anti tfap2α  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Loss of Adam13 affects gene expression and Tfap2 α transcription start. (A) Schematic representation of the approach used for CNC-specific RNA-seq. Control (NI) and Adam13KD (MO13) CNC were dissected at the same apparent stage (Stage17) in 3 independent experiments. (B) Heatmap visualization of RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO131, MO132, MO133), higher Z-score in red representing increase expression and blue representing decreased expression. (C) Schematic representation of the Tfap2α.L gene. Three putative transcription start sites (alternate exon1) connecting to exon2 and producing three different proteins differing at the N-terminus. (D) Sashimi plot analysis of transcript junctions detected for the Tfap2α.L gene in RNA-sequencing data between Control (NI1) and Adam13KD (MO131), circles mark the major transcript junction between corresponding exons. (E) Quantification of exon-specific counts for the three Tfap2α.L alternate starts (S1, S2 and S3) normalized to the total transcript counts for the Tfap2α.L gene. Student t-test was performed for statistical analysis, * represents p-value<0.05; ns represents p-value>0.05. (F) Histogram depicting relative fluorescence intensity of Tfap2α in Sox9 positive cells between Control (NI) and Adam13KD (MO13) CNC explants. Student t-test was performed for statistical analysis, * represents p-value<0.05. (G) ClustalW protein sequence alignment for Tfap2α-S1 and Tfap2α-S3 to highlight the sequence difference in exon1 and the yellow highlighted sequence is Exon2 which is common between both proteins. (H) Immunofluorescence images of CNC explants were dissected at stage 17 and grown on fibronectin substrate for 3 hours. CNC explants were fixed and subsequently immuno-stained for Sox9 (red), Tfap2α (green) and DAPI (blue) and imaged using confocal microscopy, all images are Max-IP.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Expressing, RNA Sequencing Assay, Control, Fluorescence, Sequencing, Immunofluorescence, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: (A) Sashimi plot analysis of transcript junctions detected for gene Tfap2α.L in RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO13-1, MO13-2, MO13-3), numbers on the line are the major transcript junction between corresponding exons, arrow points to the number of transcript junction detected between Start1 and Exon2. (B) Sashimi plot analysis of transcript junctions detected for gene Tfap2α.S in RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO13-1, MO13-2, MO13-3), numbers on the line are the major transcript junction between corresponding exons, arrow points to the number of transcript junction detected between Start1 and Exon2. (C) Quantification of exon specific counts for Tfap2α.S-start1, start2 and start3 normalized to total transcript counts for Tfap2α.S gene. Student t-test was performed for statistical analysis, * represents p-value<0.05, ** represents p-value<0.01, ns represents p-value>0.05.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: RNA Sequencing Assay, Control

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Adam13 controls Arid3a binding and histone methylation at the Tfap2 α. L promoter. Chromatin-Immunoprecipitation-qPCR analysis of DNA associated with the chromatin binding proteins. (A, B, C, D) Histogram depicting the relative abundance of DNA bound to H3K4me3 (A), H3K9me2/3 (B), Arid3a (C), and Adam13 (D) in WT and Adam13KO embryos at the promoter region of Tfap2α-S1 and Tfap2α-S3 based on ChIP-qPCR analysis. ChIP was performed at stage 20. Student t-test was performed for statistical analysis, * represents p-value<0.05, ** p-value<0.01, *** p-value<0.001, **** p-value<0.0001, ns represents p-value>0.05. (E-G) Histogram depicting the relative luciferase expression using the luciferase reporter containing Tfap2α-S1 or Tfap2α-S3 promoter region in HEK293T cells that were transfected with CS2 (empty vector), Arid3a, Adam13, Arid3a+Adam13, alone or in combination with the Human Arid3a-SH (AridSh, F-G), along with the CMV renilla reporter. One-way ANOVA was performed for statistical analysis. * Represent p-value<0.05, ** p-value<0.01, **** p-value<0.0001.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Binding Assay, Methylation, Chromatin Immunoprecipitation, Luciferase, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Chromatin-Immunoprecipitation-qPCR analysis of DNA associated with the chromatin binding proteins. (A, B, C, D) Histogram depicting the relative abundance of DNA bound to H3K4me3 (A), H3K9me2/3 (B), Arid3a (C), Adam13 (D) in WT and KO embryos at the promoter region of Tfap2α-S1 and Tfap2α-S3 based on ChIP-qPCR analysis. ChIP was performed at stage 20 (Independent biological replicate from ). Student’s t-test was performed for statistical analysis, * represents p-value<0.05, ** represents p-value<0.01, *** represents p-value<0.001, **** represents p-value<0.0001, ns represents p-value>0.05. (E) Schematic representation of the Luciferase reporters for the S1 and S3 transcription starts.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Luciferase

