Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Signature of chemokine ligands and (B) chemokine receptors in human ovarian cancer cell lines. After isolating total RNA from each cell line, PCR array was performed using a customized PCR array plate containing complementary sequences for human chemokine genes. Different colors indicate average cycle threshold with expression ranges from >35 to <25. (C) Protein expression of p53 and Mdm2 in ovarian cancer cell lines. Whole cell lysates were prepared and Western blot was carried out using antibodies specific to p53, Mdm2 and β-actin as loading control. Experiments were performed in duplicate and a representative result is shown. OV, OVCAR-3 cells; SK, SKOV-3 cells; A, A2780 cells; Ca, CaOV-3 cells; TOV, TOV-21G cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) TNF-induced chemokines in SKOV-3 cells. After isolating total RNA, PCR array was performed using a human chemokine PCR array plate. Dotted line indicates 2-fold increase; chemokines with a greater than 2-fold increase are recognized as TNF-induced chemokines. (B) Confirmation of p53 protein expression after transient transfection in SKOV-3 cells. After transfection of empty vector (EM) and p53 expression vector (p53), whole cell lysates were prepared and p53 expression was confirmed by Western blot. β-actin is used as a loading control. (C) Effect of p53 on TNF-induced chemokines. After overnight transfection of vectors, cells were treated with TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Different letters indicate significant differences (P≤0.05) within each chemokine group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Nucleotide sequences of promoters for TNF-induced chemokines such as CCL20, CXCL1, 2, 3 and 8. These chemokine promoters contain one NF-κB site at the proximal region, except for CCL20, which has two NF-κB sites at the distal and proximal region. (B) Effect of p53 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p65, cells were treated with TNF (10 ng/ml) for 4 h. (C) Effect of p53 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 (p53 null), OVCAR-3 (p53 mutant) and A2780 (p53 wild-type), cells were treated with TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown; numbers below are relative density values.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Accumulated effect of p53 on ubiquitylated proteins. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to ubiquitin, p21, IκB, p53 and β-actin (as loading control). Experiments were performed in duplicate and a representative result is shown. (B) Confirmation of p53 activity after transient transfection of p53. ELISA was performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (C) The effect of p53 on proteasome activity. Assays were performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (D) Effects of p53 on ubiquitination of IκB. Immunoprecipitated IκB was immunoblotted using ubiquitin antibody. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Effect of p53 on ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) obtained from comparison between empty vector and p53 vector transfected ovarian cancer cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Ubiquitin Proteomics, Comparison, Plasmid Preparation, Transfection

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of p53 on Mdm2 expression. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to Mdm2; and β-actin served as loading control. (B) Effects of p53 expression on p53 binding to p65 and IκB. After transient transfection of p53, immunoprecipitated (IP) p53 was immunoblotted (IB) using p65 or IκB antibody. (C) Effect of p53 on expression of various IKK isoforms. After transient transfection of p53, whole cell lysates were prepared and Western blot was carried out using antibodies specific to IKKα, IKKβ, IKKγ, IKKε; β-actin served as loading control. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Western Blot, Control, Binding Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of nutlin-3 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p53, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 4 h. Different letters indicate significant differences (P≤0.05) within each group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE. (B) Effect of nutlin-3 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 cells, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown. (C) Effect of nutlin-3 on TNF-induced chemokines. After overnight transfection of vectors, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Asterisk indicates significant differences (P≤0.05, paired Student's t -test) when compared to the presence of nutlin-3. Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Chronic inflammation promotes ovarian cancer progression via NF-κB signaling. Wild-type p53 reduces activity of the ubiquitin-proteasome system, resulting in low IκB degradation (blue line). This reduces NF-κB activity, inhibiting proinflammatory chemokine expression and attenuating the proinflammatory tumor microenvironment (blue arrow). On the other hand, p53 increases Mdm2 expression (dark arrow) in a feedback loop to compensate for the reduced activity of the ubiquitin-proteasome system. Loss of p53 observed frequently in advanced ovarian cancer triggers high proinflammatory chemokines by increasing NF-κB signaling which is composed of IκB and p65/p50 followed by a high IκB degradation (red arrow). Enhanced NF-κB activity results in potentiation of the proinflammatory tumor microenvironment for ovarian cancer progression such as peritoneal tumor dissemination and massive ascites. The imbalance between p53 and Mdm2 also contributes to increasing NF-κB signaling via the ubiquitin-proteasome system.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Ubiquitin Proteomics, Expressing

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Loss of p53, rather than beta-catenin overexpression, induces survivin-mediated resistance to apoptosis in an esophageal cancer cell line.

doi: 10.1016/j.jtcvs.2009.11.038

Figure Lengend Snippet: FIGURE 4. TE7 cells exhibit a loss of p53 with resulting increased survi- vin transcription. A, Representative immunoblots of p53 protein expression in nhEso and TE7 cells. B, Schematic representation of the survivin pro- moter-luciferase reporter construct that included 2 TCF-b-catenin binding

Article Snippet: Co-transfection assays were carried out in nhEso and TE7 cells using 0.3 mg of survivin luciferase reporter gene plasmid, 0.3 mg of either an empty expression vector or an overexpression vector encoding the tumor protein p53 (OriGene Technologies Inc, Rockville, Md), in 0.7 mL Lipofectamine reagent.

