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: Cancer Cell International

Article Title: Long non-coding RNA GAS5 accelerates oxidative stress in melanoma cells by rescuing EZH2-mediated CDKN1C downregulation

doi: 10.1186/s12935-020-01167-1

Figure Lengend Snippet: Overexpression of GAS5 prevents H3K27 trimethylation and upregulates CDKN1C expression to accelerate oxidative stress in melanoma by inhibiting EZH2. a Correlation analysis of lncRNA GAS5 expression and EZH2 expression. b RIP assay of the recruitment of transcription factor E2F4 by lncRNA GAS5. c ChIP assay of the enrichment of E2F4 in EZH2 promoter region. d RNA pull-down assay of the direct interaction of lncRNA GAS5 and E2F4. A375 cells were treated with oe-GAS5 alone or in the presence of oe-EZH2. e RT-qPCR assay of lncRNA GAS5 expression and mRNA expression of EZH2 and CDKN1C. f Western blot assay of the protein expression of E2F4, EZH2, H3K27me3 and CDKN1C. g Western blot assay of the protein expression of MDA5, IRE1α and SOD-1. h ELISA analysis of the content of ROS. i CCK-8 assay of cell viability. j Flow cytometric analysis of cell apoptosis. * p < 0.05, compared with the IgG group, cells transduced with oe-NC, Bio-probe NC group, cells transduced with oe-G-NC or cells transduced with oe-GAS5 + oe-E-NC. The above measurement data are expressed as mean ± standard deviation. The unpaired t -test is used for comparison of the data between two groups. Data among multiple groups are analyzed by one-way ANOVA, followed by Tukey’s post hoc test. The statistical analysis concerning time-based measurements within each group is realized using ANOVA of repeated measurements, followed by a Bonferroni’s post hoc test. Pearson correlation analysis is performed for correlation analysis

Article Snippet: The supernatants were collected and divided into three tubes, incubated overnight at 4 °C with the NC mouse antibody to IgG and target protein specific antibody, rabbit polyclonal antibody to EZH2 (ab195409, Abcam Inc., Cambridge, UK), rabbit monoclonal antibody to E2F4 (#40291, Cell Signaling Technologies, Beverly, MA, USA), rabbit monoclonal antibody to H3K27me3 (ab192985, Abcam Inc., Cambridge, UK).

Techniques: Over Expression, Expressing, Pull Down Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Transduction, Standard Deviation, Comparison

Journal: Cell reports

Article Title: Two Distinct E2F Transcriptional Modules Drive Cell Cycles and Differentiation

doi: 10.1016/j.celrep.2019.05.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-E2F4 , Millipore , Cat# MABE160; RRID: AB_10845939.

Techniques: Recombinant, Protease Inhibitor, Western Blot, Imaging, Purification, RNA HS Assay, Reverse Transcription, SYBR Green Assay, Staining, Plasmid Preparation, Software