Journal: eLife

Article Title: Formation of retinal direction-selective circuitry initiated by starburst amacrine cell homotypic contact

doi: 10.7554/eLife.34241

Figure Lengend Snippet:

Article Snippet: Antibody , AP-2a: mouse, 1:200 , Developmental Studies Hybridoma Bank , 3B5 , .

Techniques: Plasmid Preparation, Electron Microscopy, Fluorescence, Membrane, Blocking Assay, Immunoprecipitation, Protease Inhibitor, DC Protein Assay, Recombinant, Construct, Software, Sequencing

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A,B ) Human pluripotent stem cells (hPSC)-derived pluripotent cyst (hPSC-pluripotent cyst, Glass-3D in A ) and amnion cyst (Glass-3D +BMP in B ) assays: immunofluorescence (IF) images show hPSC-pluripotent ( A ) and -amnion ( B ) cysts stained for PODXL (lumen, magenta) and DNA (nuclear shape, blue). BMP4 treatment (20 ng/mL) leads to uniformly squamous amnion cysts by day 4. ( C ) Confocal optical sections of d2 cysts harvested prior to (0 min) and 10, 20, 30, and 60 min after BMP4 treatment, stained with indicated markers. Staining for phosphorylated SMAD1/5 reveals a prominent nuclear pSMAD1/5 enrichment within 10 min of BMP4 treatment in all cells. ( D ) Quantification of nuclear pSMAD1/5: five independent samples were counted, more than 50 cells were counted per sample. * indicates statistically significant changes (p<0.05) compared to 0 min timepoint. ( E ) Abundant GATA3 expression (in green), but not TFAP2A (red), is seen by 3 hr after BMP4 treatment while SOX2 expression is maintained. ( F–H ) Pluripotency (NANOG, SOX2) and amnion (ISL1/2, GATA3, TFAP2A) marker expression analyses of d4 cysts grown in the Glass-3D (top) or in Glass-3D +BMP (bottom) conditions. ( I, J ) Confocal micrographs of d4 cysts grown in the Glass-3D +BMP system stained for phosphorylated EZRIN, RADIXIN, and MOESIN (pERM), an apical membrane marker, as well as with indicated amnion markers (top – cysts were treated with BMP4 for 24 hr between d2 and d3; bottom – treated with BMP4 for 48 hr from d2 to d4). ( K, L ) Quantification of GATA3 and ISL1/2 fluorescent intensities in 24 (blue, n=115 cells) and 48 hr (red, n=76 cells) BMP4 treatment samples ( K ), as well as of nuclear aspect ratios of d4 hPSC-cyst (black, n=63 cells), and 24 (blue, n=45 cells) and 48 hr (red, n=52) BMP4-treated cysts (harvested at d4), ( L ). ( M ) pSMAD1/5 staining (green) shows similar nuclear enrichment between 24 and 48 hr BMP4 treatment at d4 (also stained with indicated markers). Scale bars = 50 μm. BMP, bone morphogenetic protein.

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Derivative Assay, Immunofluorescence, Staining, Expressing, Marker, Membrane

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A ) Timeline of the Glass-3D +BMP hPSC-amnion assay. Twenty four hr BMP treatment is sufficient to generate uniformly squamous hPSC-amnion cysts. ( B,C ) Optical sections of time-course Glass-3D +BMP hPSC-amnion cysts (harvested at indicated timepoints after BMP4 treatment) stained with indicated markers (inverted black and white fluorescent signals shown for individual channels, to aid visualization). GATA3, an immediate BMP target is seen by 3 hr (yellow arrowheads). hPSC-amnion cysts with prominent nuclear TFAP2A ( B ) and ISL1/2 ( C ) are seen by 12 and 24 hr, respectively (yellow arrowheads), while SOX2 shows gradual decrease. Consistent changes are also observed at mRNA transcript levels using quantitative RT-PCR ( D–F ), also see the expression of additional markers: HAND1 (amniotic) and NANOG (pluripotent); * indicates statistically significant changes (p<0.05) compared to 0 hr timepoint. ( G ) Relative mRNA expression analyses of indicated pluripotency (top) and amniotic (bottom) markers between d4 Glass-3D hPSC-pluripotent cyst (black), and d4 Glass-3D +BMP hPSC-amnion with 24 hr (blue) or 48 hr (red) BMP treatment; * indicates statistically significant changes (p<0.05). ( H ) BMP inhibitor (LDN-193189) treatment outline as well as confocal micrographs of d4 Glass-3D +BMP hPSC-amnion (top, control – 24 hr BMP4 treatment between d2 and d3) and hPSC-amnion treated with LDN-193189 between d3 and d4 after BMP4 treatment (between d2 and d3). Scale bars = 50 μm. BMP, bone morphogenetic protein.

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Staining, Quantitative RT-PCR, Expressing, Control

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A ) A Uniform Manifold Approximation and Projection (UMAP) plot displaying the original single-cell transcriptome coordinates of the CS7 human embryo described in Tyser et al., shown with the original annotations. The inset ( i ) is used for expression analyses in ( B ). ( B ) Expression of amnion markers – GATA3 , TFAP2A, and ISL1 – superimposed onto the UMAP plot in ( A – i ). ( C ) Transcriptome similarity analysis comparing the CS7 human embryo single-cell dataset and the Glass-3D +BMP time-course bulk RNA sequencing dataset, showing the human CS7 embryo UMAP plot (inset) with colored cells that are transcriptomically similar to each timepoints as indicated (orange – pluripotent cysts/monolayer and 0, 0.5, 1, 3, and 6 hr post-BMP4; green – 12 hr post-BMP4; blue – 24 hr; magenta – 48 hr). While pluripotent and earlier timepoints (orange colored cells) overlap with Epiblast cells, some 12 hr (green) and all 24 hr (blue) samples are similar to a subpopulation of Primitive Streak cells, and 48 hr (magenta) samples overlap exclusively with Ectoderm (see Materials and methods for detailed bioinformatics pipeline). ( D ) TBXT expression transposed onto the CS7 human embryo UMAP plot, revealing TBXT low subpopulation that show transcriptomic similarities to 24 hr post-BMP samples (indicated by dotted circle). ( E ) An integrated UMAP plot for two in vitro cultured gestation date (GD) 14 (8 days after culturing 6-day-old blastocyst) cynomolgus macaque peri-gastrula (replotted and reclustered from Yang et al.). ( F ) Transcriptome similarity analysis comparing the GD14 cynomolgus macaque peri-gastrula dataset, and the Glass-3D +BMP time-course dataset, showing the Yang et al. GD14 UMAP plot (inset ‘ i’ ) with colored cells that are transcriptomically similar to each timepoints as indicated (orange – pluripotent cysts/monolayer and 0, 0.5, 1, 3, 6, and 12 hr post-BMP4; blue – 24 hr; magenta – 48 hr). While pluripotent and early datapoints (orange colored cells) overlap with epiblast cells, the 24 hr samples share similarities with TBXT low population, and the 48 hr samples overlap with ISL1 high amnion cells ( G ), expression analysis of indicated markers. Gradient scales indicate expression level (orange = high, gray = low). Uncropped plots are found in . BMP, bone morphogenetic protein. Created with BioRender.com .