Techniques: Western Blot, Expressing, Luciferase, Construct, Binding Assay

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Loss of p53, rather than beta-catenin overexpression, induces survivin-mediated resistance to apoptosis in an esophageal cancer cell line.

doi: 10.1016/j.jtcvs.2009.11.038

Figure Lengend Snippet: FIGURE 5. Effect of p53 overexpression on survivin promoter activity and survivin mRNA transcription in nhEso and TE7 cells. A, Reporter gene activity in nhEso and TE7 cells after overexpression of p53. Cells were co-transfected with the survivin reporter construct and the expression vector containing human p53 cDNA (p53) or control vector lacking p53 (control) using Lipofectamine (Invitrogen, Carlsbad, Calif). Twenty-four

Article Snippet: Co-transfection assays were carried out in nhEso and TE7 cells using 0.3 mg of survivin luciferase reporter gene plasmid, 0.3 mg of either an empty expression vector or an overexpression vector encoding the tumor protein p53 (OriGene Technologies Inc, Rockville, Md), in 0.7 mL Lipofectamine reagent.

Techniques: Over Expression, Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: RASSF10 is Epigenetically Inactivated and Suppresses Cell Proliferation and Induces Cell Apoptosis by Activating the p53 Signalling Pathway in Papillary Thyroid Carcinoma Cancer.

doi: 10.1159/000464386

Figure Lengend Snippet: Fig. 5. RASSF10 and p53 regulated PTC cell apoptosis. Apoptosis was determined using flow cytometry analysis of Annexin V/PI double-stained K1 cells. *P < 0.05 versus control group, #P < 0.05 versus RASSF10 group.

Article Snippet: Expression vector construction and transfection The open reading frame (ORF) of the RASSF10 gene was generated by RT-PCR, and p53 siRNA and shRASSF10 were purchased from OriGene Technologies (Rockville, USA).

Techniques: Flow Cytometry, Staining, Control

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Targeting strategy. The wildtype (WT) Trp53 gene is within a 17-kb-long EcoRI (RI) fragment (black boxes are for coding sequences and white boxes for UTRs). The targeting construct contains: (1) a 1.5-kb-long 5’ homology region; (2) a Lox-Stop-Lox (LSL) cassette with a neomycin selection gene (Neo), four transcriptional stops (STOP) and an EcoRI site, flanked by LoxP sites (arrowheads); (3) p53 coding exons, including the Y217C (YC) missense mutation in exon 6 (asterisk) and an additional BanII site; (4) a 2.8-kb-long 3’ homology region; and (5) the diphteria α-toxin (DTA) gene for targeting enrichment. Proper recombinants with a Trp53 LSL-Y217C allele, resulting from the described crossing-overs, were G418 resistant. They were identified by a 2.4-kb-long band after PCR with primers a and b, and confirmed by bands of 635 and 224 bp after PCR with primers c and d and BanII digestion. They were also verified by Southern blot with the indicated probe as containing a 10.5 kb EcoRI band. Two recombinant ES clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping with primers c and d and BanII digestion. Excision of the LSL cassette was performed in vivo, by breeding Trp53 +/LSL-Y217C male mice with females carrying the PGK- Cre transgene, to obtain mice with a Trp53 Y217C allele. ( B–D ) Screening of recombinant ES clones (+) by PCR with primers a and b ( B ); PCR with primers c and d then BanII digestion ( C ); Southern blot ( D ). ( E ) Genotyping of mouse embryonic fibroblasts (MEFs) from an intercross of Trp53 +/Y217C mice, by PCR with primers c and d and BanII digestion. ( F ) Trp53 Y217C sequence around codon 217. The introduced Y217C missense mutation and the silent mutation creating an additional BanII restriction site are highlighted (asterisks). ( G ) WT and Trp53 Y217C/Y217C (YC/YC) MEFs express similar p53 mRNA levels. Total RNA was extracted, then p53 mRNAs were quantified by real-time qPCR, normalized to control mRNAs and the amount in WT cells was assigned the value of 1. Means + SEM (n=3) are shown. Primer sequences are listed in . Figure 1—source data 1. Labeled files for gels and blots in . Figure 1—source data 2. Raw and unedited gels and blots for .

