Journal: Genes to Cells

Article Title: Attenuation of Oxygen‐Induced Neovascularization and Inflammation by Neutralizing VEGFA and/or ANG ‐2 With an Antibody

doi: 10.1111/gtc.70107

Figure Lengend Snippet: Nature of photoreceptor, amacrine, and bipolar cells and ONL in the OIR retina. The antibodies were intravitreally injected into OIR mice at P14, and the retinas were harvested at P17 or P19 and frozen sectioned. The sections were immunostained with anti‐ARR3 (cone), CHX10 (bipolar cells), and AP2α (amacrine) antibodies, and nuclei were visualized with DAPI staining (A, B, E, F). Numbers of ARR3‐positive processes are counted (C). ONL thickness at around 100 μm from the optic nerve is measured (D). Numbers of CHX10 or AP2α‐positive cells/100‐μm section were counted (G, H). Scale bars are 50 μm (A, B, E, F). Data are averages of 8 samples with standard deviation. * p < 0.05; *** p < 0.001.

Article Snippet: The primary antibodies used were anti‐ARR3 (Millipore, AB15282), AP2α (DSHB, 3B5‐s), CHX10 (Exalpha, X1190P164), and GFAP (Sigma‐Aldrich, G3893) antibodies.

Techniques: Injection, Staining, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2A is functionally associated with TP63-binding sites. ( A ) Effect on the IRF6 enhancer activity of the addition of exogenous TP63 in H1299 cells as measured by luciferase activity. All luciferase graphs represent the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05. ( B ) Luciferase assay measuring the effect of the addition of increasing amounts of TFAP2A on TP63-mediated activation of the IRF6 enhancer region in H1299 cells. ( C ) Western blot analysis of exogenous TP63 and TFAP2A expression in H1299 cells. ( D ) Luciferase assay measuring the effect of the addition of increasing amounts of TFAP2A on TP63-mediated activation of the PVRL1 enhancer region in H1299 cells. ( E ) Quantitative ChIP-PCR comparing TP63 and TFAP2A binding to IRF6, FGFR2, JAG2, PVRL1 and PDGFC associated binding sites, expressed as fold enrichment compared to a negative control region within the same ChIP. Data represent the mean of two independent biological replicates. ( F ) Western blot validation of TP63 and TFAP2A depletion in HFKs used in recruitment ChIPs. ( G ) Quantitative ChIP-PCR comparing TP63 and TFAP2A binding to IRF6, FGFR2, JAG2 and PVRL1 associated binding sites in HFKs depleted for TP63 or TFAP2A compared to scrambled control. Results calculated as fold enrichment compared to a negative control region within the same ChIP and normalized to scrambled control to compare between antibodies and experiments. Data represent the mean of two independent biological replicates. ( H ) Quantitative ChIP-PCR of second negative control binding site (BCL2 upstream) in HFKs depleted for TP63 or TFAP2A expressed as fold change compared to scrambled control. Data represent the mean of two independent biological replicates. ( I ) Quantitative RT-PCR quantification of expression of CL/P associated genes in TFAP2A-depleted human foreskin keratinocytes compared to scrambled control. Graph represents the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05, ** P < 0.01.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Activity Assay, Luciferase, Activation Assay, Western Blot, Expressing, Negative Control, Biomarker Discovery, Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: Identification of TFAP2A as a potential TP63 co-factor. ( A ) Table of results from transcription factor motif enrichment analysis of 7574 TP63-binding sites. ( B ) Cumulative frequency distribution plots comparing distance of predicted TFAP2A sites and TP63-binding motifs from the centre of TP63-binding sites/peaks. ( C ) Comparison of localization of TP63-binding sites with or without predicted AP-2 sites. ( D and E ) Quantitative PCR ChIP validation of TP63 (D) and TFAP2A (E) binding to TP63 sites associated with CL/P genes (data represent the mean of three biological replicates expressed as % input ± SEM). ( F ) Semi-quantitative PCR results for sequential re-ChIP assay for TP63 and TFAP2A for the IRF6 , FGFR2, TGFB1 and PVRL1 associated TP63-binding sites, showing both factors co-ChIP.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Comparison, Real-time Polymerase Chain Reaction, Biomarker Discovery

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2C interacts with a subset of TP63-binding sites. ( A ) Quantitative PCR estimation of TFAP2A and TFAP2C mRNA copy number in cycling HFKs. Data represent the mean of three independent biological replicates ± SEM. ( B ) Quantitative PCR ChIP validation comparing TFAP2A (3B5) and TFAP2C (H77) interaction with a subset of TP63-binding regions associated with CL/P genes. Data represent the mean of three biological replicates expressed as % input ± SEM). ( C and D ) Luciferase assay measuring the effect of the addition of TFAP2A or TFAP2C to six TP63 isoforms on activation of the IRF6 (C) or PVRL1 (D) enhancer region in H1299 cells. ( E ) Luciferase assay measuring the effect of the addition of TFAP2A or TFAP2C on TP63-mediated activation of the IRF6 enhancer region in primary human foreskin keratinocytes (HFKs). Luciferase graphs represent the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05, ** P < 0.01.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Biomarker Discovery, Luciferase, Activation Assay

