mouse monoclonal antibodies for oct4  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos

doi: 10.7554/elife.100735

Figure Lengend Snippet: Figure 1. Generation of maternal-zygotic knockout (KO) embryos. (A) Schemes of mKO2-labeled Pou5f1-KO (Pou5f1mKO2) and Pou5f1 flox alleles. (B) Schemes of EGFP-labeled Sox2-KO (Sox2EGFP) and Sox2 flox alleles. (C) Mating strategy for Pou5f1 Ctrl and KO embryos. (D) Mating strategy for Sox2 Ctrl and KO embryos. (E) Immunostaining with embryos separated based on the mKO2 fluorescence. (F) Immunostaining with embryos separated based on the EGFP fluorescence. N, the number of embryos. Scalebar, 20 μm.

Article Snippet: Immunofluorescence Immunofluorescence was performed using the following primary antibodies with dilutions: mouse monoclonal anti- Oct4 (sc- 5279, Santa Cruz), 1:1000; goat anti- Sox2 (GT15098, Neuromics), 1:300; Hou et al. eLife 2024;13:RP100735.

Techniques: Knock-Out, Labeling, Immunostaining, Fluorescence

Journal: eLife

Article Title: Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos

doi: 10.7554/elife.100735

Figure Lengend Snippet: Figure 2. Oct4 and Sox2 regulate chromatin landscape and transcriptome in the inner cell mass (ICM). (A) Principal component analysis (PCA) plot of all the identified ATAC-seq peaks. (B) Number of differentially accessible ATAC-seq peaks in knockout (KO) vs Ctrl samples. Cutoff, adjusted p-value<0.05. (C) k-Means clustering of all the significantly differential ATAC-seq peaks in KO vs Ctrl in B. The heatmap is sorted by clusters, motifs, and transcription start sites (TSSs). Peaks located within 100 bp from TSS were considered as TSS peaks. OCT-SOX, OCT, or SOX motifs indicate that the peak contains the canonical OCT-SOX, OCT, or SOX motif, respectively, while OCT&SOX motif indicates that separate OCT and SOX motifs were discovered in one peak. Cutoff, adjusted p-value<0.05. (D) Number of differentially expressed genes in KO vs Ctrl samples. Cutoff, adjusted p-value<0.05 and log2 fold change≥1. (E) Gene set enrichment analysis (GSEA) shows the correlation between significantly changed ATAC-seq peaks and the transcription of genes whose TSSs are located within 10 kb of the peak centers in Pou5f1- or Sox2-KO late ICMs. NES, normalized enrichment score. (F) Examples of down- and upregulated genes. The underlined numbers represent the adjusted p-values. (G) The ATAC-seq profiles surrounding the genes in E. Boxes mark the differentially accessible peaks, and red boxes specifically mark those with the OCT-SOX motif.

Article Snippet: Immunofluorescence Immunofluorescence was performed using the following primary antibodies with dilutions: mouse monoclonal anti- Oct4 (sc- 5279, Santa Cruz), 1:1000; goat anti- Sox2 (GT15098, Neuromics), 1:300; Hou et al. eLife 2024;13:RP100735.

Techniques: Knock-Out

Journal: eLife

Article Title: Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos

doi: 10.7554/elife.100735

Figure Lengend Snippet: Figure 3. Oct4 and Sox2 activate epiblast (EPI)-specific genes and suppress trophectoderm (TE)-specific genes in inner cell mass (ICM). (A) A scatter plot shows the log2 fold change of ATAC-seq signals in the Pou5f1-KO and Sox2-KO late ICM. Colored dots represent the significantly changed peaks. Cutoff, adjusted p-value<0.05. (B) GREAT ontology enrichment analysis of the significantly changed peaks shared in the Pou5f1-KO and Sox2-KO late ICM. Red terms are related to the pluripotency and preimplantation embryonic development, and green ones are related to the development of embryonic and extraembryonic lineages in the post-implantation embryos. (C) A scatter plot shows the log2 fold changes of RNA-seq signals in the Pou5f1-KO and Sox2-KO late ICM. Cutoff, adjusted p-value<0.05 and log2 fold change≥1. (D) Violin plots of RNA-seq rlog values for Nanog, Esrrb, and Klf4 in the embryos. The underlined numbers represent the adjusted p-values. (E) Immunostaining of Pecam1 in the late blastocysts (E2.5+2 days).

Article Snippet: Immunofluorescence Immunofluorescence was performed using the following primary antibodies with dilutions: mouse monoclonal anti- Oct4 (sc- 5279, Santa Cruz), 1:1000; goat anti- Sox2 (GT15098, Neuromics), 1:300; Hou et al. eLife 2024;13:RP100735.