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Tfap2 α variants have distinct biological functions. (A) Fluorescence tracking of CNC cell migration. Embryos were injected with fluorescent Membrane Cherry (MbC) mRNA at the 8-cell stage in one dorsal animal blastomere and scored at stages 22-24 for the presence or absence of fluorescence in the branchial arches to quantify migration. The absence of fluorescence in the branchial arches is considered either defective migration or missed targeting. The value obtained for MbC alone give the missed targeting value for each experiment and is used to normalize between biological repeats (Typically 5 to 20% depending on embryo pigmentation). (B) Histogram representing the percentage proper migration in Control (MbC), Adam13KD (MO13), MO13+Tfap2α-S1, MO13+Tfap2α-S3. One-way ANOVA was performed for statistical analysis. ** represents p-value<0.01, **** represents p-value<0.0001. (C) Schematic representation of the strategy for luciferase assay in embryos. Embryos were injected at the 8-cell stage in the dorsal blastomere with Calpain-8 luciferase reporter and the CMV renilla reporter to target prospective CNC. Embryos with an expression of MbC in the CNC region at stage 18 were selected to perform luciferase assay. Individual embryos were used for luciferase reading. (D) Histogram depicting relative calpain-8 luciferase reporter expression normalized to CMV renilla expression in Control and Adam13KD (MO13) embryos. Student’s t-test was performed for statistical analysis. ** represents p-value<0.01. Each experiment was repeated 3 times with 5 individual embryos per case in each experiment. (E) Histogram depicting the relative calpain-8 luciferase reporter expression in cells transfected with CS2 (empty vector), Tfap2α.L-S1-Flag, and Tfap2α.L-S3-Flag along with Calpain-8 luciferase reporter plasmid and the CMV renilla plasmid. One-way ANOVA was performed for statistical analysis. * Represents p-value<0.05, ** represents p-value<0.01, ns represents p-value>0.05.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Fluorescence, Migration, Injection, Membrane, Control, Luciferase, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Tfap2 α. L-S1 and Adam13 are key regulators of splicing. (A) Venn diagram representing proteins detected following IP of Tfap2α.L-S1-Flag and Tfap2α.L-S3-Flag after removing proteins found in the RFP-Flag negative control. We used T-test to identify proteins that were significantly enriched in either of the 2 samples. Proteins that associate significantly more with the S1 variant (65) are highlighted in yellow. (B) Dot plot representing the gene set enrichment pathway analysis for biological processes of proteins that were found significantly associated with Tfap2α-S1-Flag. The red color represents higher significance (lower p-value), and the size of the dot represents the number of genes identified in the set. (C) Dot plot representing the gene set enrichment pathway analysis for biological processes of differentially expressed genes in Adam13KD (MO13) CNC. The red color represents higher significance (lower p-value), and the size of the dot represents the number of genes identified in the set. (D) Schematic representation of the different types of alternative splicing events detected by rMATS analysis on CNC RNA sequencing data. (E) Venn diagram depicting the genes found to have significant differences in splicing in CNC lacking Adam13. Genes having Alternate 5’ splice site (A5SS), Alternate 3’ splice site (A3SS), and mutually exclusive exons (MXE) are compared to differentially expressed genes (DEG). (F) Venn diagram depicting the genes found statistically differentially expressed versus genes having skipped exon (SE).

