Journal: Oncotarget

Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

doi: 10.18632/oncotarget.2587

Figure Lengend Snippet: Figure 3: Mutant p53 protein binds to BRCA1 and RAD17 gene promoters. (A) Schematic representation of BRCA1 and RAD17 promoter gene regions containing E2F4 consensus box sequences analyzed in ChIP assays. BRCA1 promoter: two regions at 2593 bp and 1633 bp upstream the first exon of BRCA1 gene. E2FA and E2FB is the region previously characterized by the Glazer’s group [43]. RAD17 promoter: two regions at 3590 bp and 1819 bp upstream the first exon of RAD17 gene. The TSS is indicated for each gene by Eponine software prediction [63].

Article Snippet: For each immunoprecipitation, 1 μg of rabbit E2F4 antibody (Santa Cruz Biotech.) and 1 μg of rabbit IgG (Santa Cruz Biotech.) as control were used.

Techniques: Mutagenesis, Software

Journal: Oncotarget

Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

doi: 10.18632/oncotarget.2587

Figure Lengend Snippet: Figure 4: Mutant p53 and E2F4 proteins bind concomitantly BRCA1 and RAD 17 promoters. (A-D) Re-ChIP assays of si-p53-MDA-MB-468 (a and b) and of si-E2F4-MDA-MB-468 (c and d) cells with their siGFP-transfected control, using the indicated antibodies. The analysis performed by qPCR was employed using specific primers for the previously indicated regions in the Figure 3A.

Article Snippet: For each immunoprecipitation, 1 μg of rabbit E2F4 antibody (Santa Cruz Biotech.) and 1 μg of rabbit IgG (Santa Cruz Biotech.) as control were used.

Techniques: Mutagenesis, Transfection, Control

Journal: Oncotarget

Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

doi: 10.18632/oncotarget.2587

Figure Lengend Snippet: Figure 4 (Continued ): (E) qRT-PCR analysis of RAD17 and BRCA1 expression of transiently transfected CAL27 cells (for 48 h with 150pmol of si-GFP and si-E2F4 oligogonucleotides) were done as biological triplicates. P-values were calculated with two tailed t-test. Statistically significant results were with p-value < 0.05. (F) The western blot analysis of 40 μg derived from protein lysates of CAL27 cells previously used in (e) was performed to evaluate the expression of the indicated proteins.

Article Snippet: For each immunoprecipitation, 1 μg of rabbit E2F4 antibody (Santa Cruz Biotech.) and 1 μg of rabbit IgG (Santa Cruz Biotech.) as control were used.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Two Tailed Test, Western Blot, Derivative Assay

Journal: Oncotarget

Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

doi: 10.18632/oncotarget.2587

Figure Lengend Snippet: Figure 5: Mutant p53 and E2F4 proteins form a protein complex in tumour cells. (A, B) H1299 cells were transfected with 2 μg of pcDNA3-p53R273H, pcDNA3HA-p53D281G (a) and pcDNA3-p53R175H (b) vectors and empty pcDNA3 as control. Immunoprecipitation of the whole cell extracts derived from these samples were performed with E2F4 antibody and preimmune rabbit serum. Cell extracts (30 μg) and immunoprecipitated samples (800 μg) were subjected to western blotting with the indicated antibodies. (C, D) Whole cell extracts (40 μg) and immunoprecipitations (800 μg) from MDA-MB-468 (c), SKBr3 and CAL27 cells respectively (d), immunoprecipitated with E2F4 and p53 antibodies were subjected to western blot analysis probed with the indicated antibodies.

Article Snippet: For each immunoprecipitation, 1 μg of rabbit E2F4 antibody (Santa Cruz Biotech.) and 1 μg of rabbit IgG (Santa Cruz Biotech.) as control were used.

Techniques: Mutagenesis, Transfection, Control, Immunoprecipitation, Derivative Assay, Western Blot

Journal: Oncotarget

Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

doi: 10.18632/oncotarget.2587

Figure Lengend Snippet: Figure 8: The depicted model proposes the molecular mechanisms underlying the transcriptional control exerted by mutp53/E2F4 repressive protein complex on BRCA1 and RAD17 gene expression. Its impact on DNA repair and tumorigenesis is also depicted.