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2A and TFAP2C are required for efficient differentiation of organotypic raft cultures. ( A ) Western blot analysis of TFAP2A, TFAP2C, TP63 and actin loading control of the HFKs transfected with TFAP2A, TFAP2C, TP63 targeting and scrambled control siRNA. ( B ) Sections of organotypic raft cultures generated from TFAP2A-, TFAP2C- or TP63-depleted HFKs stained for haematoxylin & Eosin (H&E) and indirect immunofluorescent staining of early [keratins 1 (KRT1), intermediate (transglutaminase-1 (TGM1)] and late [Filaggrin (FLG)] markers of differentiation (scale bar = 100 µM). ( C ) Number of bromodeoxyuridine (BrDU) incorporating cells in organotypic raft culture assessed by immunofluorescent staining. Graph represents the average number of BrDU-positive cells in the basal epithelial layer from organotypic raft cultures per 1000 µM, expressed as percentage scrambled control (mean ± SE at least 10 counts each of two independent biological replicates).

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Western Blot, Control, Transfection, Generated, Staining

Journal: Stroke

Article Title: GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na ⁺ -Ca ²⁺ Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection

doi: 10.1161/strokeaha.121.034637

Figure Lengend Snippet: Figure 1. The transcription factor GATA3 (GATA-binding protein 3) activated NCX3 (Na+-Ca2+ exchanger 3) promoter by binding a specific GATA site on NCX3 promoter sequence. A, Quantitative real-time polymerase chain reaction in neurons transfected with the following constructs: AP2, EGR1 (early growth response protein 1), GATA1, GATA2, GATA3, Sp4 (specificity protein 4). *P<0.05 vs empty vector by Student t test (n=3). B, Effect of a transient overexpression of AP2, EGR1, GATA1, GATA2, GATA3, Sp4 constructs on NCX3 promoter activity in cortical neurons, measured by luciferase assay. *P<0.05 vs pGL3-NCX3+empty vector by 1-way ANOVA analysis followed by Bonferroni post hoc test (n=3). C, JASPAR matrix representation (MA0037.1) of the consensus GATA-binding site on rat NCX3 gene. GATA sequence (TGATAG) has 100% identity between human, rat, and mouse NCX3 promoter. D, Luciferase assay in neurons under the following experimental conditions: (1) pGL3basic, (2) pGL3- ncx3, (3) pGL3-ncx3+Empty Vector, (4) pGL3-ncx3+GATA3 Vector, (5) pGL3-ncx3GATA3mut+GATA3 Vector. *P<0.05 vs pGL3-ncx3 by one-way ANOVA analysis followed by Bonferroni post hoc test (n=3). E, Chromatin immunoprecipitation analysis of NCX3 promoter in cortical neurons transfected with empty vector or with GATA3 Vector. The binding activity of GATA3 is graphically represented as the percentage of empty vector. *P<0.05 vs empty vector by Student t test (n=3).

Article Snippet: Plasmids were purchased from Addgene: AP2 ([activating

Techniques: Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Over Expression, Activity Assay, Luciferase, Chromatin Immunoprecipitation

Journal: Cell reports

Article Title: Genetic and chromatin regulation of Pvt1 monoallelic expression

doi: 10.1016/j.celrep.2025.116554

Figure Lengend Snippet: (A) Allelic ratio (AR) of Pvt1 in F 1 -23 hybrid NPC clonal lines compared to other genes. Each point represents a clonal line ( n = 120). NPC clonal lines used for other experiments are highlighted with different shapes. (B) Summary of F 1 hybrid clonal lines used in this paper and their respective Pvt1 allelic expression status based on an AR cutoff of 0.2. (C) Allele-specific H2K27ac ChIP-seq with 3 different F 1 -23 clonal lines. Top row shows SNP differences between the 129 allele and the CAST allele in relation to the H3K27ac ChIP-seq signals across three samples. Tfap2a SNP is highlighted in the black box, the second of the two SNPs. (D) Allele-specific ATAC-seq and ChIP-seq from mE6 NPCs, clonal line with Pvt1 CAST monoallelic expression (mE6 NPC, blue). (E) Allele-specific ATAC-seq and ChIP-seq from Ch8 NPCs, clonal line with Pvt1 129 monoallelic expression (Ch8 NPC, pink). (F) ChIP input tracks (black) with allele-specific ChIP-seq tracks from mE6 NPCs. (G) ChIP input track (black) with allele-specific ChIP-seq tracks from Ch8 NPCs.

Article Snippet: Antibodies used: H3K27ac (Abcam, 4729), H3K27ac (Active Motif, 39133), H3K4me2 (Abcam, ab7766), H3K4me3 (Active Motif, 39159), H3K9me3 (Abcam, ab8898), H2AK199ub (Cell signaling, D27C4), H3K27me3 (Cell Signaling, C36B11), RNA Pol II-RPB1 (Bio-legend, 664906), and TFAP2a (Novus, NB100-74359).