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Negative Control, Variant Assay, Alternative Splicing, RNA Sequencing Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Transcriptional and post-transcriptional regulation of CARMN and its anti-tumor function in cervical cancer through autophagic flux blockade and MAPK cascade inhibition

doi: 10.1186/s13046-024-03229-y

Figure Lengend Snippet: CARMN inactivated MAPK13-mediated MAPK cascade by repressing the transcriptional activation of MAPK13 via its sense chain. a The expression of MAPK cascade in the HeLa cells with or without CARMN overexpression. b Restoration of the MAPK signaling pathway in CARMN-overexpressing HeLa cells using honokiol. c CCK-8 assay results showed that honokiol treatment enhanced the proliferation ability of CARMN-overexpressing HeLa cells. d The expression of eight dysregulated genes related to MAPK signaling pathway in the HeLa-CARMN and -NC cells validated by qPCR. e MAPK13 expression of the HeLa-CARMN and -NC cells. f MAPK13 expression in the RNA-seq data of 30 cervical tissues. g MAPK13 expression in the cytoplasm and nucleus of the CC cells with or without CARMN overexpression. h The effect of MAPK13 on the expression of branches of MAPK cascade in the HeLa-CARMN and -NC cells. i The proliferation of HeLa-CARMN cells with or without MAPK13 overexpression by CCK8. j Luciferase reporter assay detecting the influence of TFAP2α on the transcriptional activity of MAPK13 promoter in the HeLa cells. k Luciferase reporter plasmids containing varied length of MAPK13 promoter regions were constructed to determine the potential binding sites of TFAP2α. l The levels of immunoprecipitated chromatin by anti-TFAP2α and control IgG in HeLa cells by ChIP assay. m RIP assay revealed the directly binding of CARMN and TFAP2α (HeLa). ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: ChIP was performed as previously reported [ ] with a slight modification using antibody against SP1 (#9389, CST, USA), TFAP2α (#13019-3-AP, Proteintech, USA) or control IgG.

Techniques: Activation Assay, Expressing, Over Expression, CCK-8 Assay, RNA Sequencing, Luciferase, Reporter Assay, Activity Assay, Construct, Binding Assay, Immunoprecipitation, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Transcriptional and post-transcriptional regulation of CARMN and its anti-tumor function in cervical cancer through autophagic flux blockade and MAPK cascade inhibition

doi: 10.1186/s13046-024-03229-y

Figure Lengend Snippet: Schematic representation of the CARMN mechanism involving transcriptional and post-transcriptional regulation. This figure illustrated the molecular mechanism of CARMN regulation. At the transcriptional level, SNP rs12517403 (T > C) influenced the binding affinity of SP1 to the CARMN promoter region, modulating CARMN transcriptional activity, and was significantly associated with CC risk. At the post-transcriptional level, YBX1 and DHX9 bound to CARMN, synergistically enhancing its stability in cells. Functionally, CARMN inhibited MAPK13 expression by repressing TFAP2α transcription, thereby suppressing the MAPK signaling pathway. Concurrently, CARMN promoted Keap1-Nrf2 binding, facilitating Nrf2 ubiquitination and degradation, which indirectly inhibited Nrf2 nuclear translocation and antioxidant factor expression. This led to ROS accumulation, inhibiting the Akt-mTOR signaling pathway and suppressing the malignant progression of CC cells

Article Snippet: ChIP was performed as previously reported [ ] with a slight modification using antibody against SP1 (#9389, CST, USA), TFAP2α (#13019-3-AP, Proteintech, USA) or control IgG.

Techniques: Binding Assay, Activity Assay, Expressing, Ubiquitin Proteomics, Translocation Assay

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Loss of Adam13 affects gene expression and Tfap2 α transcription start. (A) Schematic representation of the approach used for CNC-specific RNA-seq. Control (NI) and Adam13KD (MO13) CNC were dissected at the same apparent stage (Stage17) in 3 independent experiments. (B) Heatmap visualization of RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO131, MO132, MO133), higher Z-score in red representing increase expression and blue representing decreased expression. (C) Schematic representation of the Tfap2α.L gene. Three putative transcription start sites (alternate exon1) connecting to exon2 and producing three different proteins differing at the N-terminus. (D) Sashimi plot analysis of transcript junctions detected for the Tfap2α.L gene in RNA-sequencing data between Control (NI1) and Adam13KD (MO131), circles mark the major transcript junction between corresponding exons. (E) Quantification of exon-specific counts for the three Tfap2α.L alternate starts (S1, S2 and S3) normalized to the total transcript counts for the Tfap2α.L gene. Student t-test was performed for statistical analysis, * represents p-value<0.05; ns represents p-value>0.05. (F) Histogram depicting relative fluorescence intensity of Tfap2α in Sox9 positive cells between Control (NI) and Adam13KD (MO13) CNC explants. Student t-test was performed for statistical analysis, * represents p-value<0.05. (G) ClustalW protein sequence alignment for Tfap2α-S1 and Tfap2α-S3 to highlight the sequence difference in exon1 and the yellow highlighted sequence is Exon2 which is common between both proteins. (H) Immunofluorescence images of CNC explants were dissected at stage 17 and grown on fibronectin substrate for 3 hours. CNC explants were fixed and subsequently immuno-stained for Sox9 (red), Tfap2α (green) and DAPI (blue) and imaged using confocal microscopy, all images are Max-IP.

