comparison

Journal: Nature Communications

Article Title: Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

doi: 10.1038/s41467-021-23320-8

Figure Lengend Snippet: a Representative FACS analysis of the percentages of OCT4 + or NANOG + cells in xeno-free hEPS cells. b Hierarchical clustering of global gene expression of EPS cells cultured in XF-LCDM and F-LCDM. Distance is calculated by 1-Spearman correlation coefficient. c GSEA analysis of enrichment of module C and module D in xeno-free and feeder-cultured hEPS cells. Bulk RNA-seq data for xeno-free and feeder-cultured hEPS cells were used. Gene signatures for module C and module D were defined in our previous study (Yang et al.) . As the control, dataset for pre-implantation human embryos (GSE36552, Yan et al.) were chosen. d GSEA analysis of enrichment of gene signatures for E5/E6/E7 epiblasts or naive pluripotent stem cells in xeno-free and feeder-cultured hEPS cells. Bulk RNA-seq data for xeno-free and feeder-cultured hEPS cells were used. Gene signatures for E5/E6/E7 epiblasts were defined in the study performed by Petropoulos et al. , and naive signatures were defined in the study performed by Liu et al. . As the control, dataset for pre-implantation human embryos (GSE36552, Yan et al.) , and naive or primed hPSCs (E-MTAB-4461, Guo et al.; E-MTAB-5674, Guo et al.) , were chosen. e Mean FPKM values at TE marker genes in XF-LCDM, F-LCDM and mTeSR1 cultured primed cells. The signal density of given region (2 kb up- and down- streams of TSS) and randomly selected genome regions of the same length were analyzed. TE marker genes were defined in the study performed by Petropoulos et al. .

Article Snippet: To evaluate human OCT4 DE transcriptional regulation in primed hPSCs and hEPS cells cultured in different conditions, OCT4 -DE luciferase plasmid (Addgene, 52414) was transfected into the cells by nucleofection (4D-NucleofectorTM System, Lonza).

Techniques: Gene Expression, Cell Culture, RNA Sequencing, Control, Marker

Journal: Nature Communications

Article Title: Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

doi: 10.1038/s41467-021-23320-8

Figure Lengend Snippet: a Representative images showing histological analysis of teratomas generated from xeno-free hEPS cells. Scale bars, 100 µm. Similar images were obtained in at least 3 independent experiments. b Phase contrast images of primed and XF-EPS cells. To derive hTSC-like cells, primed hPSCs and XF-EPS cells were cultured in hTSC medium. Primed H1 and XF H1-EPS cells were used. Scale bars, 100 µm. Similar images were obtained in at least three independent experiments. c Representative immune-staining images showing the marker expression of XF-EPS cells and hTSC-like cells. XF H1-EPS cells were used. Scale bars, 100 µm. Similar images were obtained in at least three independent experiments. d Representative images showing chimerism of xeno-free hEPS cells in E4.5 mouse blastocysts. GFP, anti-GFP antibody. White and yellow arrows indicate GFP + cells with OCT4 and CDX2 expression respectively. Scale bar, 100 µm. Similar images were obtained in at least 5 independent experiments. e Chimerism of XF-EPS cells, naive hPSCs and primed hPSCs in E6.5 mouse conceptuses. Representative images of chimerism were shown. A summary of the chimeric experiment was also shown. Scale bar, 500 µm. f Immunofluorescent staining of E6.5 chimeric embryos showing lineage contributions of xeno-free hEPS derivatives to epiblast or trophoblast lineages. mclover+ ES1-EPS cells were used. Embryos were stained for OCT4 (epiblast), CK18 (trophoblast compartment) and mclover (GFP). White and yellow arrows indicate GFP + cells with OCT4 and CK18 expression respectively. Scale bars, 50 µm. Similar images were obtained in at least 5 independent experiments. Experiments in a – f were all independently repeated at least three times with similar results.

Article Snippet: To evaluate human OCT4 DE transcriptional regulation in primed hPSCs and hEPS cells cultured in different conditions, OCT4 -DE luciferase plasmid (Addgene, 52414) was transfected into the cells by nucleofection (4D-NucleofectorTM System, Lonza).