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Expressing, RNA Sequencing, In Vitro, Cell Culture

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A, B ) Original UMAP expression plots of the Tyser et al. Carnegie stage (CS)7 human embryo single-cell RNA sequencing dataset used in this study (GATA3, TFAP2A, ISL1, TBXT, and DLX5, A ) as well as time-course transcriptome similarity UMAP plot ( B ). ( C ) Confocal micrographs of control Glass-3D +BMP cysts stained for ISL1/2 (green), SOX2 (red), and TBXT (magenta). ( D ) Time-course plot of normalized expression values for TBXT. ( E, F ) Original time-course transcriptome similarity UMAP plot of the Yang et al. GD14 cynomolgus macaque dataset ( E ) as well as expression plots ( F ). ( G ) Optical sections of a CS6/7 cynomolgus macaque embryo stained for TBXT and SOX2. TBXT expression is enriched in primitive streak as well as in the primitive streak-derived disseminating cells.

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Expressing, RNA Sequencing, Control, Staining, Derivative Assay

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A ) RNA velocity analysis (scVelo) of the Tyser et al. CS7 human embryo dataset, showing predicted lineage progression trajectories from Epiblast to Ectoderm traversing the TBXT low population. ( B–G ) Optical sections of a CS6/7 cynomolgus macaque embryo stained for indicated transcription factors (GATA3, ISL1/2, pSMAD1/5, and SOX2, B, D, F ), as well as quantitation for relative fluorescent intensity in proximal (closer to epiblast) to distal (closer to amnion) cells of indicated markers at the amnion-epiblast boundary ( C , E , and G are quantitation of images in B , D , and F , respectively). Each dot represents a single cell. ( i ), ( ii ), and ( iii ) indicate insets in ( F ). Half of the amniotic sac shown. Quantitation shows that, while GATA3 + /TFAP2A + cells are also SOX2 + , ISL1 + cells are only seen after SOX2 expression is no longer detected: ISL1 expression is exclusive to squamous amnion cells. Moreover, the expression of pSMAD1/5 and TFAP2A is reduced in most distal amnion ( ii , iii ). Scale bars = 200 μm. ( H ) Nuclear aspect ratio quantitation of amnion-epiblast boundary cells in ( B ), ( C ), and ( D ), mean + standard error of the mean (SEM) shown. Dotted vertical line in each plot indicates amnion-epiblast boundary ( C, E, G, H ). ( I ) A schematic representation of the CS6/7 cynomolgus macaque amniotic sac: the confocal micrographs in ( B ) were edited to represent SOX2 + epiblast (pseudocolored in blue), primitive streak-derived disseminating (green), GATA3 + /TFAP2A + /SOX2 low transition boundary (magenta) and GATA3 + /TFAP2A + /ISL1 + lineage specified amnion (red) cells.

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Staining, Quantitation Assay, Expressing, Derivative Assay

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A,B ) Expression analyses for ISL1 ( A ) and DLX5 ( B ) in the time-course bulk RNA sequencing (top) and in the single-cell Carnegie stage (CS)7 human embryo (bottom) datasets. ( C ) Confocal optical sections of Glass-3D +BMP human pluripotent stem cell (hPSC)-amnion cysts (at 0 hr, 24 hr ( d3 ) and d4) stained with indicated markers. ( D ) Histological section of a cynomolgus monkey amniotic sac at CS7 stained with indicated markers (SOX2 + epiblast; TFAP2A + /DLX5 + /SOX2 - amnion). Insets ( i and ii ) indicate the amnion-epiblast boundaries. DLX5 and TFAP2A share a similar expression pattern at the boundary. Similar to pSMAD1/5 and TFAP2A (shown in ), most distal amnion cells show reduced DLX5 expression. Surface ectoderm bridging neural ectoderm and amniotic ectoderm is not seen at this stage. Amniotic mesenchyme is observed underlying the TFAP2A + /DLX5 + amniotic epithelium at this stage. Scale bars = 500 μm.

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Expressing, RNA Sequencing, Staining

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A–C ) Sequenced genotyping results of GATA3 -KO #1 and #2 lines ( A ), representative sequence traces for each mutation are shown in ( B ). Western blot analysis for GATA3 in control and GATA3 -KO #1 and #2 lysates ( C , β-actin is used as a loading control, triplicates shown). ( D–F ) Genotyping results of TFAP2A -KO #1 and #2 lines ( D ), representative sequence traces for each mutation are shown in ( E ). Western blot analysis for TFAP2A in control and TFAP2A -KO #1 and #2 lysates ( F , β-actin is used as a loading control, duplicates shown).

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Sequencing, Mutagenesis, Western Blot, Control

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: ( A,B ) Optical sections of control and TFAP2A -KO cysts in Glass-3D ( A , pluripotent) as well as in Glass-3D +BMP ( B ). While control-like pluripotent cysts are formed in Glass-3D, several NANOG + cells are still seen in Glass-3D +BMP in the absence of TFAP2A . ( C ) Confocal micrographs of d4 control, TFAP2A -KO and TFAP2A -KO carrying a doxycycline (DOX)-inducible human TFAP2A transgenic construct with or without DOX treatment, stained with indicated markers. ( D ) Transcriptome similarity analysis comparing the Carnegie stage (CS)7 human embryo single-cell dataset with the time-course (0, 12, 24, and 48 hr post-BMP) bulk dataset from control and TFAP2A -KO Glass-3D +BMP hPSC-amnion. Orange, green, blue, and magenta colored cells are transcriptomically similar to 0, 12, 24, and 48 hr post-BMP bulk RNA sequencing samples, respectively. While a clear differentiation trajectory from Epiblast to Ectoderm is seen in control (left), a halted amnion lineage progression is seen in the TFAP2A -KO background. ( E ) Optical sections of control and TFAP2A -KO cysts in Glass-3D +BMP stained for NANOG (green), ISL1/2 (red), and TBXT (magenta). Scale bars = 50 μm. BMP, bone morphogenetic protein.

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Control, Transgenic Assay, Construct, Staining, RNA Sequencing

Journal: eLife

Article Title: Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification

doi: 10.7554/eLife.89367

Figure Lengend Snippet: Uncropped transcriptional similarity Uniform Manifold Approximation and Projection (UMAP) plots for control and TFAP2A -KO shown in .

Article Snippet: The TFAP2A gRNA targeted site of human TFAP2A construct (gift of Robert Tjian, Addgene #12100, ) was mutated using Q5 site directed mutagenesis kit (NEB, primers: SP (RSV) AP2 (hTFAP2A)-CRISPR RES-fw and SP (RSV) AP2 (hTFAP2A)-CRISPR RES-rv).