Article Snippet: Mouse monoclonal to TFAP2α (DSHB Cat# 3B5, RRID:AB_2202275).

Techniques: Gene Expression, RNA Sequencing, Control, Expressing, Fluorescence, Sequencing, Immunofluorescence, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: (A) Sashimi plot analysis of transcript junctions detected for gene Tfap2α.L in RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO13-1, MO13-2, MO13-3), numbers on the line are the major transcript junction between corresponding exons, arrow points to the number of transcript junction detected between Start1 and Exon2. (B) Sashimi plot analysis of transcript junctions detected for gene Tfap2α.S in RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO13-1, MO13-2, MO13-3), numbers on the line are the major transcript junction between corresponding exons, arrow points to the number of transcript junction detected between Start1 and Exon2. (C) Quantification of exon specific counts for Tfap2α.S-start1, start2 and start3 normalized to total transcript counts for Tfap2α.S gene. Student t-test was performed for statistical analysis, * represents p-value<0.05, ** represents p-value<0.01, ns represents p-value>0.05.

Article Snippet: Mouse monoclonal to TFAP2α (DSHB Cat# 3B5, RRID:AB_2202275).

Techniques: RNA Sequencing, Control

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Adam13 controls Arid3a binding and histone methylation at the Tfap2 α. L promoter. Chromatin-Immunoprecipitation-qPCR analysis of DNA associated with the chromatin binding proteins. (A, B, C, D) Histogram depicting the relative abundance of DNA bound to H3K4me3 (A), H3K9me2/3 (B), Arid3a (C), and Adam13 (D) in WT and Adam13KO embryos at the promoter region of Tfap2α-S1 and Tfap2α-S3 based on ChIP-qPCR analysis. ChIP was performed at stage 20. Student t-test was performed for statistical analysis, * represents p-value<0.05, ** p-value<0.01, *** p-value<0.001, **** p-value<0.0001, ns represents p-value>0.05. (E-G) Histogram depicting the relative luciferase expression using the luciferase reporter containing Tfap2α-S1 or Tfap2α-S3 promoter region in HEK293T cells that were transfected with CS2 (empty vector), Arid3a, Adam13, Arid3a+Adam13, alone or in combination with the Human Arid3a-SH (AridSh, F-G), along with the CMV renilla reporter. One-way ANOVA was performed for statistical analysis. * Represent p-value<0.05, ** p-value<0.01, **** p-value<0.0001.

Article Snippet: Mouse monoclonal to TFAP2α (DSHB Cat# 3B5, RRID:AB_2202275).

Techniques: Binding Assay, Methylation, Chromatin Immunoprecipitation, ChIP-qPCR, Luciferase, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Chromatin-Immunoprecipitation-qPCR analysis of DNA associated with the chromatin binding proteins. (A, B, C, D) Histogram depicting the relative abundance of DNA bound to H3K4me3 (A), H3K9me2/3 (B), Arid3a (C), Adam13 (D) in WT and KO embryos at the promoter region of Tfap2α-S1 and Tfap2α-S3 based on ChIP-qPCR analysis. ChIP was performed at stage 20 (Independent biological replicate from ). Student’s t-test was performed for statistical analysis, * represents p-value<0.05, ** represents p-value<0.01, *** represents p-value<0.001, **** represents p-value<0.0001, ns represents p-value>0.05. (E) Schematic representation of the Luciferase reporters for the S1 and S3 transcription starts.

Article Snippet: Mouse monoclonal to TFAP2α (DSHB Cat# 3B5, RRID:AB_2202275).