Techniques: RNA Sequencing, Immunostaining

Journal: eLife

Article Title: Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos

doi: 10.7554/elife.100735

Figure Lengend Snippet: Figure 4. Oct4 and Sox2 activate OCT-SOX enhancers cooperatively and independently in inner cell mass (ICM). (A) Motif enrichment analysis of significantly changed ATAC-seq peaks in the Pou5f1-KO and Sox2-KO ICMs. (B) Occurrence of the motifs in the ranked peaks. The decreased enhancers were ranked by the fold reduction. The cumulative percentages of peaks containing at least one sequence for a given motif are plotted against ranks of peaks. (C) Average profiles of the 8993 OCT-SOX peaks across all the Ctrl and KO ICM samples. (D) Scatter plots show the log2 fold change of 8993 OCT-SOX peaks in the Pou5f1- and Sox2-KO late ICMs. (E) The profiles of ATAC-seq in early embryos, Sox2 CUT&RUN in E4.5 EPI (Li et al., 2023), and Oct4 and Sox2 ChIP-seq in embryonic stem cells (ESCs) (Marson et al., 2008) around the known OCT-SOX enhancers of Klf4 and Dppa3. The red boxes mark the OCT-SOX enhancers.

Article Snippet: Immunofluorescence Immunofluorescence was performed using the following primary antibodies with dilutions: mouse monoclonal anti- Oct4 (sc- 5279, Santa Cruz), 1:1000; goat anti- Sox2 (GT15098, Neuromics), 1:300; Hou et al. eLife 2024;13:RP100735.

Techniques: Sequencing, ChIP-sequencing

Journal: eLife

Article Title: Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos

doi: 10.7554/elife.100735

Figure Lengend Snippet: Figure 5. Oct4 and Sox2 promote the developmental trajectory from the morula to the inner cell mass (ICM). (A) Bar graphs show the dynamics of the decreased peaks (left) and genes (right) in Pou5f1- and Sox2-KO early ICMs from the morula to the early ICM. (B and D) Alluvial plots show the dynamics of chromatin accessibility (B) and transcriptome (D) from morula to late ICM in the Ctrl embryos. Green, gray, and red lines represent the decreased, unchanged, and increased peaks/genes, respectively. In D, only genes significantly up- or downregulated in both Pou5f1 Ctrl and Sox2 Ctrl embryos were considered as up- or downregulated genes, while all the rest were considered as unchanged genes. (C and E) Gene set enrichment analysis (GSEA) plots show the enrichment of the 21,731 ATAC-seq peaks (C) and 1115 genes (E) in Pou5f1-KO and Sox2-KO early ICMs. NES, normalized enrichment score. (F) The bar chart illustrates the GSEA Wikipathway enrichment in WebGestalt. The log2 fold change values of the 1115 upregulated genes (D) in Sox2-KO vs Ctrl early ICM were used in this analysis. False discovery rate (FDR)≤0.05. FC, fold change. (G) GSEA enrichment plot of the term PluriNetWork in F. NES, normalized enrichment score. (H) Upper panel: IGV tracks displaying ATAC-seq and Sox2 CUT&RUN profiles (Li et al., 2023), along with ChIP-seq profiles of Oct4 and Sox2 in embryonic stem cells (ESCs) (Marson et al., 2008), centered around the genomic locus of Il6st. Red box marks the OCT-SOX enhancer. Lower panel: violin plot showing the rlog values of Il6st. The underlined numbers represent the adjusted p-values. (I) Model of the activation of pluripotency-related genes in the early embryos.

Article Snippet: Immunofluorescence Immunofluorescence was performed using the following primary antibodies with dilutions: mouse monoclonal anti- Oct4 (sc- 5279, Santa Cruz), 1:1000; goat anti- Sox2 (GT15098, Neuromics), 1:300; Hou et al. eLife 2024;13:RP100735.

Techniques: ChIP-sequencing, Activation Assay

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 1. Expression of OCT4 and VCC-1 in clinical lung adenocarcinoma tissues and lung cancer cell lines. (A-C) Immunohistochemical staining (A) and quantification (B, C) of OCT4 and VCC-1 expression in lung adenocarcinoma patients (Stage I, n = 3; Stage II, n = 3; Stage III, n = 3) and normal lung tissues (Normal), (Scale bars, 100 μm for OCT4; 20 μm for VCC-1). (D) Positive correlation between OCT4 and VCC-1 intensities (r = 0.4789, p = 0.0126, Pearson’s correlation coefficient). (E) Expression of OCT4 and VCC-1 in six lung cancer cell lines and HEL299 normal lung fibroblasts detected by immunoblot analysis. Expression of β-actin served as the loading control.