Techniques: Control

Journal: Cancer cell international

Article Title: Polymorphism rs2327430 in TCF21 predicts the risk and prognosis of gastric cancer by affecting the binding between TFAP2A and TCF21.

doi: 10.1186/s12935-024-03343-z

Figure Lengend Snippet: Fig. 3 The C allele of rs2327430 played a role in GC by blocking the binding of TFAP2A to the TCF21 promoter. Notes: (A and B) The potential of the bind ing between tagSNPs and transcription factor was analyzed and displayed according to the JASPAR database. (C) Correlation analysis between TFAP2A and TCF21 mRNA was performed in 120 GC patients and analyzed with Spearman’s correlation test. (D) Schematic illustration of the reporter gene con taining rs2327430 C or T allele constructs and reporter plasmids with the different alleles of rs2327430 were transfected into AGS and MKN45 cells. Then, the results were expressed as relative luciferase activity (Firefly luciferase/Renilla luciferase). Both cells were also co-transfected with TFAP2A plasmid, re spectively. (E) CHIP assay evaluated by qRT-PCR was performed with control IgG or antibody against TFAP2A in the AGS and MKN45 cells, results of which are normalized to the input group and shown as means ± SEM in 3 independent experiments. (F and G) The knock-down efficiency of siRNAs on TCF21 and TFAP2A was determined by qRT-PCR and western blots, respectively. (H) The rescue experiment was carried out to verify whether the expression of TCF21 is affected by TFAP2A through qRT-PCR and western blot. (I–N) It is shown that TCF21 knockdown could partially rescue functional phenotypes caused by TFAP2A knockdown including proliferation, invasion, metastasis, and apoptosis. (O) The effect of TFAP2A knockdown on AKT/Bcl-xL signaling pathway could be rescued partially by TCF21 knockdown. (P) Images of the subcutaneous xenografts were presented. tumor growth curves were drawn and the tumor weight was calculated. (Q) IHC was used to examine the expression of TCF21 and Ki67 in xenografts

Article Snippet: Broadly, we sonicated the cross-linked chromatin DNA into 200–1000 bp fragments that were immunoprecipitated with an anti-TFAP2A antibody (Proteintech, China), while normal rabbit IgG was used as the reference group.

Techniques: Blocking Assay, Binding Assay, Construct, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Quantitative RT-PCR, Control, Knockdown, Western Blot, Expressing, Functional Assay

Journal: Cancer cell international

Article Title: Polymorphism rs2327430 in TCF21 predicts the risk and prognosis of gastric cancer by affecting the binding between TFAP2A and TCF21.

doi: 10.1186/s12935-024-03343-z

Figure Lengend Snippet: Fig. 4 The role of rs2327430 in modulating gastric cancer. Notes: The allele C played a protective role in gastric cancer development by inhibiting the binding with TFAP2A

Article Snippet: Broadly, we sonicated the cross-linked chromatin DNA into 200–1000 bp fragments that were immunoprecipitated with an anti-TFAP2A antibody (Proteintech, China), while normal rabbit IgG was used as the reference group.

Techniques: Binding Assay

Journal: eLife

Article Title: TFAP2 transcription factors are regulators of lipid droplet biogenesis

doi: 10.7554/elife.36330

Figure Lengend Snippet: Figure 1. The Wnt pathway and the regulation of lipid droplets. (A–B) HeLa-MZ cells were transfected with plasmids encoding wild-type or a S9A mutant of GSK3B 24 hr before the addition of Wnt-3a- or control-conditioned media (CM) for a further 24 hr. Cells were fixed, labeled with BODIPY (lipid droplets, green) and Hoechst 33342 (nuclei, magenta), and imaged by light microscopy. In (B) the number of lipid droplets was quantified by automated microscopy (bar graph), and the data are presented as the mean number of lipid droplets per cell of five independent experiments ± SEM, Figure 1 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.36330 12 of 24 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Mus musculus) L Cells American Type Culture Collection Cat #: CRL-2648; RRID:CVCL_4536 PMID:14056989 Cell line (Mus musculus) L Wnt3A Cells American Type Culture Collection Cat #: CRL-2647; RRID:CVCL_0635 PMID:12717451 Cell line (Homo sapiens) HeLa-MZ other Clone of HeLa (American Type Culture Collection Cat#: CCL-2) provided by Prof. Lucas Pelkmans (University of Zurich) Cell line (Homo sapiens) CRISPR DDIT3 this paper Cell line (Homo sapiens) CRISPR TFAP2A this paper Transfected construct (Homo sapiens) GSK3B Addgene Cat #: 49491 Transfected construct (Homo sapiens) GSK3BS9A Addgene Cat #: 49492 Transfected construct (Homo sapiens) Wnt Project plasmid library Addgene Kit # 1000000022 Transfected construct (Homo sapiens) pCMV-SREBP-1a(460 ) American Type Culture Collection Cat #: 99637 PMID:9062341 Transfected construct (Homo sapiens) pCMV-SREBP-1c(436 ) American Type Culture Collection Cat #: 99636 PMID:9062341 Transfected construct (Homo sapiens) pCMV-SREBP-2(468) American Type Culture Collection Cat #: 63452 PMID:9062341 Transfected construct (Homo sapiens) DDIT3-mCherry this paper Transfected construct (Homo sapiens) mCherry-TFAP2A this paper Transfected construct (Homo sapiens) mCherry-TFAP2B this paper Transfected construct (Homo sapiens) mCherry-TFAP2C this paper Transfected construct (Homo sapiens) V5-TFAP2A this paper Biological sample (Bos taurus) Lipoprotein-depleted serum PMID:13252080 Antibody Rabbit anti-AP2 alpha; anti-TFAP2A Abcam Cat #: ab52222 Antibody Mouse anti-CHOP (L63F7); anti-DDIT3 Cell Signaling Cat #: 2895 Antibody Mouse anti-V5 Tag ThermoFisher Scientific Cat #: R960-25 Continued on next page Scott et al. eLife 2018;7:e36330.

Techniques: Transfection, Mutagenesis, Control, Labeling, Light Microscopy, Microscopy

Journal: British Journal of Cancer

Article Title: Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

doi: 10.1038/sj.bjc.6604685

Figure Lengend Snippet: Cooperation between the hypoxia-inducible factor (HIF)-2 and Ets-1 transcription factor. ( A ) HeLa cells were co-transfected with the −1091/+43 or −919/+43 pGL3-basic promoter construct (1 μ g), pRL-TK plasmid (50 ng) and HIF-2 α and/or Ets-1 expression vectors (1 μ g each). Empty pcDNA3.1 was used to adjust total DNA content in all samples to 3 μ g. The transfected cells were incubated in hypoxia and the promoter activity was assessed as described in . Simultaneous expression of HIF-2 α and Ets-1 showed synergic effect on endosialin promoter activity. ( B ) HeLa cells were transfected with the −235/+43 pGL3-basic promoter construct and its mutated variants with deletions of one (Δ−174/−147) or both (Δ−174/−91) EBS elements, respectively, and pRL-TK, HIF-2 α and/or Ets-1 plasmids were co-transfected as above. The graph shows the promoter activity obtained in hypoxic cells only, as a similar profile was observed in the normoxic counterparts. ( C ) Chromatin immunoprecipitation assay was performed on FIB-3 cells under same conditions as described in , except that primers flanking the ETS transcription factor-binding site (EBS) elements were used to detect binding of the HIF subunits. HIF-2 α clearly bound to the EBS-containing region.