Techniques: Chromatin Immunoprecipitation, Binding Assay, ChIP-qPCR, Luciferase

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Tfap2 α variants have distinct biological functions. (A) Fluorescence tracking of CNC cell migration. Embryos were injected with fluorescent Membrane Cherry (MbC) mRNA at the 8-cell stage in one dorsal animal blastomere and scored at stages 22-24 for the presence or absence of fluorescence in the branchial arches to quantify migration. The absence of fluorescence in the branchial arches is considered either defective migration or missed targeting. The value obtained for MbC alone give the missed targeting value for each experiment and is used to normalize between biological repeats (Typically 5 to 20% depending on embryo pigmentation). (B) Histogram representing the percentage proper migration in Control (MbC), Adam13KD (MO13), MO13+Tfap2α-S1, MO13+Tfap2α-S3. One-way ANOVA was performed for statistical analysis. ** represents p-value<0.01, **** represents p-value<0.0001. (C) Schematic representation of the strategy for luciferase assay in embryos. Embryos were injected at the 8-cell stage in the dorsal blastomere with Calpain-8 luciferase reporter and the CMV renilla reporter to target prospective CNC. Embryos with an expression of MbC in the CNC region at stage 18 were selected to perform luciferase assay. Individual embryos were used for luciferase reading. (D) Histogram depicting relative calpain-8 luciferase reporter expression normalized to CMV renilla expression in Control and Adam13KD (MO13) embryos. Student’s t-test was performed for statistical analysis. ** represents p-value<0.01. Each experiment was repeated 3 times with 5 individual embryos per case in each experiment. (E) Histogram depicting the relative calpain-8 luciferase reporter expression in cells transfected with CS2 (empty vector), Tfap2α.L-S1-Flag, and Tfap2α.L-S3-Flag along with Calpain-8 luciferase reporter plasmid and the CMV renilla plasmid. One-way ANOVA was performed for statistical analysis. * Represents p-value<0.05, ** represents p-value<0.01, ns represents p-value>0.05.

Article Snippet: Mouse monoclonal to TFAP2α (DSHB Cat# 3B5, RRID:AB_2202275).

Techniques: Fluorescence, Migration, Injection, Membrane, Control, Luciferase, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Tfap2 α. L-S1 and Adam13 are key regulators of splicing. (A) Venn diagram representing proteins detected following IP of Tfap2α.L-S1-Flag and Tfap2α.L-S3-Flag after removing proteins found in the RFP-Flag negative control. We used T-test to identify proteins that were significantly enriched in either of the 2 samples. Proteins that associate significantly more with the S1 variant (65) are highlighted in yellow. (B) Dot plot representing the gene set enrichment pathway analysis for biological processes of proteins that were found significantly associated with Tfap2α-S1-Flag. The red color represents higher significance (lower p-value), and the size of the dot represents the number of genes identified in the set. (C) Dot plot representing the gene set enrichment pathway analysis for biological processes of differentially expressed genes in Adam13KD (MO13) CNC. The red color represents higher significance (lower p-value), and the size of the dot represents the number of genes identified in the set. (D) Schematic representation of the different types of alternative splicing events detected by rMATS analysis on CNC RNA sequencing data. (E) Venn diagram depicting the genes found to have significant differences in splicing in CNC lacking Adam13. Genes having Alternate 5’ splice site (A5SS), Alternate 3’ splice site (A3SS), and mutually exclusive exons (MXE) are compared to differentially expressed genes (DEG). (F) Venn diagram depicting the genes found statistically differentially expressed versus genes having skipped exon (SE).

Article Snippet: Mouse monoclonal to TFAP2α (DSHB Cat# 3B5, RRID:AB_2202275).

Techniques: Negative Control, Variant Assay, Alternative Splicing, RNA Sequencing