Techniques: Generated, Cell Culture, Staining, Marker, Expressing

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 1 OCT4 was overexpressed in the SP of ovarian cancer cells. Notes: (A–C) Western blotting and RT-PCR were carried out to analyze the protein and mRNA expressions of OCT4 in the SP and NSP population of SKOV3 and A2780 cells. **P< 0.01; ***P<0.001. Abbreviations: NSP, non-SP; SP, side population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 2 Downregulation of OCT4 reduced cell drug resistance and inhibited cell proliferation and tumorigenesis in the SP of ovarian cancer cells. Notes: (A, B) Western blotting analysis of the knockdown efficiency of OCT4 after 48 hours of the cells were transfected with sh-OCT4. (C, D) Different concentrations of DDP were added in the SP of SKOV3 and A2780 cells after 48 hours of the cells were transfected with sh-OCT4, then CCK-8 assay was performed to assess cell viability. (E, F) CCK-8 analysis of cell viability after 48 hours of cell treatments. (G, H) Flow cytometry analysis of cell cycle after 48 hours of cell treatments. (I, J) In vivo xenograft model was carried out to analyze the effect of sh-OCT4 on tumorigenesis in the SP cells. The data presented are the mean ± standard error and represent three independent experiments (*P<0.05; **P<0.01). Effects of downregulation of OCT4 on cell viability and cycle in the SP population of SKOV3 cells. Abbreviations: CCK-8, cell counting kit-8; SP, side population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: Western Blot, Knockdown, Transfection, CCK-8 Assay, Flow Cytometry, In Vivo, Cell Counting

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 3 Effects of OCT4 overexpression on cell functions and drug resistance in the NSP of SKOV3 and A2780 cells. Notes: (A, B) Western blotting was carried out to analyze the protein expressions of OCT4 after 48 hours of the SP cells were treated with Lentiv-OCT4 or Lentiv-NC, respectively. (C, D) Different concentrations of DDP were added in the NSP of SKOV3 and A2780 cells after 48 hours of the cells were treated with Lentiv-OCT4 or Lentiv- NC, and then CCK-8 assay was performed to assess cell viability. (E, F) CCK-8 analysis of cell proliferation after 48 hours of the NSP cells was infected with Lentiv-OCT4 or Lentiv-NC. (G, H) Flow cytometry was used to assess cell cycle after 48 hours of the NSP cells were infected with Lentiv-OCT4 or Lentiv-NC. The data presented are the mean ± standard error and represent three independent experiments (*P<0.05; **P<0.01). Abbreviations: CCK-8, cell counting kit-8; NSP, non-SP; SP, side population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: Over Expression, Western Blot, CCK-8 Assay, Infection, Flow Cytometry, Cell Counting

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 4 Evaluation of the effect of OCT4 on JAK/STAT signaling pathway activation in the NSP of SKOV3 and A2780 cells. Notes: (A, B) Western blotting analysis of the protein expressions of phosphorylated JAK1 (p-JAK1), p-STAT6, p-AKT, and p-NF-κB after 48 hours of the NSP cells was infected with Lentiv-OCT4 or Lentiv-NC. (C, D) Western blotting analysis of the protein expressions of p-JAK1, p-JAK2, p-JAK3, and p-Tyk2 after 48 hours of the NSP cells was infected with Lentiv-OCT4 or Lentiv-NC. (E, F) Western blotting analysis of the protein expressions of p-STAT1, p-STAT2, p-STAT3, p-STAT4, p-STAT5, and p-STAT6 after 48 hours of the NSP cells was treated with Lentiv-OCT4 or Lentiv-NC. (G, H) Immunofluorescence staining was used to determine the subcellular location of STAT6 after 48 hours of the SP SKOV3 cells were transfected Lentiv-OCT4. (I, J) Western blotting analysis of the expression of Cyclin D1, c-Myc, and Bcl-2 after 48 hours of the NSP cells was treated with Lentiv-OCT4 or Lentiv-NC. The data presented are the mean ± standard error and represent three independent experiments (*, #P<0.05). Abbreviation: NSP, nonside population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: Activation Assay, Western Blot, Infection, Immunofluorescence, Staining, Transfection, Expressing