Article Snippet: Expression plasmids CMV-Sp1 containing human SP1 cDNA and SP(RSV)AP-2 containing human TFAP2A/AP-2 cDNA were purchased from Addgene (Cambridge, MA, USA). pcDNA3.1/Neo/Ets-1 expression plasmid was created by insertion of 1571 bp human Ets-1 cDNA fragment amplified by RT–PCR into pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Incubation, Activity Assay, Chromatin Immunoprecipitation, Binding Assay

Journal: British Journal of Cancer

Article Title: Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

doi: 10.1038/sj.bjc.6604685

Figure Lengend Snippet: Schematic illustration of the molecular anatomy of the endosialin promoter and regulatory pathways induced by hypoxia and cell density. Distal part of the promoter contains adjacent hypoxia-responsive element (HRE) and ETS transcription factor-binding site (EBS) element and transmits hypoxic signalling through hypoxia-inducible factor (HIF)-2 directly binding to DNA and cooperating with Ets-1. Proximal part of the promoter contains two EBS elements (but not HRE) and hypoxia signalling is mediated by HIF-2 through Ets-1 and two EBS elements. High cell density contributes to endosialin activation by means of SP1-binding sites in the proximal promoter and partially feeds the hypoxic pathway by pericellular hypoxia. Moreover, hypoxia might influence activation of endosialin indirectly through HIF-1 that upregulates expression of Ets-1, which in turn upregulates expression of SP1. On the basis of this model, hypoxia and density cross-talk to induce endosialin transcription.

Article Snippet: Expression plasmids CMV-Sp1 containing human SP1 cDNA and SP(RSV)AP-2 containing human TFAP2A/AP-2 cDNA were purchased from Addgene (Cambridge, MA, USA). pcDNA3.1/Neo/Ets-1 expression plasmid was created by insertion of 1571 bp human Ets-1 cDNA fragment amplified by RT–PCR into pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).

Techniques: Binding Assay, Activation Assay, Expressing

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: Bioinformatics and expression analyses identify the upregulation of FAM83A in human NSCLC. A The TIMER web server depicting the expression patterns of FAM83A in pan-cancer. B , C The UALCAN-TCGA database revealing the expression patterns of FAM83A in NSCLC with different tumor stage and clinicopathological features. D , E The UALCAN-TCGA database showing the relationship between FAM83A expression and patient outcome. F Relative FAM83A mRNA expression by quantitative PCR in NSCLC samples ( n = 41) and corresponding normal lung tissues ( n = 41) in a cohort of 41 NSCLC patients. P -value based on the Mann-Whitney U test. * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: FAM83A inhibition diminishes the growth, invasiveness, and migration in NSCLC cells. A Relative FAM83A protein expression by immunoblot assay in NSCLC samples ( n = 3) and corresponding normal lung tissues ( n = 3). P -value based on a two-tailed Student’s t -test. B , C FAM83A mRNA and protein levels in H1299 and A549 NSCLC cells and non-cancerous 16HBE cells. P based on one-way ANOVA followed by Tukey-Kramer post hoc test. D – H H1299 and A549 NSCLC cells were subjected to introduction with sh-FAM83A or sh-NC. The influence on FAM83A expression ( D ), cell proliferation by CCK-8 assay ( E , F ), cell motility by wound healing assay ( G ), and cell invasiveness by transwell invasion assay ( H ) ( n = 3). Scale bars: 50–100 μm. P based on two-way ANOVA followed by Sidak’s multiple comparisons test in ( D – H ). n = 3 in ( A – H ). * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: Inhibition, Migration, Expressing, Western Blot, Two Tailed Test, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: FAM83A inhibition promotes NSCLC cell ferroptosis and DDP sensitivity. A – E H1299 and A549 NSCLC cells were subjected to introduction with sh-FAM83A or sh-NC and checked for the levels of ROS ( A ), MDA ( B ), GSH ( C ), SOD ( D ), and Fe 2+ ( E ) using corresponding assay kits. F , G H1299 and A549 cells transfected with sh-FAM83A or sh-NC were exposed to various concentrations of DDP, followed by the determination of cell viability and the IC50 value of DDP by CCK-8 assay. P based on two-way ANOVA followed by Sidak’s multiple comparisons test in ( A – G ). n = 3 in ( A – G ). * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: Inhibition, Transfection, CCK-8 Assay

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: TFAP2A transcriptionally controls FAM83A expression. A Relative FAM83A mRNA expression in H1299 and A549 cells transfected as indicated ( n = 3). P based on two-way ANOVA followed by Sidak’s multiple comparisons test. B The predicted binding region (CCGGCCCGAGGGC, WT) between TFAP2A and the FAM83A promoter and its miss-matched sequence (GGCCGGGCTCCCG, MUT). C ChIP experiment with whole cellular lysates using a specific anti-TFAP2A antibody and the subsequent quantitative PCR of the enrichment abundance of the segments of the FAM83A promoter ( n = 3). P based on two-way ANOVA followed by Sidak’s multiple comparisons test. D , E Luciferase assays in H1299 and A549 cells transfected with sh-TFAP2A or sh-NC and FAM83A promoter reporter constructs encompassing the predicted binding region (FAM83A WT) or miss-matched sequence (FAM83A MUT) ( n = 3). P based on two-way ANOVA followed by Sidak’s multiple comparisons test. F The GEPIA online web showed the expression association between TFAP2A and FAM83A in NSCLC samples. Left panel represented the association in lung adenocarcinoma (LUAD); right panel represented the association in lung squamous cell carcinoma (LUSC). G Relative TFAP2A mRNA expression by quantitative PCR in NSCLC samples ( n = 41) and corresponding normal lung tissues ( n = 41) in a cohort of 41 NSCLC patients. P -value based on the Mann-Whitney U test. H Relative TFAP2A protein expression by immunoblot assay in NSCLC samples ( n = 3) and corresponding normal lung tissues ( n = 3). P -value based on a two-tailed Student’s t -test. I , J TFAP2A mRNA and protein levels in H1299 and A549 NSCLC cells and non-cancerous 16HBE cells ( n = 3). P based on one-way ANOVA followed by Tukey-Kramer post hoc test. K Relative FAM83A protein expression in cells transfected with sh-NC + pcDNA, sh-TFAP2A + pcDNA, or sh-TFAP2A + FAM83A expression plasmid ( n = 3). P based on two-way ANOVA followed by Sidak’s multiple comparisons test. * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: Expressing, Transfection, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, Luciferase, Construct, MANN-WHITNEY, Western Blot, Two Tailed Test, Plasmid Preparation

