goat polyclonal sox17  (R&D Systems)

Journal: Nature Communications

Article Title: Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

doi: 10.1038/s41467-025-67308-0

Figure Lengend Snippet: A Evolution of chromatin accessibility measured by ATAC-seq over GATA6 binding groups. Presented and ordered as in Fig. . B Evolution of SOX17 binding across the same regions, ordered as in A and Fig. . C Average profiles of GATA6 binding, SOX17 binding, chromatin accessibility (small ATAC-seq fragments) and nucleosome positioning (MNase-seq) at regions displaying or not SOX17 recruitment, throughout GATA6 induction (colored lines). All plots are centered on the GATA6 summit. D Top. Quantification of nucleosome order using the spectral density calculated from the profiles shown in Supplementary Fig. at a period of 180 bases–nucleosome (150 bases) plus linker DNA (30 bases)—throughout GATA6 induction time-points. Bottom. Quantification of nucleosome density over 50 bp centered on the GATA6 summit calculated from the profiles shown in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Primary antibody incubation was performed overnight at 4 °C with the following antibodies diluted at 1:1000: goat polyclonal anti-GATA6 (R&D Systems AF1700), mouse anti-OCT4 (clone C10, Santa Cruz #5279), rabbit polyclonal anti-SOX2 (Active Motif #39823), mouse monoclonal anti-ESRRB (Perseus Proteomics, H6-705-00), goat polyclonal α-SOX17 (R&D Systems AF1924–Fig. ) or Rabbit Monoclonal α-SOX17 (Abcam #ab224637) and rat monoclonal anti-PDGFRA (APA5, Thermo Fisher #14-1401-82).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

doi: 10.1038/s41467-025-67308-0

Figure Lengend Snippet: A Binding dynamics of OCT4, SOX2 and ESRRB upon GATA6 induction, both at GATA6 peaks showing pluripotency TF binding before or during differentiation (i.e., GATA6 peaks lacking pluripotency TF binding are omitted) and at other pluripotency TF binding sites in undifferentiated ES cells. B Correlations between categories of GATA6 binding segregated by pluripotency TF binding ( X axis) and by GATA6 dynamics (top; Y axis) or by SOX17 binding (bottom; Y axis). The dependency between variables was assessed with two-sided Chi-square tests ( p < 2.2e-16). C Log2 odds ratio of the proportion of regions displaying ectopic pluripotency TF binding across different GATA6 binding groups shown on the left. The statistical significance of the enrichments/depletions was assessed with two-sided Fisher's Exact tests ( p < 1.3e-155). D Average profiles of GATA6 binding, SOX17 binding, chromatin accessibility (small ATAC-seq fragments) and nucleosome positioning (MNase-seq) at regions displaying no pluripotency TF binding at all (noPTF) or an ectopic recruitment (ectopic), throughout GATA6 induction (colored lines). All plots are centered on the GATA6 summit. E Statistical association of GATA6 binding regions divided by pluripotency TF binding groups, with the six groups of differentially expressed genes shown in Fig. , measured with two-sided Fisher Exact tests at increasing distances. Source data are provided as a Source Data file.

Article Snippet: Primary antibody incubation was performed overnight at 4 °C with the following antibodies diluted at 1:1000: goat polyclonal anti-GATA6 (R&D Systems AF1700), mouse anti-OCT4 (clone C10, Santa Cruz #5279), rabbit polyclonal anti-SOX2 (Active Motif #39823), mouse monoclonal anti-ESRRB (Perseus Proteomics, H6-705-00), goat polyclonal α-SOX17 (R&D Systems AF1924–Fig. ) or Rabbit Monoclonal α-SOX17 (Abcam #ab224637) and rat monoclonal anti-PDGFRA (APA5, Thermo Fisher #14-1401-82).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