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Construct, Selection, Mutagenesis, Southern Blot, Recombinant, Clone Assay, Injection, Transmission Assay, In Vivo, Sequencing, Control, Labeling

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: Portions of the DNA-binding domains from the mouse (residues 208–228) and human (residues 211–231) p53 proteins are shown, with identical residues in bold, and mouse Tyrosine 217 and human Tyrosine 220 in red.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Binding Assay

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Increased p53 protein levels in Trp53 YC/YC and Trp53 +/YC mouse embryonic fibroblasts (MEFs). MEFs of the indicated genotypes were treated or not with 10 μM Nutlin 3a for 24 hr, then protein extracts were immunoblotted with antibodies against Mdm2, p53, p21, and actin. ( B ) The transactivation of classical p53 target genes Cdkn1a and Mdm2 is impaired in Trp53 YC/YC cells. Wildtype (WT), Trp53 YC/YC , and Trp53 -/- MEFs were treated as in ( A ), then (top) mRNAs were quantified in five to six independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed WT cells assigned a value of 1; or (bottom) ChIP assays were performed at the Cdkn1a and Mdm2 promoters in two to three independent experiments with an antibody against p53 or rabbit IgG as a negative control. Immunoprecipitates were quantified using real-time PCR, normalized to data over an irrelevant region, and the amount in unstressed WT cells was assigned a value of 1. Error bars: SEM. ( C ) Assessment of p53 WT and p53 Y217C subcellular localization by cellular fractionation. WT and Trp53 YC/YC MEFs were treated or not with 1 μΜ doxorubicin (Doxo) for 24 hr, submitted to cellular fractionation, then protein extracts were immunoblotted with antibodies against p53 or the fraction controls Tubulin for cytoplasm (Cp.), Nup98 for nucleoplasm (Np.), and histone H3 for chromatin (χin). ( D ) Assessment of p53 WT and p53 Y217C subcellular localization by immunofluorescence. WT, Trp53 YC/YC and Trp53 -/- MEFs were treated with 10 μM Nutlin 3a for 24 hr, then stained with antibodies against p53 (red) or actin (green) and DNA was counterstained with DAPI (blue). ( E ) Absence of a cell cycle arrest response in Trp53 YC/YC MEFs. Asynchronous cell populations of Trp53 +/+ , Trp53 YC/YC , and Trp53 -/- MEFs were analyzed 24 hr after 0, 3, or 12 Gy γ-irradiation. Means + SEM from three independent experiments. ( F ) Absence of a p53-dependent apoptotic response in Trp53 YC/YC thymocytes. Age-matched mice of the indicated genotypes were left untreated or submitted to 10 Gy whole-body γ-irradiation then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS. Means + SEM from two independent experiments. ( G ) Increased chromosomal instability in Trp53 YC/YC fibroblasts. Metaphase spreads were prepared from WT, Trp53 YC/YC , and Trp53 -/- MEFs at passage 4, then aberrant metaphases (with chromosome breaks, radial chromosomes, or double-minute chromosome [DMs]) were scored. Left: distribution of aberrant metaphases. Data from 110 WT, 97 Trp53 YC/YC , or 119 Trp53 -/- complete diploid metaphases, independently observed by two experimenters. Right: examples of two aberrant Trp53 YC/YC metaphases: one with a DM, a chromosome break (Br) and a radial chromosome (R), the other with multiple DMs. Enlargements of regions of interest are presented between the two metaphases. Scale bars ( D, G ): 5 μm. ***p<0.001, **p<0.01, *p<0.05, °p=0.09, ns: non-significant by Student’s t ( B, E, F ) or Fisher’s ( G ) tests. Figure 2—source data 1. Labeled files for gels and blots in . Figure 2—source data 2. Raw and unedited gels and blots for .

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Real-time Polymerase Chain Reaction, Control, Negative Control, Cell Fractionation, Immunofluorescence, Staining, Irradiation, Labeling

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Transactivation of the classical p53 target genes Cdkn1a (alias p21 ), Mdm2, and Pmaip1 (alias Noxa ) in response to Nutlin or Doxorubicin. WT, Trp53 +/YC , and Trp53 +/- mouse embryonic fibroblasts (MEFs) were treated or not with 10 μM Nutlin 3a (Nut) or 1 μM Doxorubicin (Doxo) for 24 hr, then mRNAs were quantified in ≥4 independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed WT cells assigned a value of 1. Means + SEM are shown. For each condition and gene, a dominant-negative effect (DNE) would lead to significant decrease in transactivation in Trp53 +/YC MEFs compared to both WT and Trp53 +/- MEFs, a result that was not observed. ( B ) Cell cycle arrest responses to γ-irradiation. Asynchronous cell populations of WT, Trp53 +/YC , and Trp53 +/- MEFs were analyzed 24 hr after 0, 3, or 12 Gy γ-irradiation. Means + SEM from three independent experiments. The comparison of cells submitted to identical irradiation doses revealed similar arrest responses in cells of all genotypes. ( C ) Apoptotic responses to γ-irradiation. WT, Trp53 +/YC , and Trp53 +/- MEFs age-matched mice were left untreated or submitted to 10 Gy whole-body γ-irradiation, then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS. Means + SEM from two independent experiments. The percentage of apoptotic cells was significantly higher in irradiated WT thymocytes compared to irradiated Trp53 +/YC or Trp53 +/- cells, whereas Trp53 +/YC and Trp53 +/- cells were not significantly different. **p<0.01, *p<0.05, ns: non-significant by Student’s t-test.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Real-time Polymerase Chain Reaction, Control, Dominant Negative Mutation, Irradiation, Comparison, Staining