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: FAM83A restoration counteracts sh-TFAP2A-imposed alterations in cell growth, motility, invasiveness, ferroptosis, and DDP sensitivity. A – I H1299 and A549 cells were transfected with sh-NC + pcDNA, sh-TFAP2A + pcDNA, or sh-TFAP2A + FAM83A expression plasmid. The effects of different transfection groups on cell proliferation by CCK-8 assay ( A and B ), cell motility by wound healing assay ( C ), cell invasiveness by transwell invasion assay ( D ), and the levels of ROS ( E ), MDA ( F ), GSH ( G ), SOD ( H ), and Fe 2+ ( I ) using corresponding assay kits. Scale bar: 50 μm. P based on two-way ANOVA followed by Sidak’s multiple comparisons test in ( A – I ). J , K H1299 and A549 cells subjected to transfection with sh-NC + pcDNA, sh-TFAP2A + pcDNA, or sh-TFAP2A + FAM83A expression plasmid were exposed to various concentrations of DDP, followed by the detection of cell viability and the IC50 value of DDP by CCK-8 assay. P based on one-way ANOVA followed by Tukey-Kramer post hoc test. n = 3 in ( A – K ). * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: TFAP2A depletion decreases the growth of A549 xenografts in vivo. A – D Subcutaneous xenografts were generated by A549 cells transduced with sh-TFAP2A lentivirus or sh-NC controls. Each group included five mice ( n = 5). After 35 days, subcutaneous xenografts were harvested. Tumor growth curves ( A ), representative images and average weight ( B ), and TFAP2A and FAM83A levels by immunoblot and immunohistochemical assays ( C and D ) of subcutaneous xenografts. Scale bar: 100 μm. P based on one-way ANOVA followed by Tukey-Kramer post hoc test in ( A , C , and D ). P -value based on a two-tailed Student’s t -test in ( B ). * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: In Vivo, Generated, Transduction, Western Blot, Immunohistochemical staining, Two Tailed Test

Journal: Cell Division

Article Title: TFAP2A upregulates FAM83A to suppress ferroptosis and diminish cisplatin sensitivity in non-small cell lung cancer

doi: 10.1186/s13008-025-00162-0

Figure Lengend Snippet: TFAP2A depletion inactivated the PI3K/AKT signaling through the downregulation of FAM83A. A – D H1299 and A549 cells were subjected to transfection with sh-NC, sh-FAM83A, sh-NC + pcDNA, sh-TFAP2A + pcDNA, or sh-TFAP2A + FAM83A expression plasmid, followed by detection of PI3K, p-PI3K, AKT, and p-AKT by immunoblot assay ( n = 3). E , F H1299 and A549 cells were transfected with oe-NC or oe-FAM83A and treated with or without LY294002 or XAV939, followed by detection of cell viability ( E ) and Fe 2+ levels ( F ) ( n = 3). P based on two-way ANOVA followed by Sidak’s multiple comparisons test. * P < 0.05

Article Snippet: The fixed, paraffin-embedded xenografts were sliced into 4 μm sections, which were subjected to immunohistochemistry methods [ ] to detect FAM83A and TFAP2A using anti-FAM83A (rabbit polyclonal, #20618-1-AP, Proteintech, Wuhan, China, 1:400) or anti-TFAP2A (mouse monoclonal, #67076-1-Ig, Proteintech, 1:800) antibody.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A ) Schematic diagrams of chicken embryos at different developmental stages from gastrulation to neurulation. Dashed line in whole mount indicates level of section. Dashed boxes indicate area displayed in ( B–H ). ( B–B’’ ) Sox2 and Tfap2a in whole mount embryo at HH4+. ( B’ ) Confocal image of npb (white box in B ) shows Sox2 (blue) on medial (left) side and Tfap2a (magenta) on lateral (right) side of npb. B’’ zoom of box in B’ shows an example of a cell coexpressing Sox2 and Tfap2a (arrow) as well as a cell expressing either Sox2 (blue arrowhead) or Tfap2a (magenta arrowhead) only. ( C–D ) Transverse sections of embryos at HH8 to HH10 with co-immunostaining for Sox2 (blue), Tfp2a (magenta) and Pax7 (yellow). Arrowheads indicate examples of cells coexpressing all three markers. ( E–F ) Transverse sections of embryos at HH6 to HH7 with co-immunostaining for Sox2 (blue) and Msx1/2 (yellow). Arrowheads indicate examples of cells coexpressing both markers. ( G–H ) Transverse sections of embryos at HH8 and HH10 with co-immunostaining for Sox2 (blue) and Msx1/2 (yellow) and Six1 (magenta). Arrowheads in ( G ) indicate examples of cells coexpressing all three markers. White arrowheads in ( H ) indicate cells coexpressing Sox2 and Msx1/2 and green arrowhead indicates cell in epidermis co-expressing all three markers. All sections are oriented with medial to left and lateral to right side. Scale bars in wholemounts = 100 µm in sections = 20 µm (except 1B’’ = 10 µm). DOI: http://dx.doi.org/10.7554/eLife.21620.002

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Expressing, Immunostaining

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A ) Schematic diagrams of sections staged accordingly to those used in ( B–E ). Box indicates area that was imaged in ( B–E ). ( B–E ) Transverse sections immunostained for Pax7 (yellow), Sox2 (blue) and Six1 (magenta) at HH5 ( B ), HH6 ( C ), HH8 ( D ) and HH9 ( E ). Grey lines outline the embryo borders. Dashed colored lines indicate borders between strong and weak/no expression of corresponding markers. Sections are oriented medial (left) to lateral (right). See also . ( F–H ) Scatterplots of quantification of all cells that express Sox2 ( F’ ), Pax7 ( G’ ) and Six1 ( H’ ) and the fraction of cells expressing only Sox2 ( F’’ ), Pax7 ( G’’ ) or Six1 ( H’’ ) at HH5 to HH9. ( I ) Scatterplots representing the fraction of cells that coexpress Sox2 and Pax7, but not Six1 or ( J ) Sox2, Pax7 and Six1 at HH5 to HH9. See also . On top of scatterplots are sample images of a HH6 section with dots indicating cells expressing the corresponding marker. ( K ) Combination of medians of F’’ , G’’ , H’’ and I to illustrate difference of single (Sox2-blue, Pax7-yellow) or coexpressing cells (Sox2/Pax7-grey) at different stages. Scale bars = 20 µm. Asterisks indicate significance as calculated using a Student’s t-test with p-values displayed. Error bars indicate standard deviation. DOI: http://dx.doi.org/10.7554/eLife.21620.003 10.7554/eLife.21620.004 Figure 2—source data 1. Quantification of marker coexpression in single cells. DOI: http://dx.doi.org/10.7554/eLife.21620.004

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Expressing, Marker, Standard Deviation

