anti hif 3α  (Proteintech)

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: The associations with NSCPO risk of the three variants in discovery and replication stages

Article Snippet: The lentiviral Hif3a constructs were purchased from GENECHEM (Shanghai, China).

Techniques:

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: The variant rs3826795 at 19q13.3 regulates transcriptional activity of HIF3A by modulating the binding affinity of VEZF1 to the promoter Allele-specific constructs containing the putative promoter sequence flanking rs3826795 were cloned into the pGL3-basic luciferase reporter vector and transfected into the (A) HEK-293, (B) HEPM, and (C) HOK. The results of luciferase activity were normalized to those of pGL3 basic ( n = 6). (D) EMSA with biotin-labelled oligonucleotides containing the rs3826795[A] or rs3826795[G] allele and nuclear extracts from HEPM and HOK. Lanes 1 showed the mobility of the labeled oligonucleotides without nuclear extracts; lanes 2 and 4 showed the mobility of the labeled oligonucleotides with nuclear extracts in the absence of the competitor oligonucleotide and lanes 3 and 5 showed the mobility of the labeled oligonucleotides with nuclear extracts in the presence of unlabeled competitors. The arrow indicates a DNA–protein complex. (E) In silico predicted preferential binding of VEZF1 to the non-risk allele C of rs3826795. (F) The transcription factor VEZF1 was analyzed for correlation with Hif3a in mouse embryo craniofacial tissues, lip and palate tissues, palate shelves (from left to right). (G) The ChIP enrichment of VEZF1 as determined by ChIP-qPCR. IgG was used as a negative control. (H) Supershift assays using the VEZF1 antibody in HEPM and HOK. IgG was used as a negative control. The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001, and ∗∗∗∗ indicates p < 0.0001. EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. SD, standard deviation.

Article Snippet: The lentiviral Hif3a constructs were purchased from GENECHEM (Shanghai, China).

Techniques: Variant Assay, Activity Assay, Binding Assay, Construct, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Labeling, In Silico, ChIP-qPCR, Negative Control, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Standard Deviation

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: In vitro functional roles of HIF3A in HEPM and HOK (A–D) Quantitative analysis of cell apoptosis by flow cytometry between the two groups ( n = 6). The rate of apoptosis is lower with HIF3A knockdown and is higher than those in the corresponding negative controls in HEPM and HOK. (E and F) Photos showing the migration of HEPM and HOK with knockdown and overexpression of HIF3A in 0h, 12h, and 24h after scratch. Overexpression of HIF3A significantly reduced the migration ability in HEPM and HOK (scale bar = 200 μm). (G) Transwell assays was used to assess the migration of HEPM and HOK after transfection with HIF3A siRNAs or overexpression plasmids. The rate of migration is lower with HIF3A overexpression groups in HEPM and HOK (scale bar = 200 μm). (H) EdU proliferation analysis before or after knockdown/overexpression of HIF3A in HEPM and HOK. High HIF3A expression significantly reduced the proliferation rate of HEPM and HOK (scale bar = 200 μm). The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗ indicates p < 0.05, ∗∗∗ indicates p < 0.001.

Article Snippet: The lentiviral Hif3a constructs were purchased from GENECHEM (Shanghai, China).

Techniques: In Vitro, Functional Assay, Flow Cytometry, Knockdown, Migration, Over Expression, Transfection, Expressing

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: Effect of Hif3a overexpression on proliferation and apoptosis in vivo (A) Schematic diagram of the mice experiment. (B) Proliferation analysis before or after overexpressing of Hif3a in E13.5d mouse palate shelves (scale bar = 100 μm). High Hif3a expression significantly reduced the proliferation rate. (C) Apoptosis analysis before or after overexpressing of Hif3a in E13.5d mouse palate shelves (scale bar = 100 μm). The rate of apoptosis is higher with Hif3a overexpression. (D) Proliferation analysis before or after overexpressing of Hif3a in E14.5d mouse palate shelves (scale bar = 100 μm). High Hif3a expression significantly reduced the proliferation rate. (E) Apoptosis analysis before or after overexpressing of Hif3a in E14.5d mouse palate shelves (scale bar = 100 μm). The rate of apoptosis is higher with Hif3a overexpression.

Article Snippet: The lentiviral Hif3a constructs were purchased from GENECHEM (Shanghai, China).

Techniques: Over Expression, In Vivo, Expressing

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: Pathway analysis with overexpression of Hif3a in mouse embryo (A) Heatmap of differentially expressed genes between controls and overexpression of Hif3a . (B) Volcano plot of differentially expressed genes between controls and overexpression of Hif3a . (C) KEGG and (D) GSEA pathway enrichment analysis showed the association function of the genes reside on the susceptible region. The term/pathway were ranked by -log 10 ( P ). The relative mRNA expression levels of all differentially expressed genes in GABAergic synapse pathway were assessed by qRT-PCR with (E) HEPM and (F) HOK. The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, and ∗∗∗ indicates p < 0.001. KEGG, Kyoto encyclopedia of genes and genomes; GSEA, gene set enrichment analysis.

Article Snippet: The lentiviral Hif3a constructs were purchased from GENECHEM (Shanghai, China).

Techniques: Over Expression, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet:

Article Snippet: The lentiviral Hif3a constructs were purchased from GENECHEM (Shanghai, China).

Techniques: Virus, Over Expression, Recombinant, Bicinchoninic Acid Protein Assay, Luciferase, Reporter Gene Assay, Extraction, Control, Chromatin Immunoprecipitation, In Vitro, TUNEL Assay, Sequencing, Software

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: The associations with NSCPO risk of the three variants in discovery and replication stages

Article Snippet: Lentivirus Hif3a overexpression , GENECHEM , N/A.