Article Snippet: For immunoprecipitation, the chromatin solution was incubated with mouse monoclonal anti-human OCT4 antibody (sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4° C on a rotator.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Control

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 2. Overexpression of OCT4 increases, whereas knockdown of OCT4 decreases, VCC-1 expression in lung cancer cells. (A-C) Detection of OCT4 and VCC-1 expression. H1299 cells were transfected with 4 µg of pSin-EF2-OCT4-Pur (OCT4) or pSin-EF2-GFP-Pur (GFP), or mock-transfected (A), or with 1, 2, and 4 µg of pSin-EF2-OCT4-Pur or pSin-EF2-GFP-Pur (4 µg) (B), or transduced with lentiviral vectors expressing shRNAs specific to OCT4 (#1 or #2) or luciferase (Luc) (C). After 48 h, levels of OCT4 and VCC-1 mRNA were examined by qPCR (A, n = 4), and their protein levels were detected by immunoblotting (B, C, n = 3). Expression of β-actin served as the loading control. Representative immunoblots from three independent experiments (left panels) and quantitative analysis of OCT4 (middle panels) and VCC-1 (right panels) are shown. Ratios between the intensity of the bands corresponding to the indicated protein and those corresponding to β-actin were calculated, and ratios of control cells were

Article Snippet: For immunoprecipitation, the chromatin solution was incubated with mouse monoclonal anti-human OCT4 antibody (sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4° C on a rotator.

Techniques: Over Expression, Knockdown, Expressing, Transfection, Transduction, Luciferase, Western Blot, Control

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 3. OCT4 and VCC-1 promote TGF-β production in lung cancer cells. (A-C) Detection of TGF-β production by ELISA. H1299 cells were transfected with pCMV-Tag2B (Vector, 6 µg), pCMV-Tag2B-OCT4 (OCT4, 1, 2, 4, and 6 µg), pCMV-Tag2B-VCC-1 (VCC-1, 1, 2, 4, and 6 µg), or mock-transfected (A, B). A549 cells were transduced with lentiviral vectors expressing shRNAs specific to VCC-1 (#1 to #4) or luciferase (Luc) (C) for 48 h. Dose-dependent overexpression of OCT and VCC-1 in H1299 cells transfected with Flag-tagged OCT4 and VCC-1 expression vectors were verified by immunoblotting with the anti-Flag antibody, respectively (lower panels, A, B). The culture medium was analyzed for TGF-β production by ELISA (upper panels, A-C, n = 3). (D) IL-4 and VCC-1 proteins enhance TGF-β production. THP-1 cells were treated with PMA (5 ng/ml) for 48 h, and stimulated with recombinant IL-4 (90 ng/ml) or VCC-1 (5 nM) proteins for 24 h. Levels of TGF-β in the culture medium were determined by ELISA at 48 h post-treatment (n = 3). (E) Detection of VEGF in H1299 cells that had been transfected with pCMV-Tag2B-OCT4 (OCT4), pCMV-Tag2B (Vector), or mock-transfected for 48 h. The culture medium was analyzed for VEGF production by ELISA (n = 3). Note that overexpression of OCT4 does not affect VEGF expression.

Article Snippet: For immunoprecipitation, the chromatin solution was incubated with mouse monoclonal anti-human OCT4 antibody (sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4° C on a rotator.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Transduction, Expressing, Luciferase, Over Expression, Western Blot, Recombinant

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 4. Lung cancer cells overexpressing OCT4 or VCC-1 attract the migration of macrophage-like THP-1 cells. (A, B) H1299 cells plated in the lower wells in the Boyden chambers were transfected with pCMV-Tag2B (Vector, 6 µg), pCMV-Tag2B-OCT4 (OCT4, 1, 2, 4, and 6 µg), pCMV-Tag2B-VCC-1 (VCC-1, 1, 2, 4, and 6 µg), or

Article Snippet: For immunoprecipitation, the chromatin solution was incubated with mouse monoclonal anti-human OCT4 antibody (sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4° C on a rotator.

Techniques: Migration, Transfection, Plasmid Preparation

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 5. Knockdown of VCC-1 in A549 lung cancer cells decreases tumor growth in a human tumor xenograft model. (A) Cell proliferative assay of VCC-1-knockdown (shVCC-1-1 or shVCC-1-2), shRNA control (shLuc), and parental A549 cells (n = 4). (B) Tumor volumes of mice bearing VCC-1-knockdown (shVCC-1-1 or shVCC-1-2) or control A549 tumors. Groups of four NOD/SCID mice were subcutaneously inoculated with 1 × 106 cells of VCC-1-knockdown or control A549 cells. Tumor volumes of the mice were monitored and measured to elucidate the influence of VCC-1 on tumor development. (C) A schematic representation of the OCT4-VCC-1 axis involved in lung cancer progression. OCT4 overexpression in lung cancer cells upregulates VCC-1, which drives tumor aggressiveness through TGF-β secretion and tumor-associated macrophage (TAM) recruitment. OCT4 overexpression in lung cancer cells also promotes M2 macrophage polarization by increasing macrophage colony-stimulating factor (M-CSF) production and enhancing tumor migration, growth, and metastasis. The impact of OCT4 on the upregulation of M-CSF (the pathways in gray color) has been described in our previous paper [4].

Article Snippet: For immunoprecipitation, the chromatin solution was incubated with mouse monoclonal anti-human OCT4 antibody (sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4° C on a rotator.

Techniques: Knockdown, shRNA, Control, Over Expression, Migration