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Distribution of weaned mice obtained from Trp53 +/- or Trp53 +/YC intercrosses. Obs: observed numbers of mice at weaning (P21); exp: expected numbers assuming a Mendelian distribution without sex distortion; f/m: observed female/male ratios. Consistent with previous reports, the observed distribution of weaned mice from Trp53 +/- intercrosses did not conform to values expected for a Mendelian distribution without sex distortion (U=5; χ 2 =16.31>15.09), indicating a significant deficit in female Trp53 -/- mice (top). The distribution of weaned mice from Trp53 +/YC intercrosses diverged even more from values for a Mendelian distribution without sex distortion (U=5; χ 2 =104.23>15.09), due to a striking deficit in female Trp53 YC/YC mice (bottom). Differences between the frequencies of Trp53 YC/YC (4/677) and Trp53 -/- (8/196) females in the progeny, or between the female to male ratios for Trp53 YC/YC (4/75) and Trp53 -/- (8/28) animals, are statistically significant (p=0.0012 and p=0.0087 in Fisher’s tests, respectively). ( B ) Exencephaly is frequently observed in p53 YC/YC female embryos. Top: the distribution of E12.5–16.5 embryos from heterozygous ( Trp53 +/YC ) intercrosses or heterozygous-homozygous ( Trp53 +/YC × Trp53 YC/YC ) crosses is shown. f or m exenc.: number of female or male embryos with exencephaly; o.a.: embryos with other abnormalities. Below, examples of female Trp53 YC/YC embryos at E12.5, E13.5, and E16.5 exhibiting exencephaly (arrows) are each shown (center) together with a normal embryo from the same litter (bottom). Scale bars : 1 mm. ( C ) Distribution of mice at birth from the indicated crosses. Of note, out of five Trp53 YC/YC females observed at birth, only one remained alive at weaning age. Thus, the female/male ratio for weaned Trp53 YC/YC animals from these crosses was 1/22, a ratio similar to the one observed in A (4/75).

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques:

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A–B ) In homozygous males, p53 Y217C leads to accelerated tumor-induced death ( A ), and aggressive metastatic tumors ( B ); n=cohort size. ( C ) Hematoxylin and eosin (H&E) staining of sections from the lung (top) and spleen (bottom) of Trp53 -/- and Trp53 YC/YC male mice, showing metastases in Trp53 YC/YC animals. Normal organ structures are shown, with ‘A’ indicating pulmonary alveoli, and ‘WP’ and ‘RP’ standing for splenic white and red pulp, respectively. In the lung section of the Trp53 YC/YC mouse, the rectangle indicates a lymphoma area. In the spleen section of the Trp53 YC/YC mouse, the typical splenic structures are absent due to massive tissue homogenization of the spleen by lymphoma cells.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Staining, Tissue Homogenization

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Heat-map plot, with 717 differentially expressed genes suggestive of a loss of function (LOF), a separation of function (SOF), or a gain of function (GOF) for the p53 Y217C mutant, ranked according to log 2 fold change (n=number of genes). ( B ) Evidence of LOF in Trp53 YC/YC cells for genes encoding Puma, p21, and Zmat3. Data from three mice per genotype. Means + SEM are shown. ***p<0.001, **p<0.01, *p<0.05, °p=0.07, ns: non-significant by Student’s t-tests.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Mutagenesis

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: Effects of the Trp53 Y217C mutation: a summary. The comparison between Trp53 -/- and Trp53 Y217C/Y217C mice is presented. The phenotypes observed in Trp53 -/- male (M) and female (F) mice result from p53 loss of function (LOF), whereas those observed in Trp53 Y217C/Y217C mice result from p53 LOF as well as additional effects (gain of function [GOF] in bold). The + signs denote the presence of a phenotype. Xi: X chromosome inactivation.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Mutagenesis, Comparison

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet:

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Cell Culture, Sequencing, Recombinant, SYBR Green Assay, Staining, Software

Journal: Journal of Clinical Laboratory Analysis

Article Title: High‐dimensional single‐cell proteomics analysis reveals the landscape of immune cells and stem‐like cells in renal tumors

doi: 10.1002/jcla.23155

Figure Lengend Snippet: Purified antibodies about the stem‐like cell‐centric panel

Article Snippet: p53 , 143Nd , 7F5 , Fluidigm , 3143018A.