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A–D ) Transverse sections immunostained for Pax7 (yellow)/Six1 (magenta) or Sox2 (blue)/Six1 (magenta) at HH5 ( A ), HH6 ( B ), HH8 ( C ) and HH9 ( D ). Grey lines outline the embryo borders. Dashed colored lines indicate borders of strong and weak/no expression of corresponding markers. Sections are oriented medial (left) to lateral (right). ( E ) Scatterplot of quantification of cells coexpressing Sox2 and Six1, but not Pax7 at HH5 to HH9. ( F ) Scatterplot of quantification of cells coexpressing Pax7 and Six1, but not Sox2 at HH5 to HH9. ( G ) Boxplot showing quantification of cells in the neural plate border/dorsal neural fold expressing only one of the markers examined (Sox2 only, Pax7 only, Tfap2a only) or a combination of two (Sox2/Pax7, Sox2/Tfap2a, Pax7/Tfap2a) or three markers (Sox2/Pax7/Tfap2a) at HH7 (light grey), HH8 (medium grey) to HH9 (black). For quantification, non-consecutive sections from a representative embryo were used for each stage (HH7, n = 12 sections; HH8, n = 9 sections; HH9, n = 5 sections). DOI: http://dx.doi.org/10.7554/eLife.21620.005

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Expressing

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A–B ) Chicken Df1 fibroblasts were transfected with pCI-Sox1-GFP ( A ) or pCI-Sox2H2BRFP ( B ) and immunostained against GFP or RFP respectively, as well as against Sox2. ( A ) Arrowheads point to Sox1-GFP transfected cells/nuclei that express GFP, but not Sox2. ( B ) Arrowheads point to Sox2-H2BRFP transfected cells that express nuclear RFP as well as Sox2. ( C–D ) Df1 cells transfected with pCI-Pax3-H2BGFP ( C ) or pCI-Pax7-H2BRFP ( D ), and immunostained against GFP or RFP respectively, as well as against Pax7. ( C ) Arrowheads point to Pax3-H2BGFP transfected cells that express nuclear GFP, but not Pax7. ( D ) Arrowheads point to Pax7-H2BRFP transfected cells that express nuclear RFP as well as Pax7. ( E–F ) Df1 cells transfected with either pCI-H2B-GFP or pCI-H2B-RFP as negative control with subsequent immunostaining against GFP/RFP respectively as well as Sox2 and Pax7. Cells transfected with either construct show strong GFP or RFP expression, but lack Sox2 and Pax7 expression. Dapi was used to visualize the cell nuclei. Scale bars equal 20 µm. ( G ) RT-PCR of cDNA reverse transcribed from RNA from cells transfected with either Sox1-GFP or Sox2-H2B-RFP (Line 2 and 5, respectively). As positive control plasmid DNA of pCI-Sox1-GFP (line 1) and pCI-Sox2-H2B-RFP (line 4) was used. As negative control cDNA reverse transcribed from RNA from cells transfected with either pCI-H2B-GFP (line 3) or pCI-H2B-RFP (Line 6) was used. ( H ) RT-PCR of cDNA reverse transcribed from RNA from cells transfected with either Pax3-H2B-GFP or Pax7-H2B-RFP (Line 2 and 5, respectively). Plasmid DNA of pCI-Pax3-H2B-GFP (line 1) and pCI-Pax7-H2B-RFP (line 4) were used as positive controls. Negative control as in ( G ). DOI: http://dx.doi.org/10.7554/eLife.21620.006

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Transfection, Negative Control, Immunostaining, Construct, Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Positive Control, Plasmid Preparation

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A–C ) Images of Pax7 immunostaining (yellow) in representative embryos used to measure intensity profiles across the neural plate border at HH5 ( A ), HH6 ( B ) and the dorsal neural fold at HH8 ( C ). Scale bar = 20 µm. ( D–F ) A line (yellow), as illustrated in this example, was drawn across the neural plate border/neural fold to measure intensity. ( G–I ) Intensity profiles of Sox2 (blue) and Pax7 (yellow) protein expression across the neural plate border from medial to lateral. Grey bars indicate extent of Pax7 expression at the neural plate border/dorsal neural fold. See also – . ( J ) Box plot of intensity of Sox2 (blue) and Pax7 (yellow) protein expression in dorsal neural fold (dNF) at HH8 compared to dorsal neural tube (dNT) and migrating neural crest (mNC) at HH9. Asterisks indicate significance as calculated using a Student’s t-test with p-values displayed. Error bars indicate standard deviation. See also . ( K–L ) Line plots of relative intensity of certain markers in combination with Sox2 (blue) and Pax7 (yellow) at HH8: ( K ) Tfap2a (green) (2–8 sections per embryo, n = 4 embryos) ( L ) Six1 (magenta) (5–8 sections per embryo, n = 5 embryos). ( M ) Combination of all markers Tfapa2 (green), Six1 (magenta) Sox2 (blue) and Pax7 (yellow). See also and and . ( A–D ) Dark line indicates average across all embryos, with standard deviation indicated by shaded region. Dotted line is polynomial trend line (order 6). M-medial, L-lateral. Intensities are displayed as grey values as measured by Fiji. DOI: http://dx.doi.org/10.7554/eLife.21620.008 10.7554/eLife.21620.009 Figure 3—source data 1. Spatial distribution of transcription factor expression at the neural plate border at HH5. DOI: http://dx.doi.org/10.7554/eLife.21620.009 10.7554/eLife.21620.010 Figure 3—source data 2. Spatial distribution of transcription factor expression at the neural plate border at HH6. DOI: http://dx.doi.org/10.7554/eLife.21620.010 10.7554/eLife.21620.011 Figure 3—source data 3. Spatial distribution of transcription factor expression at dorsal neural fold at HH8. DOI: http://dx.doi.org/10.7554/eLife.21620.011 10.7554/eLife.21620.012 Figure 3—source data 4. Comparison of Sox2 and Pax7 protein expression at HH8 vs HH9. DOI: http://dx.doi.org/10.7554/eLife.21620.012 10.7554/eLife.21620.013 Figure 3—source data 5. Spatial distribution of Tfap2a expression at the neural plate border at HH8. DOI: http://dx.doi.org/10.7554/eLife.21620.013 10.7554/eLife.21620.014 Figure 3—source data 6. Spatial distribution of Six1 expression at the neural plate border at HH8. DOI: http://dx.doi.org/10.7554/eLife.21620.014

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Immunostaining, Expressing, Standard Deviation, Comparison

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A ) Line plots of relative intensity of Msx1/2 (grey) (7 sections of one embryo) in combination with Sox2 (blue) and Pax7 (yellow) (see also ): ( B ) Combination of all measured markers: Tfap2a (green), Msx1/2 (grey), Six1 (magenta) Sox2 (blue) and Pax7 (yellow). ( A , B ) Dark line indicates average across all embryos, with standard deviation indicated by shaded region. Dotted line is polynomial trend line (order 6). ( C ) Simplified version of ( B ) with only trendline of the corresponding markers. M-medial, L-lateral. DOI: http://dx.doi.org/10.7554/eLife.21620.015

