Journal: Cells

Article Title: Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression

doi: 10.3390/cells13100826

Figure Lengend Snippet: ( A ) Representative overview of transcription factor impact on ADGB promoter-driven luciferase activity. Results are displayed in relative luminescence units (RLU) as ratio of firefly to Renilla luciferase activities. ( B ) Confirmation of overexpression of transcription factors. We performed qPCR analysis to assess expression intensity of all factors following transfection. RNA samples were DNase-treated before synthesis of cDNA to reduce vector contamination. Results are displayed as relative mRNA expression in fold change. The white bar shows the negative control; black bar shows FOXJ1 as positive control. MYBL2 and PITX2 are highlighted in blue. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: In the public databases of the human protein atlas, evidence exists for an occurrence of ADGB mRNA in myeloid dendritic cells derived from this lineage ( https://www.proteinatlas.org/ENSG00000118492-ADGB/immune+cell ; [ ]).

Techniques: Luciferase, Activity Assay, Over Expression, Expressing, Transfection, Plasmid Preparation, Negative Control, Positive Control

Journal: Cells

Article Title: Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression

doi: 10.3390/cells13100826

Figure Lengend Snippet: MYBL2 increases promoter activity in the upstream region close to the ADGB transcriptional start site (TSS). Luciferase reporter gene assays in HEK293T cells transfected with empty pGL3b vector or various ADGB promoter fragments with and without co-overexpression of MYBL2. ( A ) Schematic fragmentation of the ADGB promoter. The ADGB promoter was systematically fragmented into non-overlapping promoter segments. ( B ) Luciferase reporter gene assay on ADGB promoter (AP) fragments in three different lengths: AP431 (−1 bp to −431 bp), AP1032 (−432 bp to −1032 bp), and AP1981 (−1033 bp to −1981 bp) upstream of the ADGB TSS. ( C ) Luciferase reporter gene assay on AP sub-fragments of AP431 in three different lengths: AP431_A (−431 bp to −237 bp), AP431_B (−236 bp to −141 bp), and AP431_C (−140 bp to −1 bp). ( D ) Luciferase reporter gene assay on AP sub-fragments of AP431_C: AP431_C1 (−140 bp to −71 bp) and AP431_C2 (−70 bp to −1 bp). Results are displayed in relative luminescence units (RLU) as ratio of firefly to Renilla luciferase activities. Data are represented as means ± S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant).

Article Snippet: In the public databases of the human protein atlas, evidence exists for an occurrence of ADGB mRNA in myeloid dendritic cells derived from this lineage ( https://www.proteinatlas.org/ENSG00000118492-ADGB/immune+cell ; [ ]).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Over Expression, Reporter Gene Assay

Journal: Cells

Article Title: Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression

doi: 10.3390/cells13100826

Figure Lengend Snippet: PITX2 increases promoter activity in the upstream region close to the ADGB transcriptional start site (TSS). Luciferase reporter gene assays in HEK293T cells transfected with empty pGL3b vector or various ADGB promoter fragments with and without co-overexpression of PITX2. ( A ) Schematic fragmentation of the ADGB promoter. The ADGB promoter was systematically fragmented into non-overlapping promoter segments. ( B ) Luciferase reporter gene assay on ADGB promoter (AP) fragments in three different lengths: AP431 (−1 bp to −431 bp), AP1032 (−432 bp to −1032 bp), and AP1981 (−1033 bp to −1981 bp) upstream of the ADGB TSS. ( C ) Luciferase reporter gene assay on AP sub-fragments of AP431 in three different lengths: AP431_A (−431 bp to −237 bp), AP431_B (−236 bp to −141 bp), and AP431_C (−140 bp to −1 bp). ( D ) Luciferase reporter gene assay on AP sub fragments of AP431_C: AP431_C1 (−140 bp to −71 bp) and AP431_C2 (−70 bp to −1 bp). Results are displayed in relative luminescence units (RLU) as ratio of firefly to Renilla luciferase activities. Data are represented as means ± S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant).