Techniques: Purification

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: Western blot analyses of lamin A/C and progerin, full-length p53, Δ133p53α, and p21 Waf1/Cip1 were performed in four 15-week-old Δ133p53α-expressing Group-1 mice ( CAG-133 Tam/+ ; Cre Tg/+ ; Lmna G609G/+ ), along with four each of age-matched, non-expressing control Group-2 ( CAG-133 LSL/+ ; Cre Tg/+ ; Lmna G609G/+ ) and Group-4 mice ( CAG-133 +/+ ; Cre +/+ ; Lmna G609G/+ ), as well as two age-matched wild-type mice ( CAG-133 +/+ ; Cre +/+ ; Lmna +/+ ). Results from skin ( a ), skeletal muscle ( b ), kidney ( c ), spleen ( d ), and lung ( e ) are presented. An inter-blot control (liver from a Group-1 mouse) was included in all blots. F, female; M, male. GAPDH was a loading control and used for normalization of p21 Waf1/Cip1 , progerin, and full-length p53 expression levels. Quantitative data summaries of p21 Waf1/Cip1 , progerin, and full-length p53 in Group-1, -2 and -4 mice are shown as relative values to Group-2 mice (mean ± s.d. from n = 4; open circles indicate two females, and closed circles indicate two males). P values were determined by Welch’s t -test. Two wild-type mice were used only as references and not for statistical comparisons.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: a,b , Top Hallmark pathways identified by Gene set enrichment analysis (GSEA). The bulk RNA-seq data were obtained from the heart ( a ) and kidney ( b ) in 9-10-month-old Group-1 and Group-3 mice (n = 5 each). Pathways are ranked by normalized enrichment score. False discovery rate < 0.10. c-g, Enrichment plots illustrate significant downregulation of the p53 pathway ( c,d ) and upregulation of the oxidative phosphorylation ( e,f ) in both heart and kidney, as well as upregulation of the glycolysis in the kidney ( g ). Enrichment plots depict running enrichment scores (ES) and the distribution of genes within each Hallmark gene set. All leading edge genes in each pathway are listed in Extended Data Table 2. h,i, qRT-PCR assays of mRNA expression of genes in the oxidative phosphorylation pathway (Ndufs6, Ndufc2, Uqcrq, Hsd17b10, and Gpx4) and an antioxidant gene Prdx1 in the heart ( h ) and kidney ( i ) of 9-10-month-old Group-1 and Group-3 mice (mean ± s.d. from n = 5, each with technical triplicate; open circles, females; closed circles, males). P values were calculated by Welch’s t -test.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: RNA Sequencing, Phospho-proteomics, Quantitative RT-PCR, Expressing

Journal: Journal of Bioscience and Applied Research

Article Title: 8-hydroxy-2'-deoxyguanosine and TP53 in Egyptian Patients with Hepatitis C Viral Chronic Liver Diseases: Insight into the Pathogenesis and Predictive Force

doi: 10.21608/jbaar.2022.223518

Figure Lengend Snippet: Figure 2 Receiver operator characteristic (ROC) curve of 8-OHdG (A) and TP53 (B) for discriminating HCC

Article Snippet: Assessment of Human TP53 (Tumor protein p53) Serum TP53 concentrations were quantitatively measured by ELISA kit using the Sandwich-ELISA principle (Elabscience Biotechnology Inc., USA).

Techniques:

Journal: Cell Death Discovery

Article Title: Transcriptomic profiling reveals p53 as a key regulator of doxorubicin-induced cardiotoxicity

doi: 10.1038/s41420-019-0182-6

Figure Lengend Snippet: a The transcription factor encoded by TP53 increases or decreases the transcription of genes involved in apoptotic ( TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, FAS, PMAIP1, BBC3, BAX ) and cell cycle progression pathways ( CDK1, GADD45A, CDKN1A ), respectively. Genes in gray depict overlap with Reyes et al., genes depicted in gray with red outline indicate diffirent isoforms of genes identified in Reyes et al.; Yellow = increased expression, Blue = decreased expression. b Damage caused by interaction of doxorubicin into the DNA activated p53, which then increases transcription of DRs and stalls cell cycle progression. If the damage is deemed irreparable by DNA repair mechanisms, apoptosis is triggered

Article Snippet: A 1:2000 dilution of anti-p53 antibody (R&D Systems, Minneapolis, MN, cat. no: HAF1355) was used and a 1:1000 of each of the following antibodies was used: anti-phosphorylated-S15-p53 (Cell Signaling Technology, Danvers, MA, cat. no: 9286); anti-DR4 (Cell Signaling Technology, Danvers, MA, cat. no: 42533); anti-DR5 (Cell Signaling Technology, Danvers, MA, cat. no: 8074); anti-Fas (Santa Cruz Biotechnology, Dallas, TX, cat. no: sc-715); anti-TNFR1 (Cell Signaling Technology, Danvers, MA, cat. no: 3736).