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Standard Deviation

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A–C ) Sox2 protein expression compared to Pax7 and Six1. Low Sox2 levels are detectable in early ( A , HH10) and late ( B , HH11; C , HH12) migrating neural crest. All sections are oriented medial (left) to lateral (right). White arrowheads indicate delaminated neural crest cells that coexpress Pax7 and Sox2, but not Six1. Green arrowheads (in A ) indicate epidermal cells that coexpress Pax7 and Sox2 and Six1. White line in ( A ) demarcates the embryonic midline and dashed line represents the border between neural tube and migrating neural crest. Scale bars = 20 µm. DOI: http://dx.doi.org/10.7554/eLife.21620.016

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Expressing

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A ) Sox2N1N2-H2B-eGFP construct was electroporated in HH4 chicken embryos. At HH12, eGFP reporter expression is visible not only in the neural tube, but also in migrating neural crest cells (box). Dashed line indicates level of transversal section in ( A’ ). Arrowheads indicate N1N2-reporter positive migrating neural crest cells. ( B ) N1N2-reporter expression is maintained in HH14 cranial crest. Dashed line indicates level of section ( B’ ). Arrowheads indicate cells positive for N1N2-reporter and endogenous Sox2 protein. ( C , D ) In ovo electroporation of Sox2-N1N2-H2B-GFP 2ss ( C ) and 4ss ( D ). Note that the number of electroporated cells decreases at progressively later stages. Arrowheads indicate migrating neural crest cells positive for reporter expression. ( E ) N1N2-eGFP-PEST was electroprated into HH4 embryos. At HH12, N1N2-eGFP-PEST-reporter is expressed in neural tube, but barely visible in its dorsal most portion (bracket in E’ ), although endogenous Sox2 protein expression is maintained as visualized by immunostaining (blue). Arrowheads indicate migrating neural crest that express low levels of endogenous Sox2 protein but lack the N1N2-destabilized reporter expression. ( F–I ) Sox2 mRNA expression. Dashed lines indicate levels of transverse sections ( G’ , H’ , H’’ , I’ , I’’ ). Transverse sections of ( F ) HH7 and ( G ) HH8 embryos showing strong Sox2 mRNA expression in the dorsal neural fold that overlaps with Pax7 protein expression (box). ( H ) At HH9 Sox2 mRNA is barely detectable in the dorsal neural tube and premigratory crest ( H’ , H’’ ). Dashed line in H’ and H’’ demarcates boundary of dorsal neural tube and pre-migratory neural crest. ( I ) At HH13 Sox2 mRNA is barely detectable in the dorsal neural tube ( I’ ), but seen at very low levels in the rhombomere 4 neural crest stream (bracket in I’’ ). ( J , K ) mRNA expression of Msx1 in wholemounts. Dashed lines indicate levels of sections ( J’ and K’ ). ( J’ ) HH7 embryo showing strong Msx1 mRNA expression (blue arrowheads) in the dorsal neural fold that is nested within the Pax7 protein domain (red arrowheads). ( K ) HH9 embryo with overlapping Msx1 mRNA expression and Pax7 protein expression in the dorsal neural tube and the delaminated neural crest. ( L ) mRNA expression of Tfap2a in wholemount at HH8 with dashed line indicating approximate level of section ( L’ ). Scale bars on whole mounts = 100 µm; on sections = 20 µm, except (2B’ = 100 µm; F’’ = 40 µm). DOI: http://dx.doi.org/10.7554/eLife.21620.017

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Construct, Expressing, In Ovo, Electroporation, Immunostaining

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: HH4 embryos were electroporated with ( A , B ) control vector pCIG or ( C , D ) pCAGGS-cSox2 on right side of embryo and immunostained for Sox2 (blue) and Pax7 (magenta). Embryos displayed as whole mount ( A , C ) or transversal sections ( B , D ). ( E ) Quantification of numbers of pCIG or pCaggs-Sox2 treated embryos with a strong or mild loss or unchanged expression of Pax7 versus control side of same embryo. Asterisk indicates significant difference as calculated by contingency table followed by Chi-Square test. Scale bar = 100 µm in whole mount and 20 µm in sections. See also . ( F ) Transversal sections of embryos overexpressing Sox2 at HH8. White lines outline neural tube. Box indicates area of enlarged inset. Cells with strong Sox2 (blue) overexpression (arrowheads) in dorsal neural fold show very low levels of Pax7 (magenta). ( G ) Sections of embryo where Sox2 was knocked down with 0.5 mM Fitc-labeled Sox2 MO on right side and control MO on left side of HH4 embryo (inset). Immunostaining for Sox2 (blue) and Pax7 (magenta). Circles in dorsal neural fold indicate area of measurement for ( H ). ( H ) Quantification of Sox2 and Pax7 protein expression in dorsal neural fold upon Sox2 knockdown. Sox2 is reduced by 23.4 ± 9.9% when comparing control to experimental side. This causes increase of Pax7 expression of 33.3 ± 28.0%. Asterisks indicate significance as calculated using a Student’s t-test. p-values are indicated in graphs. Error bars signify standard deviation. See also . ( I, J ) Sox2 knock down with 0.5 mM Fitc-labeled Sox2 MO on right side or 0.5 mM Fitc-labeled control MO on left side of HH4 embryo (inset) with subsequent ISH for ( I ) Sox10 mRNA or for ( J ) FoxD3 mRNA at HH9. ( K ) Quantification of Sox10 and FoxD3 mRNA expression level changes upon Sox2 knockdown. Scale bar = 100 µm. See also . DOI: http://dx.doi.org/10.7554/eLife.21620.019 10.7554/eLife.21620.020 Figure 6—source data 1. Quantification/Analysis of Sox2 overexpression experiments. DOI: http://dx.doi.org/10.7554/eLife.21620.020 10.7554/eLife.21620.021 Figure 6—source data 2. Quantification/Analysis of Sox2 knock down experiments. DOI: http://dx.doi.org/10.7554/eLife.21620.021 10.7554/eLife.21620.022 Figure 6—source data 3. Quantification of Sox10 and FoxD3 mRNA expression upon Sox2 knock down. DOI: http://dx.doi.org/10.7554/eLife.21620.022

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Control, Plasmid Preparation, Expressing, Over Expression, Labeling, Immunostaining, Knockdown, Standard Deviation

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A ) Section of embryo electroporated with Sox2 MO in combination with ( A ) empty vector pCIG or ( B ) with pCaggs-Sox2 expression construct. ( C ) Quantification of embryos with upregulation of Pax7 expression upon Sox2 MO knock down (5–8 sections/embryo, n = 8 embryos) compared to rescue experiment (4–7 sections/embryo, n = 16 embryos). Asterisks indicate significance as calculated using a Student’s t-test. p-values are indicated in graphs. Error bars signify standard deviation. Scale bar = 20 µm. DOI: http://dx.doi.org/10.7554/eLife.21620.023

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Plasmid Preparation, Expressing, Construct, Knockdown, Standard Deviation