Techniques:

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: The variant rs3826795 at 19q13.3 regulates transcriptional activity of HIF3A by modulating the binding affinity of VEZF1 to the promoter Allele-specific constructs containing the putative promoter sequence flanking rs3826795 were cloned into the pGL3-basic luciferase reporter vector and transfected into the (A) HEK-293, (B) HEPM, and (C) HOK. The results of luciferase activity were normalized to those of pGL3 basic ( n = 6). (D) EMSA with biotin-labelled oligonucleotides containing the rs3826795[A] or rs3826795[G] allele and nuclear extracts from HEPM and HOK. Lanes 1 showed the mobility of the labeled oligonucleotides without nuclear extracts; lanes 2 and 4 showed the mobility of the labeled oligonucleotides with nuclear extracts in the absence of the competitor oligonucleotide and lanes 3 and 5 showed the mobility of the labeled oligonucleotides with nuclear extracts in the presence of unlabeled competitors. The arrow indicates a DNA–protein complex. (E) In silico predicted preferential binding of VEZF1 to the non-risk allele C of rs3826795. (F) The transcription factor VEZF1 was analyzed for correlation with Hif3a in mouse embryo craniofacial tissues, lip and palate tissues, palate shelves (from left to right). (G) The ChIP enrichment of VEZF1 as determined by ChIP-qPCR. IgG was used as a negative control. (H) Supershift assays using the VEZF1 antibody in HEPM and HOK. IgG was used as a negative control. The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001, and ∗∗∗∗ indicates p < 0.0001. EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. SD, standard deviation.

Article Snippet: Lentivirus Hif3a overexpression , GENECHEM , N/A.

Techniques: Variant Assay, Activity Assay, Binding Assay, Construct, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Labeling, In Silico, ChIP-qPCR, Negative Control, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Standard Deviation

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: In vitro functional roles of HIF3A in HEPM and HOK (A–D) Quantitative analysis of cell apoptosis by flow cytometry between the two groups ( n = 6). The rate of apoptosis is lower with HIF3A knockdown and is higher than those in the corresponding negative controls in HEPM and HOK. (E and F) Photos showing the migration of HEPM and HOK with knockdown and overexpression of HIF3A in 0h, 12h, and 24h after scratch. Overexpression of HIF3A significantly reduced the migration ability in HEPM and HOK (scale bar = 200 μm). (G) Transwell assays was used to assess the migration of HEPM and HOK after transfection with HIF3A siRNAs or overexpression plasmids. The rate of migration is lower with HIF3A overexpression groups in HEPM and HOK (scale bar = 200 μm). (H) EdU proliferation analysis before or after knockdown/overexpression of HIF3A in HEPM and HOK. High HIF3A expression significantly reduced the proliferation rate of HEPM and HOK (scale bar = 200 μm). The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗ indicates p < 0.05, ∗∗∗ indicates p < 0.001.

Article Snippet: Lentivirus Hif3a overexpression , GENECHEM , N/A.

Techniques: In Vitro, Functional Assay, Flow Cytometry, Knockdown, Migration, Over Expression, Transfection, Expressing

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: Effect of Hif3a overexpression on proliferation and apoptosis in vivo (A) Schematic diagram of the mice experiment. (B) Proliferation analysis before or after overexpressing of Hif3a in E13.5d mouse palate shelves (scale bar = 100 μm). High Hif3a expression significantly reduced the proliferation rate. (C) Apoptosis analysis before or after overexpressing of Hif3a in E13.5d mouse palate shelves (scale bar = 100 μm). The rate of apoptosis is higher with Hif3a overexpression. (D) Proliferation analysis before or after overexpressing of Hif3a in E14.5d mouse palate shelves (scale bar = 100 μm). High Hif3a expression significantly reduced the proliferation rate. (E) Apoptosis analysis before or after overexpressing of Hif3a in E14.5d mouse palate shelves (scale bar = 100 μm). The rate of apoptosis is higher with Hif3a overexpression.

Article Snippet: Lentivirus Hif3a overexpression , GENECHEM , N/A.

Techniques: Over Expression, In Vivo, Expressing

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet: Pathway analysis with overexpression of Hif3a in mouse embryo (A) Heatmap of differentially expressed genes between controls and overexpression of Hif3a . (B) Volcano plot of differentially expressed genes between controls and overexpression of Hif3a . (C) KEGG and (D) GSEA pathway enrichment analysis showed the association function of the genes reside on the susceptible region. The term/pathway were ranked by -log 10 ( P ). The relative mRNA expression levels of all differentially expressed genes in GABAergic synapse pathway were assessed by qRT-PCR with (E) HEPM and (F) HOK. The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, and ∗∗∗ indicates p < 0.001. KEGG, Kyoto encyclopedia of genes and genomes; GSEA, gene set enrichment analysis.

Article Snippet: Lentivirus Hif3a overexpression , GENECHEM , N/A.

Techniques: Over Expression, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Functional variant at 19q13.3 confers nonsyndromic cleft palate susceptibility by regulating HIF3A

doi: 10.1016/j.isci.2025.111829

Figure Lengend Snippet:

Article Snippet: Lentivirus Hif3a overexpression , GENECHEM , N/A.

Techniques: Virus, Over Expression, Recombinant, Bicinchoninic Acid Protein Assay, Luciferase, Reporter Gene Assay, Extraction, Control, Chromatin Immunoprecipitation, In Vitro, TUNEL Assay, Sequencing, Software