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: Transcriptomic profiling reveals p53 as a key regulator of doxorubicin-induced cardiotoxicity

doi: 10.1038/s41420-019-0182-6

Figure Lengend Snippet: a Immunoblot and b densitometric quantification demonstrate after exposing cardiomyocytes to 150 nM or 500 nM of doxorubicin for 48 h the phosphorylation of p53 and total expression of p53, TNFR1, DR4, DR5, and Fas increase. TNFR1 protein expression increased only after exposure to 500 nM of doxorubicin. The protein expression of p53 targets, DR4 and DR5, decrease after a 7-day washout period. Fas remains slightly upregulated after 7-day washout

Article Snippet: A 1:2000 dilution of anti-p53 antibody (R&D Systems, Minneapolis, MN, cat. no: HAF1355) was used and a 1:1000 of each of the following antibodies was used: anti-phosphorylated-S15-p53 (Cell Signaling Technology, Danvers, MA, cat. no: 9286); anti-DR4 (Cell Signaling Technology, Danvers, MA, cat. no: 42533); anti-DR5 (Cell Signaling Technology, Danvers, MA, cat. no: 8074); anti-Fas (Santa Cruz Biotechnology, Dallas, TX, cat. no: sc-715); anti-TNFR1 (Cell Signaling Technology, Danvers, MA, cat. no: 3736).

Techniques: Western Blot, Phospho-proteomics, Expressing

Journal: Cell Death Discovery

Article Title: Transcriptomic profiling reveals p53 as a key regulator of doxorubicin-induced cardiotoxicity

doi: 10.1038/s41420-019-0182-6

Figure Lengend Snippet: Doxorubicin, a DNA intercalating agent, induces DNA strand breaks and triggers the activation of p53 to initiate DNA damage responses. Activation of p53 induces increased transcription of genes involved in the extrinsic and intrinsic apoptotic pathway. In the presence of circulating ligands, the extrinsic apoptotic pathway is activated

Article Snippet: A 1:2000 dilution of anti-p53 antibody (R&D Systems, Minneapolis, MN, cat. no: HAF1355) was used and a 1:1000 of each of the following antibodies was used: anti-phosphorylated-S15-p53 (Cell Signaling Technology, Danvers, MA, cat. no: 9286); anti-DR4 (Cell Signaling Technology, Danvers, MA, cat. no: 42533); anti-DR5 (Cell Signaling Technology, Danvers, MA, cat. no: 8074); anti-Fas (Santa Cruz Biotechnology, Dallas, TX, cat. no: sc-715); anti-TNFR1 (Cell Signaling Technology, Danvers, MA, cat. no: 3736).

Techniques: Activation Assay

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 1: Characterization of WT, null, and mutated P53 CRC cell lines response to DNA damaging agents. A. HCT116 P53+/+ cells were treated with dose-increase of Dox for 24hr and checked for PARP cleavage, also activation and stabilization of P53 were analyzed by western blotting. B. Comparison of PARP cleavage upon Dox 1μM between HCT116 P53+/+ and HCT116 P53-/-. C. Assessment of PARP cleavage in SW480 and HT-29cells lines upon a dose-increase of Dox. D. HCT116 P53+/+, HCT116 P53-/-, and HT- 29 cells were treated with 1μM Dox and proteins cell lysate were separated using SDS-PAGE and detected by WB analysis. Quantification of PARP cleavage detected by western blotting was performed by ImageJ software and based on the ratios of normalized cleaved PARP by βactin and normalized uncleaved PARP. (Fold changes are the average of three independent of three independent experiments (n=3)). E. HCT116 P53+/+ cells, HCT116 P53-/- and HT-29 cells were treated with chemotherapeutic drugs for 24hr. Cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. The cleavage of PARP was assessed using the appropriate antibodies. F. HT-29 cell death investigated by propidium iodide (PI) staining and flow cytometry analysis (mean ± S.E.M. of three independent experiments (n=3)), the cell death following treatments with (5-FU and Oxa) were not significant in comparison with the control (P=0.55), and this supports the results western blot for these treatments (no PARPc). In all the western blotting experiments βactin was used as a loading control.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: Activation Assay, Western Blot, Comparison, SDS Page, Software, Staining, Flow Cytometry, Control

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 2: Combined treatment with distinct HDAC inhibitors and DNA damaging agents induces different levels of sensitivity in CRC cells. A. HCT116 p53+/+, HCT116 p53-/- cells were treated with a single treatment of Dox (0.5μM) and Camptothecin-11 (CPT-11) (5μM) or combined treatment with VPA (1 mM), SAHA (0.5μM), and sodium butyrate (NaB) (1 mM) for 24 h and then cells were prepared for the cell death analysis by flow cytometry using Propidium iodide (PI) staining. We calculated the combination index (CI) according to the Chou-Talalay method using Calculsyn software (Biosoft, Cambridge, UK). Chou-Talalay method for drug combination was based on the median-effect to defined synergy and antagonism [38]. This method offered synergistic (CI < 1), and antagonistic effect (CI > 1) in drug combinations. Error bars represent ± S.E.M of three independent experiments (n=3). B. HT-29 cell death analysis by flow cytometry after 24 hours treatment with (Dox, CPT, 5FU), SAHA or their combination. Cells were harvested and stained with Propidium iodide (PI) to determine cell death using FACS analysis. * denotes a synergistic effect and X denotes an antagonistic effect. Error bars represent ± S.E.M of three independent experiments (n=3).