Article Snippet: In the public databases of the human protein atlas, evidence exists for an occurrence of ADGB mRNA in myeloid dendritic cells derived from this lineage ( https://www.proteinatlas.org/ENSG00000118492-ADGB/immune+cell ; [ ]).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Over Expression, Reporter Gene Assay

Journal: Cells

Article Title: Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression

doi: 10.3390/cells13100826

Figure Lengend Snippet: Schematic overview on dCas9-mediated interference strategy and mutation of AP431_C. Schematic overview of both experimental strategies (Variant A and B) to investigate potential MYBL2 binding sites on AP431_C. Variant A, CRISPR/dCas9 gRNAs (α, β, and γ) targeting specific regions on AP431_C (between −120 bp and −37 bp) were used to abolish MYBL2 binding to this region upstream of the ADGB TSS. gRNA α (−120 bp to −100 bp), gRNA β (−86 bp to −66 bp), and gRNA γ (−57 bp to −37 bp) are displayed in green and underlined. Variant B shows the locations of the used mutations on AP431_C (−100 bp to −40 bp upstream of the ADGB TSS). Substitution-based mutation at −96 to −92 bp (mut1), at −73 to −68 bp (mut2), −57 to −52 bp (mut3), and −51 to −46 bp (mut4), are highlighted in bold. For both variants, two evolutionarily conserved regions (Cons1 and Cons2) are highlighted with a grey background.

Article Snippet: In the public databases of the human protein atlas, evidence exists for an occurrence of ADGB mRNA in myeloid dendritic cells derived from this lineage ( https://www.proteinatlas.org/ENSG00000118492-ADGB/immune+cell ; [ ]).

Techniques: Mutagenesis, Variant Assay, Binding Assay, CRISPR

Journal: Cells

Article Title: Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression

doi: 10.3390/cells13100826

Figure Lengend Snippet: MYBL2-binding to the ADGB promoter is impaired by dCas9-mediated interference or mutation of the binding site. ( A ) Luciferase reporter gene assays in HEK293T cells transfected with empty pGL3b vector or AP431 with and without co-transfection of dCas9 and gRNAs (α, β, and γ) and with and without co-overexpression of MYBL2. The gRNA binding sites and conserved sequences are schematically depicted on top. ( B ) Luciferase reporter gene assays in HEK293T cells transfected with empty pGL3b vector or mutated AP431_C with and without co-overexpression of MYBL2. The position of the different mutations and the conserved sites are schematically depicted on top. Results are displayed in relative luminescence units (RLU) as ratio of firefly to Renilla luciferase activities. All statistically significant comparisons are shown. Data are represented as means ± S.E.M; ** p < 0.01, *** p < 0.001, ns = not significant).

Article Snippet: In the public databases of the human protein atlas, evidence exists for an occurrence of ADGB mRNA in myeloid dendritic cells derived from this lineage ( https://www.proteinatlas.org/ENSG00000118492-ADGB/immune+cell ; [ ]).

Techniques: Binding Assay, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Cotransfection, Over Expression

Journal: Cells

Article Title: Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression

doi: 10.3390/cells13100826

Figure Lengend Snippet: MYBL2 ( left panel) and PITX2 ( right panel) interact with the endogenous ADGB promoter via direct binding. ( A ) Chromatin immunoprecipitation (ChIP) experiments in HEK293T cells transiently transfected with MYBL2 or FLAG-PITX2. Coprecipitated chromatin derived from the ADGB promoter was determined by qPCR using a primer pair covering +21 bp to −184 bp upstream of the ADGB TSS. Control regions were targeted upstream (5′ end, ADGB 5′) and downstream (3′ end, ADGB 3′) of ADGB on chromosome 6 and two independent AURKA loci (as positive and negative control). Due to the high differences in intensity, statistics for both loci were conducted separately. ( B ) Relative mRNA expression of endogenous ADGB following transient overexpression of MYBL2 or PITX2 compared to empty vector in A375 cells or HEK293T cells. ( C ) Overexpression of MYBL2 ( left panel) and PITX2 ( right panel) was verified by immunoblotting using specific antibodies against MYBL2 and PITX2. Data are represented as means ± S.E.M; * p < 0.05, ** p < 0.01).

Article Snippet: In the public databases of the human protein atlas, evidence exists for an occurrence of ADGB mRNA in myeloid dendritic cells derived from this lineage ( https://www.proteinatlas.org/ENSG00000118492-ADGB/immune+cell ; [ ]).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Transfection, Derivative Assay, Control, Negative Control, Expressing, Over Expression, Plasmid Preparation, Western Blot