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Loss of Adam13 affects gene expression and Tfap2 α transcription start. (A) Schematic representation of the approach used for CNC-specific RNA-seq. Control (NI) and Adam13KD (MO13) CNC were dissected at the same apparent stage (Stage17) in 3 independent experiments. (B) Heatmap visualization of RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO131, MO132, MO133), higher Z-score in red representing increase expression and blue representing decreased expression. (C) Schematic representation of the Tfap2α.L gene. Three putative transcription start sites (alternate exon1) connecting to exon2 and producing three different proteins differing at the N-terminus. (D) Sashimi plot analysis of transcript junctions detected for the Tfap2α.L gene in RNA-sequencing data between Control (NI1) and Adam13KD (MO131), circles mark the major transcript junction between corresponding exons. (E) Quantification of exon-specific counts for the three Tfap2α.L alternate starts (S1, S2 and S3) normalized to the total transcript counts for the Tfap2α.L gene. Student t-test was performed for statistical analysis, * represents p-value<0.05; ns represents p-value>0.05. (F) Histogram depicting relative fluorescence intensity of Tfap2α in Sox9 positive cells between Control (NI) and Adam13KD (MO13) CNC explants. Student t-test was performed for statistical analysis, * represents p-value<0.05. (G) ClustalW protein sequence alignment for Tfap2α-S1 and Tfap2α-S3 to highlight the sequence difference in exon1 and the yellow highlighted sequence is Exon2 which is common between both proteins. (H) Immunofluorescence images of CNC explants were dissected at stage 17 and grown on fibronectin substrate for 3 hours. CNC explants were fixed and subsequently immuno-stained for Sox9 (red), Tfap2α (green) and DAPI (blue) and imaged using confocal microscopy, all images are Max-IP.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Expressing, RNA Sequencing Assay, Control, Fluorescence, Sequencing, Immunofluorescence, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: (A) Sashimi plot analysis of transcript junctions detected for gene Tfap2α.L in RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO13-1, MO13-2, MO13-3), numbers on the line are the major transcript junction between corresponding exons, arrow points to the number of transcript junction detected between Start1 and Exon2. (B) Sashimi plot analysis of transcript junctions detected for gene Tfap2α.S in RNA-sequencing data between Control (NI1, NI2, NI3) and Adam13KD (MO13-1, MO13-2, MO13-3), numbers on the line are the major transcript junction between corresponding exons, arrow points to the number of transcript junction detected between Start1 and Exon2. (C) Quantification of exon specific counts for Tfap2α.S-start1, start2 and start3 normalized to total transcript counts for Tfap2α.S gene. Student t-test was performed for statistical analysis, * represents p-value<0.05, ** represents p-value<0.01, ns represents p-value>0.05.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: RNA Sequencing Assay, Control

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Adam13 controls Arid3a binding and histone methylation at the Tfap2 α. L promoter. Chromatin-Immunoprecipitation-qPCR analysis of DNA associated with the chromatin binding proteins. (A, B, C, D) Histogram depicting the relative abundance of DNA bound to H3K4me3 (A), H3K9me2/3 (B), Arid3a (C), and Adam13 (D) in WT and Adam13KO embryos at the promoter region of Tfap2α-S1 and Tfap2α-S3 based on ChIP-qPCR analysis. ChIP was performed at stage 20. Student t-test was performed for statistical analysis, * represents p-value<0.05, ** p-value<0.01, *** p-value<0.001, **** p-value<0.0001, ns represents p-value>0.05. (E-G) Histogram depicting the relative luciferase expression using the luciferase reporter containing Tfap2α-S1 or Tfap2α-S3 promoter region in HEK293T cells that were transfected with CS2 (empty vector), Arid3a, Adam13, Arid3a+Adam13, alone or in combination with the Human Arid3a-SH (AridSh, F-G), along with the CMV renilla reporter. One-way ANOVA was performed for statistical analysis. * Represent p-value<0.05, ** p-value<0.01, **** p-value<0.0001.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Binding Assay, Methylation, Chromatin Immunoprecipitation, Luciferase, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Chromatin-Immunoprecipitation-qPCR analysis of DNA associated with the chromatin binding proteins. (A, B, C, D) Histogram depicting the relative abundance of DNA bound to H3K4me3 (A), H3K9me2/3 (B), Arid3a (C), Adam13 (D) in WT and KO embryos at the promoter region of Tfap2α-S1 and Tfap2α-S3 based on ChIP-qPCR analysis. ChIP was performed at stage 20 (Independent biological replicate from ). Student’s t-test was performed for statistical analysis, * represents p-value<0.05, ** represents p-value<0.01, *** represents p-value<0.001, **** represents p-value<0.0001, ns represents p-value>0.05. (E) Schematic representation of the Luciferase reporters for the S1 and S3 transcription starts.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Luciferase