doi: 10.1038/s41467-025-67308-0

Figure Lengend Snippet: A Representative photomicrographs of cells subject to GATA6 induction and to either non-targeting control siRNAs or Oct4 knockdown for 4 days ( n = 6 independent kd). The scale bar represents 100 µm. B Immunostaining of SOX17 and PDGFRa after 4 days of GATA6 induction and control or Oct4 siRNAs (representative of 6 independent kd). The scale bar represents 30 µm. C Average profile of GATA6 and SOX17 binding at regions identified as showing decreased or increased GATA6 binding (FDR < 0.1 and absolute Fold-change > 0.5) after 2 days of GATA6 induction and Oct4 knockdown. D Log2 odds ratio of the proportion of regions displaying decreased or increased GATA6 binding across groups of regions displaying different behaviors of pluripotency TF binding, shown on the bottom. The statistical significance of the enrichments/depletions was assessed with two-sided Fisher Exact tests ( p = 3.8e-174 and 8.5e-107 for regions binding pluripotency TFs from d0 or ectopically, respectively). E Statistical association of GATA6 binding regions showing increased or decreased GATA6 binding upon Oct4 knock-down, with genes displaying down or upregulated expression, measured with Fisher's Exact tests at increasing distances. F Expression of endogenous Gata6 expression, measured by quantification of its 5’UTR absent in the ectopic transgene, upon control or Oct4 knock-down during GATA6 induction (day 2; paired two-sided T test p = 0.003992; n = 6 independent kd; median—bar; 25–75% percentiles—box; 1.5-folds the inter-quartile range—whiskers). G Identification of an active enhancer based on histone marks (top) ~65 kb upstream of Gata6, showing no TF binding before differentiation but rapidly recruiting GATA6 and SOX17 and displaying ectopic OCT4/SOX2 binding during GATA6 induction. Source data are provided as a Source Data file.

Article Snippet: Primary antibody incubation was performed overnight at 4 °C with the following antibodies diluted at 1:1000: goat polyclonal anti-GATA6 (R&D Systems AF1700), mouse anti-OCT4 (clone C10, Santa Cruz #5279), rabbit polyclonal anti-SOX2 (Active Motif #39823), mouse monoclonal anti-ESRRB (Perseus Proteomics, H6-705-00), goat polyclonal α-SOX17 (R&D Systems AF1924–Fig. ) or Rabbit Monoclonal α-SOX17 (Abcam #ab224637) and rat monoclonal anti-PDGFRA (APA5, Thermo Fisher #14-1401-82).

Techniques: Control, Knockdown, Immunostaining, Binding Assay, Expressing

Journal: Nature Communications

Article Title: Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

doi: 10.1038/s41467-025-67308-0

Figure Lengend Snippet: A Photomicrographs of cells subject to chemical PrE induction for 7 days, either untreated or IAA-treated cells to deplete OCT4 from the onset of the chemical PrE induction (d0), from day 1 (d1) or day 2 (d2). The PrE-like cells shown for d1-treated cells represent one of the rare colonies that did not die. The experiment was repeated 2 independent times. The scale bar represents 100 µm. B Immunostaining of GATA6, PDGFRa, SOX17 and GATA4, after 7 days of chemical PrE induction, for untreated (top) and IAA-treated cells from day 2 onwards (bottom). The scale bar represents 150 µm. C Quantification of all immuno-stainings in control ES cells (ctl in gray), after 1 day of IAA treatment (d1 in gray), and in cells differentiated for 7 days (in red), either wild-type to control for IAA effects or in Oct4-AID cells in the absence of IAA (ctl) or having experienced OCT4 depletion from day 1, day 2 or day 3 onwards. The boxplots show Z-score fluorescence measurements (median—bar; 25-75% percentiles—box; 1.5-folds the inter-quartile range—whiskers; the number of cells analyzed for each factor and condition are, in the same order as in the boxplots, for GATA6: 96243, 78118, 53408, 78178, 14895, 44722, 56419; for PDGFRa: 33965, 38644, 29457, 38204, 25878, 36252, 45315; for SOX17: 78506, 79183, 58912, 64119, 25878, 42948, 62884; for GATA4: 53214, 41156, 31082, 44788, 22362, 41880, 41470, all derived from 2 independent experiments). Source data are provided as a Source Data file.

Article Snippet: Primary antibody incubation was performed overnight at 4 °C with the following antibodies diluted at 1:1000: goat polyclonal anti-GATA6 (R&D Systems AF1700), mouse anti-OCT4 (clone C10, Santa Cruz #5279), rabbit polyclonal anti-SOX2 (Active Motif #39823), mouse monoclonal anti-ESRRB (Perseus Proteomics, H6-705-00), goat polyclonal α-SOX17 (R&D Systems AF1924–Fig. ) or Rabbit Monoclonal α-SOX17 (Abcam #ab224637) and rat monoclonal anti-PDGFRA (APA5, Thermo Fisher #14-1401-82).

Techniques: Immunostaining, Control, Fluorescence, Derivative Assay

Journal: Nature Communications

Article Title: Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

doi: 10.1038/s41467-025-67308-0

Figure Lengend Snippet: Summary of the molecular observations reported in this study, highlighting different modes of GATA6 binding (opportunistic or pioneer), different dynamics (early versus late and stable versus transient) and different outcomes regarding: recruitment of SOX17 or of OCT4/SOX2; chromatin structure; gene regulatory influences at early, mid or late responsive genes; the occurrence of DNA motifs. A caption and a diagram of the epistatic interactions emerging from kinetic analyses and OCT4-depleted cells are shown.