Article Snippet: For each immunoprecipitation, 1 μg of rabbit E2F4 antibody (Santa Cruz Biotech.) and 1 μg of rabbit IgG (Santa Cruz Biotech.) as control were used.

Techniques: Control, Gene Expression

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: A FACS analysis of the percentage of GFP + (Cas9 + ) cells within the mCherry + (sgRNA + ) and mCherry - (sgRNA - ) HSPCs on day two (top) and six (bottom) post transduction. B Heatmap of survival/proliferation scores in HSPCs treated with the indicated sgRNAs ( n = 2, biological replicates). The color indicates decreased (orange) and increased (blue) survival/proliferation scores. C Western blot of TFDP1 and E2F4 three days post targeting with sgRNAs against Tfdp1 and E2f4 . SgRosa26-1 was used as a negative control. Actin was used as a loading control. D Co-immunoprecipitation (Co-IP) using TFDP1 (top) and E2F4 as precipitating antibody (bottom) ( n = 2, biological replicates).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Transduction, Western Blot, Negative Control, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: A Scheme of assessment of HSPC proliferation and apoptosis. Cas9-HSPCs were labeled with Celltrace, cultured for one day and transduced with lentiviral particles expressing sgRNAs targeting Tfdp1 , E2f4 , or the Rosa26 . B Representative FACS analysis of the percentages of Annexin V + DAPI - (early) and Annexin V + DAPI + (late) apoptotic cells within mCherry - (sgRNA - , upper panel) or mCherry + (sgRNA + , lower panel) HSPCs treated with the indicated sgRNAs. C Representative FACS analysis of active Caspase 3 + apoptotic HSPCs treated with the indicated sgRNAs three days post puromycin selection (top) and summary of the data (bottom) based on HSPCs from three mice ( n = 3). D Representative FACS analysis of the proliferation rates of mCherry - (sgRNA-) and mCherry + (sgRNA + ) HSPCs infected with the indicated sgRNAs two and four days post cell-trace labeling. The number of cell divisions is indicated. E Percentage of cell division in mCherry + (upper) and mCherry - (below) HSPC subpopulations treated with the indicated sgRNAs on day two and day four post cell-trace labeling ( n = 3 independent experiments).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Labeling, Cell Culture, Transduction, Expressing, Selection, Infection

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: A Venn diagrams depicting the overlap between the differentially expressed genes in Tfdp1 -KO HSPCs (downregulated genes in blue and upregulated genes in red) and human TFDP1- (left; GSE80661; GSE105217; GSE127368), human E2F4- (middle; GSE31477; GSE170651), and mouse E2F4- (right; GSE48666) bound target genes. B Intersection between human TFDP1- and E2F4-bound genes downregulated in Tfdp1 -KO HSPCs. C Density plots (upper panel) and heatmaps (lower panel) depicting the average tag densities around TSSs (−2/+2 kb) of up- and downregulated genes in Tfdp1 KO HSPCs. Data are derived from the ChIP-seq of RNA polymerase II S5P (RnapolII S5P; GSE34518), H3K4Me3 (GSE75426), and E2F4 (GSE48666) (together with a negative control) in mouse ES cells. Right panel: ATAC-seq signals (GSE100738) from mouse short-term (ST) HSCs in the same genomic regions. D Example of RnapolII S5P, H3K4Me3, E2F4 tracks in mouse ES cells and ATAC-seq in ST-HSCs at the mouse Cdk1 locus. E Example of E2F4 and TFDP1 ChIP-seq signals at the CDK1 locus in various human cell types. Mouse and human E2F4 and TFDP1 DNA binding sites derived from the Unibind database are shown and the core nucleotides involved in DNA binding are highlighted.

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Derivative Assay, ChIP-sequencing, Negative Control, Binding Assay