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Tfap2 α variants have distinct biological functions. (A) Fluorescence tracking of CNC cell migration. Embryos were injected with fluorescent Membrane Cherry (MbC) mRNA at the 8-cell stage in one dorsal animal blastomere and scored at stages 22-24 for the presence or absence of fluorescence in the branchial arches to quantify migration. The absence of fluorescence in the branchial arches is considered either defective migration or missed targeting. The value obtained for MbC alone give the missed targeting value for each experiment and is used to normalize between biological repeats (Typically 5 to 20% depending on embryo pigmentation). (B) Histogram representing the percentage proper migration in Control (MbC), Adam13KD (MO13), MO13+Tfap2α-S1, MO13+Tfap2α-S3. One-way ANOVA was performed for statistical analysis. ** represents p-value<0.01, **** represents p-value<0.0001. (C) Schematic representation of the strategy for luciferase assay in embryos. Embryos were injected at the 8-cell stage in the dorsal blastomere with Calpain-8 luciferase reporter and the CMV renilla reporter to target prospective CNC. Embryos with an expression of MbC in the CNC region at stage 18 were selected to perform luciferase assay. Individual embryos were used for luciferase reading. (D) Histogram depicting relative calpain-8 luciferase reporter expression normalized to CMV renilla expression in Control and Adam13KD (MO13) embryos. Student’s t-test was performed for statistical analysis. ** represents p-value<0.01. Each experiment was repeated 3 times with 5 individual embryos per case in each experiment. (E) Histogram depicting the relative calpain-8 luciferase reporter expression in cells transfected with CS2 (empty vector), Tfap2α.L-S1-Flag, and Tfap2α.L-S3-Flag along with Calpain-8 luciferase reporter plasmid and the CMV renilla plasmid. One-way ANOVA was performed for statistical analysis. * Represents p-value<0.05, ** represents p-value<0.01, ns represents p-value>0.05.

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Fluorescence, Migration, Injection, Membrane, Control, Luciferase, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

doi: 10.1101/2024.08.18.608474

Figure Lengend Snippet: Tfap2 α. L-S1 and Adam13 are key regulators of splicing. (A) Venn diagram representing proteins detected following IP of Tfap2α.L-S1-Flag and Tfap2α.L-S3-Flag after removing proteins found in the RFP-Flag negative control. We used T-test to identify proteins that were significantly enriched in either of the 2 samples. Proteins that associate significantly more with the S1 variant (65) are highlighted in yellow. (B) Dot plot representing the gene set enrichment pathway analysis for biological processes of proteins that were found significantly associated with Tfap2α-S1-Flag. The red color represents higher significance (lower p-value), and the size of the dot represents the number of genes identified in the set. (C) Dot plot representing the gene set enrichment pathway analysis for biological processes of differentially expressed genes in Adam13KD (MO13) CNC. The red color represents higher significance (lower p-value), and the size of the dot represents the number of genes identified in the set. (D) Schematic representation of the different types of alternative splicing events detected by rMATS analysis on CNC RNA sequencing data. (E) Venn diagram depicting the genes found to have significant differences in splicing in CNC lacking Adam13. Genes having Alternate 5’ splice site (A5SS), Alternate 3’ splice site (A3SS), and mutually exclusive exons (MXE) are compared to differentially expressed genes (DEG). (F) Venn diagram depicting the genes found statistically differentially expressed versus genes having skipped exon (SE).

Article Snippet: Tfap2α.L-S1 and Tfap2α.L-S3 were cloned from xenopus embryos using Takara infusion cloning.

Techniques: Negative Control, Variant Assay, Alternative Splicing, RNA Sequencing Assay

Journal: BMC Cancer

Article Title: Increased expression of transcription factor TFAP2α correlates with chemosensitivity in advanced bladder cancer

doi: 10.1186/1471-2407-11-135

Figure Lengend Snippet: Expression of TFAP2α isoforms . (A) Expression of TFAP2α isoform 1, 2 and 3 in advanced muscle invasive bladder cancer (T2-4) were determined using real-time Q-PCR. Analysis was performed on cDNA from 10 tumor specimens and each bar represents the mean from the 10 samples.(B) COS-7 cells were transiently transfected with empty pcDNA3.1/V5-His vector (lane 2, 6), pcDNA3.1/V5-His- TFAP2α isoform 1(lane 3, 7), isoform 2 (lane 4, 8) and isoform 3 (lane 5, 9). Western blot of 30 μg total protein lysate from non-transfected HU609 bladder cells (lane 1) and COS-7 transfected cells (lane 2-9) 48 h post transfection probed with anti TFAP2α antibody (lane 1-5) or anti-V5 antibody (lane 6-9).