Article Snippet: Primary antibody incubation was performed overnight at 4 °C with the following antibodies diluted at 1:1000: goat polyclonal anti-GATA6 (R&D Systems AF1700), mouse anti-OCT4 (clone C10, Santa Cruz #5279), rabbit polyclonal anti-SOX2 (Active Motif #39823), mouse monoclonal anti-ESRRB (Perseus Proteomics, H6-705-00), goat polyclonal α-SOX17 (R&D Systems AF1924–Fig. ) or Rabbit Monoclonal α-SOX17 (Abcam #ab224637) and rat monoclonal anti-PDGFRA (APA5, Thermo Fisher #14-1401-82).

Techniques: Binding Assay

Journal: Biology of Reproduction

Article Title: Maternal/zygotic knockout and droplet digital polymerase chain reaction analysis question the role of activin A in preimplantation mouse embryo development ^†

doi: 10.1093/biolre/ioaf189

Figure Lengend Snippet: Embryos deprived of maternal activin A exhibit delayed development to the blastocyst stage and compromised mitochondrial activity. Comparison of main morphokinetic parameters between maternalknockout (m-KO), maternal/zygotic knockout (mz-KO), and control – Inhba LoxP/LoxP embryos. (A) These parameters were measured as the time (in hours) from hCG injection to specific developmental milestones: tNEBD—breakdown of nuclear envelopes of pronuclei; t2–t8—completion of division to the respective number of blastomeres; tM—compaction; tC and tH—initiation of cavitation and hatching process, respectively. (B) The value of individual parameters was calculated as the number of hours between: m1 – the disappearance of pronuclei and 2-cell stage, i.e., duration of the first embryonic M-phase; cc2 and cc3—the time between 2-cell and 3-cell stage, and between 3-cell stage and 5-cell stage, i.e., duration of the second and third cell cycle, respectively; s2 and s3—the cleavage timing between progeny in the different cell generations, i.e,. synchronicity of second and third rounds of cleavage divisions, respectively; sM—compaction and cavitation, the period during which the embryo remains in the morula stage. On all graphs, the total number of embryos analyzed per genotype is indicated in parentheses. On the boxplots, the middle lines represent medians, the cross shows the mean values, the hinges indicate the interquartile ranges, the whiskers represent the minimum and maximum values within the group and the dots show outliers. Statistical analysis was performed using the Kruskal–Wallis test with post hoc Dunn test, separately for each morphokinetic parameter. * P < 0.05; ** P < 0.01; *** P < 0.001. (C) Confocal images of blastocysts immunostained with antibodies against CDX2 (TE), SOX17 (PE), SOX2 (EPI), with chromatin stained with Hoechst. Scale bars: 20 μm. (D–E) Percentage contribution of primary cell lineages, negative and bipotential cells within (D) blastocyst and (E) inner cell mass in blastocysts ( P > 0.05, Kruskal–Wallis test). Abbreviations: trophectoderm (TE), inner cell mass (ICM), primitive endoderm (PE), epiblast (EPI), negative (neg.; CDX2- SOX17- SOX2-) cells, bipotential (bipot.; SOX2+ SOX17+) cells. See also . (F) Mitochondrial membrane potential assessed by JC-1 staining. The JC-1 monomers indicate low mitochondrial membrane potential, while JC-1 aggregates form in mitochondria with high membrane potential. The fluorescent photographs are maximum orthogonal projections from ten z-stacks, whereas the bright field presents a single subset from the middle of the oocytes. Scale bars: 50 μm. (G) Ratios of JC-1 red to green fluorescence intensities, indicating mitochondrial activity. Statistical analysis using Mann–Whitney U-test revealed a significant difference in the activity of mitochondria ( *** P < 0.001).

Article Snippet: For blastocysts, the following primary antibodies were used: mouse monoclonal antibody against CDX2 (1:50, BioGenex, MU392A-UC, RRID:AB_2923402), rabbit polyclonal antibody against SOX2 (1:100, Abcam, ab97959, RRID:AB_2341193), goat polyclonal antibody against SOX17 (1:100, R&D Systems, AF1924, RRID:AB_355060).

Techniques: Activity Assay, Comparison, Knock-Out, Control, Injection, Staining, Membrane, Fluorescence, MANN-WHITNEY