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: Flow Cytometry, Staining, Software

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 3: Early effects of Dox combined to SAHA or VPA on CRC cell lines. A and B. HCT116 and HT-29 cells were treated for 24 hr treatment with dose-increase of Dox, Oxa or 5-FU. Cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. The cleavage of PARP (PARPc) and the protein level of HDAC2 were assessed and quantified using the appropriate antibodies. Actin was used as a loading control. C. The four cell lines (HCT116 p53+/+, HCT116 p53-/-, SW480, and HT-29) were treated for 6 hours with Dox, VPA or SAHA only or as a combined treatment. Cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. The PARPc and the protein level of HDAC2 were assessed using the appropriate antibodies. Actin was used as a loading control. D. Four cell lines were treated with Dox either singly or combined with SAHA or VPA for 6 hours and cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. HDAC2 protein levels were assessed using the appropriate antibodies and quantified using ImageJ software. E. Total RNA was extracted from HCT116 p53+/+ and HT-29 cells and HDAC2 mRNA expression level was measured by quantitative RT-PCR using the primer: forward primer (5’-3’) GT GAG ATT CCC AAT GAG TTG C. reverse primer (5’-3’) GGT AAC ATG CGC AAA TTT TCA A. Error bars represent ± S.E.M.; n=3 independent experiments. Test, t-test, * for p<.005, ** for p<.001, and *** for p<.0001.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: SDS Page, Control, Software, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 4: Characterisation of HDAC2 expression levels in distinct CRC cell lines and its relationship with P53 and resistance to HDACis combined with DNA damaging agent doxorubicin. A. HCT116 p53+/+, null p53, SW480 and HT-29 cells were treated with Dox either singly or combined with SAHA or VPA for 24 hours. Cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. The cleavage of PARP, a hallmark of apoptosis and the protein level of HDAC1 and HDAC2 were assessed using the appropriate antibodies. Actin was used as a loading control. B. Four cell lines were treated with Dox either singly or combined with SAHA or VPA for 24 hours and cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. HDAC2 protein levels were assessed using the appropriate antibodies and quantified using ImageJ software. C and D. P53 expression vectors were transiently transfected into HT-29 cells and HT29-ShRNA-HDAC2 cells, The cells were treated with Oxa (10μM) for 24 hours. Cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. The PARPc and the protein level of p53 were assessed using the appropriate antibodies. Actin was used as a loading control. D) P53 transfected cells were also analyzed for cell death using PI staining and FACS analysis. Cells treated with SAHA+ Oxa or illuminated by UV-C (50 mJ/m2) were used as positive controls. Error bars represent ± S.E.M. of three independent experiments (n=3) and statistical significance is depicted by (*) for p<0.05 calculated by a two-tailed Student’s T test compared to the control group.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: Expressing, SDS Page, Control, Software, Transfection, shRNA, Staining, Two Tailed Test

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 5: TP53 mutations (S15A, K120R, K373R and K381R) sensitise HT-29 to Dox treatment. A. Overexpression of mutations (S15A, K120R, K305R K320R, K372R K373R, and K381Rand K382R) in null P53 HCT-116 cells. All p53 mutants’ cells were treated with 0.5μM Dox and cleavage of PARP was detected by WB. B. Cell death analysis by flow cytometry. Stable clone HCT116 p53-/- cells were treated with 0.5μM Dox for 24 hours and cell death was assayed by incubating live cells with propidium iodide. C. Stable HT-29 cell line was generated from selected mutants (S15A, K120R, K373R and K381R). Cell death analysis was assessed by FACS after 0.5 μM Dox treatment for 24 hours and propidium iodide staining. Error bars represent ± S.E.M.; n=3 independent experiments. Test, t-test, * for p<.005, ** for p<.001.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: Over Expression, Flow Cytometry, Stable Transfection, Generated, Staining