Article Snippet: TFAP2α specific siRNA was obtained from Ambion (ABI;Foster City, CA)(Cat# s14003).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: BMC Cancer

Article Title: Increased expression of transcription factor TFAP2α correlates with chemosensitivity in advanced bladder cancer

doi: 10.1186/1471-2407-11-135

Figure Lengend Snippet: Immunohistochemistry of bladder cancer tissue with antibody against TFAP2α . TFAP2α immunoreactivity of muscle invasive bladder cancer tumors. A and D: Negative stain of nucleus and cytoplasm at 5 and 40 fold objective magnification, respectively. B and E: Negative stain of nucleus and positive stain of cytoplasm (5× and 40×, respectively). C and F: Positive stain of nucleus and cytoplasm (5× and 40×, respectively).

Article Snippet: TFAP2α specific siRNA was obtained from Ambion (ABI;Foster City, CA)(Cat# s14003).

Techniques: Immunohistochemistry, Staining

Journal: BMC Cancer

Article Title: Increased expression of transcription factor TFAP2α correlates with chemosensitivity in advanced bladder cancer

doi: 10.1186/1471-2407-11-135

Figure Lengend Snippet: The relationship between TFAP2α staining and survival after chemotherapy . TFAP2α immunoreactivity and overall survival (OS) rates and progression free survival (PFS). A and B: separation of OS (overall survival) curves based on TFAP2α nuclear and cytoplasmic staining in the lymph node invasive group, respectively. C and D: separation of PFS curves based on TFAP2α nuclear and cytoplasmic staining in the lymph node invasive group, respectively. C and F: separation of OS curves based on TFAP2α nuclear and cytoplasmic staining in the non lymph node invasive group, respectively. Red curves are high TFAP2α staining and blue curves are low TFAP2α staining.

Article Snippet: TFAP2α specific siRNA was obtained from Ambion (ABI;Foster City, CA)(Cat# s14003).

Techniques: Staining

Journal: BMC Cancer

Article Title: Increased expression of transcription factor TFAP2α correlates with chemosensitivity in advanced bladder cancer

doi: 10.1186/1471-2407-11-135

Figure Lengend Snippet: Cisplatin sensitivity of TFAP2α silenced T24 and SW780 . A and B: Transfection of 10-50 nM TFAP2α siRNA or control siRNA in T24 and SW780 cells, respectively. Real-time RT-PCR was used to determine the relative TFAP2α mRNA levels 48 h post transfektion. C and D: Transfection of 25 nM TFAP2α siRNA in T24 and SW780 cells, respectively. After 24 h incubation cisplatin or media was added to the cells. The viability of the cells was determined 96 h after transfection (48 h after the drug was added) by MTT-assay and expressed as the viability compared with the culture media control for both the TFAP2α siRNA or control siRNA transfected cells. E and F: As C and D using gemcitabine instead of cisplatin. (n = 6).

Article Snippet: TFAP2α specific siRNA was obtained from Ambion (ABI;Foster City, CA)(Cat# s14003).

Techniques: Transfection, Quantitative RT-PCR, Incubation, MTT Assay

Journal: BMC Cancer

Article Title: Increased expression of transcription factor TFAP2α correlates with chemosensitivity in advanced bladder cancer

doi: 10.1186/1471-2407-11-135

Figure Lengend Snippet: Cell proliferation of TFAP2α silenced T24 and SW780 . Real time growth curves monitoring was performed with the RT-CES system. The T24 or SW780 cells were seeded into 16-well or 96-well E-Plates, which contain electrodes integrated into the bottom surfaces of each well that measure cell index based on impedance. Cell index correlates with the area of cells attached to the bottom of the plate. A and B: Transfection of 25 nM TFAP2α siRNA in T24 and SW780 cells, respectively. (n = 3) After 24 h incubation cisplatin or media was added to the cells. C and D: Transfection of 25 nM TFAP2α siRNA in T24 and SW780 cells, respectively. After 48 h incubation CyQuant assay was performed. (n = 8)

Article Snippet: TFAP2α specific siRNA was obtained from Ambion (ABI;Foster City, CA)(Cat# s14003).

Techniques: Transfection, Incubation, CyQUANT Assay