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: ( A ) Schematic representation of expression constructs used to test MO specificity and efficiency. The MO target side containing 5’UTR and first 10 amino acids (10AA) were cloned in frame with H2B-RFP into a construct driven by the pCaggs promoter. ( B ) Representation of electroporation scheme. Targeted MO or control MO (both green) were electroporated to left and right side of same embryo, respectively, together with the corresponding expression constructs. ( C , C’ , D , D’ ) Dorsal view of embryos electroporated with Sox2 MO, left side, and control MO (contr MO), right side. ( C’ ) Embryo co-electroporated with pCaggs-Sox 2 -UTR-10AA-H2B-RFP shows downregulation on Sox2 MO electroporated side but not control MO side. ( D’ ) Embryo co-electroporated with pCaggs-Sox 3 -UTR-10AA-H2B-RFP shows same expression level on Sox2 MO and control MO side. ( E , E’ , F , F’ , G , G’ ) Dorsal view of embryos electroporated with Pax7 MO, left side, and control MO (contr MO), right side. ( E’ , F’ ) Embryos co-electroporated with (E’) pCaggs-Pax 7 -UTR-10AA-H2B-RFP or (F’) pCaggs-Pax 7 -UTR +C -10AA-H2B-RFP show downregulation on Pax7 MO electroporated side but not control MO side. ( G’ ) Embryo co-electroporated with pCaggs-Pax 3 -UTR-10AA-H2B-RFP shows same expression level on Pax7 MO and control MO side. Scale bar = 20 µm. ( H , I ) Quantification (staggered column plots) of MO-validation experiments with the four categories strongly decreased (dark grey), mildly decreased (light grey), unchanged (light blue) or increased (dark blue) expression of UTR-H2BRFP construct on targeted MO electroporated side. ( H ) Left column: Sox2 MO + pCaggs-Sox2-UTR-10AA-H2B-RFP electroporated embryos (Sox2MO Sox2UTR; n = 26 embryos) versus right column: Sox2 MO + pCaggs-Sox3-UTR-10AA-H2B-RFP electroporated embryos (Sox2MO Sox3UTR; n = 27 embryos). ( I ) Left column: Pax7 MO + pCaggs-Pax7-UTR-10AA-H2B-RFP electroporated embryos (Pax7MO Pax7UTR; n = 24 embryos) and middle column: Pax7 MO + pCaggs-Pax7-UTR-10AA-H2B-RFP electroporated embryos (Pax7MO Pax7UTR+C; n = 38 embryos) versus right column: Pax7 MO + pCaggs-Pax3-UTR-10AA-H2B-RFP electroporated embryos (Pax7MO Pax3UTR; n = 22 embryos). DOI: http://dx.doi.org/10.7554/eLife.21620.024

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Expressing, Construct, Clone Assay, Electroporation, Control, Biomarker Discovery

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: HH4 embryos were electroporated with ( A ) control vector pCI-H2B-RFP or ( B ) pCI-Pax7-H2B-RFP on right side of embryo, then immunostained for Pax7 (magenta) and Sox2 (blue) at HH8 and transverely sectioned. Circles in dorsal neural fold indicate area of measurement for C and D. ( C ) Quantification of Pax7 and ( D ) Sox2 protein expression in dorsal neural fold after pCI-H2B-RFP or pCI-Pax7-H2B-RFP overexpression. Pax7 is increased by 115.4 ± 99.3%. Sox2 is reduced by 11.7 ± 11.2% when comparing experimental to control side. Sox2 expression in embryos with Pax7-H2B-RFP treatment on right side versus embryos with H2B-RFP treatment on right side was reduced but not to a level of statistical significance. See also . ( E ) Transversal sections of embryos electroporated with H2B-RFP on left side and Pax7-H2B-RFP on right side for single cell analysis. Quantification of ( F ) Pax7 and ( G ) Sox2 expression in single cells with very high levels of RFP expression on H2B-RFP treated side to Pax7-H2B-RFP treated side. See also . ( H ) Transversal sections of embryo electroporated with 0.5 mM Fitc-labeled Pax7 MO on right side and control MO on left side at HH4. Immunostaining for Pax7 (magenta) and Sox2 (blue). Circles in dorsal neural fold indicate area of measurement for ( I ). ( I ) Quantification of Pax7 and Sox2 protein expression in dorsal neural fold upon Pax7 knockdown. Pax7 is reduced by 16.6 ± 11.8% when comparing control to experimental side. This causes an increase of Sox2 expression by 28.6 ± 24.1%. See also . ( J ) Pax7 knock down with 0.5 mM Fitc-labeled Pax7 MO on left side or 0.5 mM Fitc-labeled control MO on right side of HH4 embryo with ( G ) subsequent ISH for Sox10 mRNA at HH9. Scale bar = 100 µm. ( L ) Quantification of decreased (dark grey), unchanged (medium grey) or increased (light grey) Sox10 mRNA expression level upon Pax7 knockdown versus control side of same embryo. See also . Asterisks indicate significance as calculated using a Student’s t-test. p-values are indicated in graphs. Error bars signify standard deviation. DOI: http://dx.doi.org/10.7554/eLife.21620.025 10.7554/eLife.21620.026 Figure 7—source data 1. Quantification/Analysis of Pax7 overexpression experiments. DOI: http://dx.doi.org/10.7554/eLife.21620.026 10.7554/eLife.21620.027 Figure 7—source data 2. Quantification/Analysis of Pax7 overexpression experiments in single cells. DOI: http://dx.doi.org/10.7554/eLife.21620.027 10.7554/eLife.21620.028 Figure 7—source data 3. Quantification/Analysis of Pax7 knock down experiments. DOI: http://dx.doi.org/10.7554/eLife.21620.028 10.7554/eLife.21620.029 Figure 7—source data 4. Quantification of Sox10 and FoxD3 mRNA expression upon Pax7 knock down. DOI: http://dx.doi.org/10.7554/eLife.21620.029

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Control, Plasmid Preparation, Expressing, Over Expression, Single-cell Analysis, Labeling, Immunostaining, Knockdown, Standard Deviation

Journal: eLife

Article Title: Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

doi: 10.7554/eLife.21620

Figure Lengend Snippet: We propose that, within the neural folds, mutual repression can lead to a bistable switch with three possible solutions: an unstable state occurs when the concentration of two cross-repressing factors is similar. At a certain concentration, two different stable states are achieved. When Sox2 expression reaches a certain threshold, it causes reduction of Pax7 and induction of neural fate. When Pax7 concentration increases to a certain level, it causes reduction of Sox2 and induction of neural crest fate. DOI: http://dx.doi.org/10.7554/eLife.21620.030

Article Snippet: Primary antibodies used: Six1 (Sigma-Aldrich, St. Louis MO; HPA001893, 1:2000), rabbit anti-RFP (MBL, Woburn, MA; Cat#PM005, 1:1000) and rabbit anti-GFP (Abcam, Cambridge, MA; Cat# ab290, 1:1000) Pax7, Tfap2a and Msx1/2 (Developmental Studies Hybridoma Bank, Iowa City, Iowa; 1:5), goat polyclonal to Sox2 (Santa Cruz Biotech, Santa Cruz, CA; Y-17, 1:1500) for most figures except for and where a rabbit monoclonal to Sox2 antibody (Abcam, ab92494) was used.

Techniques: Concentration Assay, Expressing