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 6: Modulation of HDAC2 expression level by depletion or overexpression directly influences the effect of Dox as single or combined to HDACis in CRC cells. A. Stable ShRNA-HDAC2 HCT116 p53-/- cells were generated by using lentiviral vector and HDAC2 downregulation was confirmed by WB. Actin was used a loading control. Cells were exposed for 24hr to Dox as single treatment or combined with VPA. Protein cell lysates were separated by SDS-PAGE and detected by WB analysis. The PARPc protein level was assessed using the appropriate antibodies. Actin was used as a loading control. Cells were also harvested and stained by PI to determine cell death using FACS analysis. Error bars represent ± S.E.M. of three independent experiments (n=3) and statistical significance is depicted by * for p<0.05 calculated by a two-tailed Student’s T test compared to the control group. B and C. HCT16 p53+/+ and SW480 cell lines were transfected with HDAC2 vector. After 24 hours post-transfection, cells were lysed and the proteins separated using SDS-PAGE. The overexpression of HDAC2 level was detected by WB and quantified using ImageJ densitometry software. HCT16 p53+/+ and SW480 cells transiently transfected with HDAC2 vector were treated with Dox only or combined with VPA or SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. The PARPc was analyzed by WB and cell death was quantified by FACS after PI staining. Error bars represent ± S.E.M. of three independent experiments (n=3) and statistical significance is depicted by * for p<0.05 calculated by a two-tailed Student’s T test compared to the control group.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: Expressing, Over Expression, shRNA, Generated, Plasmid Preparation, Control, SDS Page, Staining, Two Tailed Test, Transfection, Software

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 8: In vivo imaging and validation of the effect of combined treatment by liposome-encapsulated SAHA/Dox in xenograft mice. A-B. HCT116 p53+/+ or HCT116 p53-/- expressing sable luciferase-reporter were transplanted intraperitoneally in male BALB/c nude/nude mice. Liposomal SAHA/Dox-was used to treat HCT116 p53+/+ (n=4) or HCT116 p53-/- (n=4) xenograft mice groups compared to each control groups (n=4). Tumor size manually measured with calipers every three days and bioluminescence imaging measurement every week. Error bars indicate ± SEM (n = 4 replicates). *P <0.005; two-tailed t-test, Imaging was performed by using LICOR. Quantitation of luciferase intensity (error bars indicate ± SEM; n = 4 replicates). P < 0.005, two-tailed t-test). C. Protein levels of HDAC2 and survivin in control and treated tumour xenografts of HCT116 P53+/+ were analyzed by immunohistochemistry (HDAC2 expression was found in the nuclei of all control cells and in <50% of treated tumour cells). D. Protein levels of HDAC2, survivin, and P53 in normal human liver tissue and liver metastasis of CRC. All antigens were strongly expressed in CRC in contrast to absent or significantly less abundant in normal liver cells. Imaging was performed by using LICOR. (Right) Quantitation of luciferase intensity (error bars indicate ± SEM; P < 0.005, two-tailed t test, n = 4 replicates).

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: In Vivo Imaging, Biomarker Discovery, Expressing, Luciferase, Control, Imaging, Two Tailed Test, Quantitation Assay, Immunohistochemistry

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 10: Dox combined with SAHA or VPA triggers induces histone hypo-acetylation. A. Histones were extracted by acid from the four cell lines (HCT 116 p53+/+, HCT116 p53-/- SW480 and HT-29) after exposure to Dox, VPA, SAHA or different combinations of these drugs for 24hours, also the cells were treated with 1μM. Extracted histones were separated using SDS-PAGE and detected by WB analysis. Histone acetylation levels at residues H3K9 and H4 (K12 and K16) were assessed using the appropriate antibodies. Total H3 and H4 protein levels were used as loading control. B. Quantifications of acetylated H3K9, H4K16, H4K12 residues in HCT116 P53+/+, SW480, HCT116 P53+/+, and HT-29 was measured by ImageJ software. Cells were treated only with Dox, VPA or SAHA or by different combinations of these drugs. The changes are presented as fold change in comparison to the control. Error bars represent ± S.E.M.; n=3 independent experiments. One-way ANOVA, Dunnett post-test, P =0.0001.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: SDS Page, Control, Software, Comparison

Journal: Oncotarget

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer.

doi: 10.18632/oncotarget.9887

Figure Lengend Snippet: Figure 9: Dox/SAHA combination decreases drastically survivin protein level in HCT 116 p53+/+, HCT116 p53-/- and SW480 but not HT-29 cell lines. Four cell lines (HCT 116 p53+/+, HCT116 p53-/- SW480 and HT-29) were treated with Dox, VPA, SAHA or different combinations of these drugs for 24hours. Cells were lysed and the proteins separated using SDS-PAGE and detected by WB analysis. The protein level of Survivin and p53 were assessed using the appropriate antibodies. Actin was used as a loading control.

Article Snippet: Generation of stable cell lines Stable cell lines were generated after transfection of p53-null HCT 116 P53-/- or HT-29 cells with wild type P53 plasmid (Origene, UK) or mutant p53 expression plasmids generated by using the QuikChange II sitedirected mutagenesis kit (Stratagene) according to the manufacturer.

Techniques: SDS Page, Control