Techniques: Expressing, ChIP-sequencing

Journal: Cell reports

Article Title: Genetic and chromatin regulation of Pvt1 monoallelic expression

doi: 10.1016/j.celrep.2025.116554

Figure Lengend Snippet: (A) Differential gene expression analysis (DESeq) between NPC clonal lines with Pvt1 CAST monoallelic expression ( n = 50) against NPC clonal lines with Pvt1 biallelic expression ( n = 58). Non-significant genes are in gray, and the significant genes based on log2 fold change and adjusted p value are in red, with the top 15 significant genes labeled. The adjusted p value cutoff is 0.01, and the log2 fold change cutoff is 0.5. (B) Tfap2a expression from F 1 -23 hybrid NPC clonal lines. Pvt1 allelic expression status is based on an AR cutoff of 0.2 ( n = 56, blue; n = 60, yellow; n = 4, red). Significance was calculated with a t test: NS p > 0.05 and ** p ≤ 0.01. (C) Summary of Tfap2a expression levels in transcript per million (TPM) between the different F 1 -23 clonal lines. The color of the clonal line is based on Pvt1 allelic status. (D) Tfap2a expression from RT-qPCR of R1-57 NPC clonal lines. Pvt1 allelic expression status is based on an AR cutoff of 0.2 ( n = 12 WT; n = 29, bi-allelic; n = 6, mutant). The relative fold change is compared to the NPC clonal line with the lowest Tfap2a expression. Significance was calculated with a t test: NS p > 0.05. (E) TFAP2a motif comparison between sequences from 129 allele (pink) and CAST allele (blue). (F) TFAP2a ChIP-seq tracks from four different samples: mE6 NPCs (blue), Ch8 NPCs (pink), Ch1 NPCs (yellow), and mESCs (green). The dotted line and black box around the SNPs demonstrate where the SNPs near the TFAP2a binding site can be found. (G) Violin plot of the AR from a non-clonal population of F 1 -23 NPCs ( n = 3) and a non-clonal population of F 1 -23 NPCs with Tfap2a overexpression ( n = 6, purple). Additionally, there is the AR of mE6 NPCs ( n = 3) and mE6 NPCs with Tfap2a overexpression ( n = 3, blue). The AR was obtained with targeted RNA-seq of Pvt1 . p values were calculated with an unpaired t test: *** p ≤ 0.001. The experimental schematic is provided in .

Article Snippet: Antibodies used: H3K27ac (Abcam, 4729), H3K27ac (Active Motif, 39133), H3K4me2 (Abcam, ab7766), H3K4me3 (Active Motif, 39159), H3K9me3 (Abcam, ab8898), H2AK199ub (Cell signaling, D27C4), H3K27me3 (Cell Signaling, C36B11), RNA Pol II-RPB1 (Bio-legend, 664906), and TFAP2a (Novus, NB100-74359).

Techniques: Gene Expression, Expressing, Labeling, Quantitative RT-PCR, Mutagenesis, Comparison, ChIP-sequencing, Binding Assay, Over Expression, RNA Sequencing

Journal: eLife

Article Title: Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment

doi: 10.7554/eLife.107161

Figure Lengend Snippet: ( A ) The expression levels of various anterior segment cell type marker genes in the integrated B6 and 129 data are depicted on a dot plot. These marker genes are generally accepted to be specific to individual cell types. Epithelial cells ( Krt14 ); neurons ( Rho ); endothelial cells ( Egfl7 ); ciliary body and iris cells ( Tyrp1 ); and immune cells ( Cd52 ). Cluster 1 expressed multiple TM marker genes including Myoc , Acta2 (encodes α-SMA), Pitx2 , and Tfap2b . Although some of the neurons may be limbal, it is possible that others are retinal contamination. ( B–C ) Heatmaps of differentially expressed genes across all limbal tissue cell clusters ( B ) and across the subclusters of cluster 1 ( C ).

Article Snippet: Antibody , Rabbit polyclonal TFAP2B , Novus Biologicals , NBP1-89063 , IF sections (1:200).

Techniques: Expressing, Marker

Journal: eLife

Article Title: Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment

doi: 10.7554/eLife.107161

Figure Lengend Snippet: Although there is variability in expression between sections, the aggregate of all sections reveals the biased distributions of TM cell subtypes as summarized in . Here we focus on TM expression. However, some of the markers are also expressed outside of the TM. For example, Edn3 is expressed in vasculature, while both Lypd1 and Inmt are expressed at low levels in scleral and iris cells (but are higher in TM cells). Markers assessed by IF: MYOC, CHIL1, CRYM, α-SMA, TFAP2B. Markers assessed by ISH: Inmt , Edn3 , Lypd1 . All scale bars: 50 µm.

Article Snippet: Antibody , Rabbit polyclonal TFAP2B , Novus Biologicals , NBP1-89063 , IF sections (1:200).

Techniques: Expressing