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 5 Assessment of the effects of OCT4/JAK/STAT on cell functions in the NSP of SKOV3 and A2780 cells. Notes: (A, B) Western blotting analysis of the protein levels of JAK1 and caspase-3 in different treated NSP cells: Lentiv-NC (48 hours), Lentiv-OCT4 (48 hours), and peficitinib (4.8 nM, 2 hours) + Lentiv-OCT4 (48 hours). (C, D) Flow cytometry was performed to assess the apoptosis of the NSP cells with different treatments: Lentiv- NC (48 hours), Lentiv-OCT4 (48 hours), and peficitinib (4.8 nM, 2 hours) + Lentiv-OCT4 (48 hours). (E, F) CCK-8 and clone formation assays were performed to assess cell viability in the SP cells with different treatments: Lentiv-NC (48 hours), Lentiv-OCT4 (48 hours), and peficitinib (4.8 nM, 2 hours) + Lentiv-OCT4 (48 hours). (G, H). Transwell assay was used to evaluate the invasion of the NSP cells with different treatments: Lentiv-NC (48 hours), Lentiv-OCT4 (48 hours), and peficitinib (4.8 nM, 2 hours) + Lentiv-OCT4 (48 hours). The data presented are the mean ± standard error and represent three independent experiments (*, #P<0.05). Abbreviations: CCK-8, cell counting kit-8; NSP, nonside population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: Western Blot, Flow Cytometry, CCK-8 Assay, Transwell Assay, Cell Counting

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 7 Graphical abstract of this study. Notes: OCT4 activates JAK/STAT signaling, then promotes the expression of Cyclin D1, c-Myc, and Bcl-2, accelerating the tumorigenesis of NSP cells in ovarian cancer. Abbreviation: NSP, nonside population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells

doi: 10.2147/cmar.s180418

Figure Lengend Snippet: Figure 6 Detection of the effect of OCT4/JAK/STAT on the tumorigenesis of the NSP of SKOV3 and A2780 cells. Notes: In vivo xenograft model analysis of the effect of OCT4/JAK/STAT on tumorigenesis. The data presented are the mean ± standard error and represent three independent experiments (*, #P<0.05). Abbreviation: NSP, nonside population.

Article Snippet: For upregulation of OCT4, the lentivirus ORF of OCT4 (No. RC211998L1, OriGene) was used.

Techniques: In Vivo

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) Effect of NFATc3 on pluripotent transcription factors (NANOG, OCT4, KLF4, LIN28, and SOX2) expression was determined by qPCR. Their levels in NOKSI/NFATc3 were plotted as fold change against those in NOKSI/EV. * P < 0.001. ( B ) Effect of NFATc3 on OCT4 promoter activity was determined by luciferase promoter assay. Cells were transfected with pGL3-Basic (promoter-less) or pGL3 vectors containing the 1.5-kb upstream (-1545∼ -24) of Oct4. * P < 0.001. ( C ) Sequence analysis reveals a consensus NFAT binding site (5′-GGAAA-3′) at -1088 ∼ -1084 indicated by star (upper diagram). OSCC cells were lysed and performed a ChIP assay. The fragment (-1191∼ -1061) containing the NFAT binding site was enriched with NFATc3, and the fragment (-2930∼ -2783) was amplified as a control. * P < 0.01. ( D ) Correlation analysis of NFATc3 and OCT4 mRNA was determined based on their expression levels in 18 human SCC cell lines by qPCR.

Article Snippet: The mammalian pCMV6-Entry Vector encoding OCT4 was purchased from OriGene (RC211998).

Techniques: Expressing, Activity Assay, Luciferase, Promoter Assay, Transfection, Sequencing, Binding Assay, Amplification

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) The effect of OCT4 knockdown on self-renewal capacity of NOKSI/NFATc3 was determined by tumor sphere formation assay. OCT4 was knocked down in NOKSI/NFATc3 using siRNA against OCT4 (Oct4i). The cells transfected with control siRNA (CTLi) were included for comparison. * P < 0.05. ( B ) The effect of OCT4 knockdown on migration ability in NOKSI/NFATc3 was determined by transwell migration assay. ** P < 0.01. ( C ) The effect of OCT4 knockdown on self-renewal capacity of SCC4 was determined by tumor sphere formation assay. ( D ) The effect of OCT4 knockdown on self-renewal capacity of SCC4 was determined by transwell migration assay. ( E ) OCT4 expression was forced in non-tumorigenic immortalized oral epithelial cells, NOKSI, by vector expressing recombinant Myc-DDK-tagged OCT4, and its ectopic expression was confirmed by Western blot analysis using anti-DDK antibody. ( F ) Effect of ectopic OCT4 expression on self-renewal capacity of NOKSI was determined by tumor sphere formation assay. Representative images of tumor spheres formed by NOKSI/EV and NOKSI/Oct4 are shown on the right. ( G ) Effect of ectopic OCT4 expression on migration ability of NOKSI was determined by transwell migration assay. ( H ) Effect of ectopic OCT4 expression on the expression of NFAT isoforms (NFATc1-c4) in NOKSI was determined by qPCR. Their levels in NOKSI/Oct4 were plotted as fold induction against those in NOKSI/EV.

Article Snippet: The mammalian pCMV6-Entry Vector encoding OCT4 was purchased from OriGene (RC211998).

Techniques: Tube Formation Assay, Transfection, Migration, Transwell Migration Assay, Expressing, Plasmid Preparation, Recombinant, Western Blot

Journal: Discover Oncology

Article Title: Quercetin suppresses endometrial cancer stem cells via ERα-mediated inhibition of STAT3 signaling

doi: 10.1007/s12672-025-03863-8

Figure Lengend Snippet: Enrichment of CSCs derived from endometrial carcinoma cells. A Representative images of spheres formed in serum-free medium on days 3, 6, 9, and 12. B , C Western blot analysis of stemness markers (ALDH1A1, c-Myc, Nanog, and Oct4) in parental cells (PCs) cultured in serum-supplemented medium and CSCs maintained in serum-free medium for 12 days. Different letters (a, b) indicate significant differences ( P < 0.05) between groups. All cytological experiments were independently repeated at least three times

Article Snippet: pGL3-human Oct4 PE-SV40-luc was purchase from Addgene; the vector pGL3 bought from Pomega was used as a control.

Techniques: Derivative Assay, Western Blot, Cell Culture

Journal: Discover Oncology

Article Title: Quercetin suppresses endometrial cancer stem cells via ERα-mediated inhibition of STAT3 signaling

doi: 10.1007/s12672-025-03863-8

Figure Lengend Snippet: The effects of Quercetin treatment on sphere formation and maintenance of stemness in CSCs. A 1 × 10 4 cells were treated with 25 or 50 µmol/L Quercetin for 12 days, and spheres were imaged. B 100 spheres with diameter > 50 μm were treated with 50 µmol/L Quercetin for 72 h, and then imaged. C 1 × 10 5 cells were treated with 25 or 50 µmol/L Quercetin for 12 days, and total protein was extracted for western blot analysis of stemness markers (ALDH1A1, c-Myc, Nanog, and Oct4). Different letters (a, b, c) indicate significant differences ( P < 0.05) between groups. All cytological experiments were independently repeated at least three times

Article Snippet: pGL3-human Oct4 PE-SV40-luc was purchase from Addgene; the vector pGL3 bought from Pomega was used as a control.

Techniques: Western Blot

Journal: Discover Oncology

Article Title: Quercetin suppresses endometrial cancer stem cells via ERα-mediated inhibition of STAT3 signaling

doi: 10.1007/s12672-025-03863-8

Figure Lengend Snippet: Quercetin inhibits STAT3’s transcriptional activity in the presence of ERα. A Luciferase reporter assay was used to evaluate the inhibitory effect of Quercetin on STAT3-mediated Oct4 promoter activity. Different letters (a, b) indicate significant differences ( P < 0.05) between groups. B mRNA expression of Oct4, Nanog, Twist, and Snai1 was measured by qPCR after Quercetin treatment. Different letters (a, b, c) indicate significant differences ( P < 0.05) between groups. The effects of Quercetin on sphere formation ( C ) and invasion ( D ) were assessed in the presence or absence of ERα. All cytological experiments were independently repeated at least three times

Article Snippet: pGL3-human Oct4 PE-SV40-luc was purchase from Addgene; the vector pGL3 bought from Pomega was used as a control.

Techniques: Activity Assay, Luciferase, Reporter Assay, Expressing