Journal: PloS one

Article Title: Sterigmatocystin-induced DNA damage triggers G2 arrest via an ATM/p53-related pathway in human gastric epithelium GES-1 cells in vitro.

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: Figure 4. The p53-p21 pathway is activated in ST-treated GES-1 cells. GES-1 cells were treated with different concentrations of ST (0.075, 0.3, 1.5, and 3 mM) or solvent for 48 h. (A) Representative immunoblots show the effect of ST treatment on the phosphorylation of p53 (Ser-15) and the expression of p53 and p21. b-actin was used as the normalization control. (B) Intensities of the immunoreactive bands were quantified by densitometric scanning and compared with those of the control (considered ‘‘1’’). The values shown represent the means 6 SD. *P,0.05 compared with the solvent-treated control group. doi:10.1371/journal.pone.0065044.g004

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse antihuman phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Solvent, Western Blot, Phospho-proteomics, Expressing, Control

Journal: PloS one

Article Title: Sterigmatocystin-induced DNA damage triggers G2 arrest via an ATM/p53-related pathway in human gastric epithelium GES-1 cells in vitro.

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: Figure 6. Silencing of p53 by specific p53 siRNA inhibited ST-induced G2 arrest. Cells were either not transfected or transfected with 100 nM p53 siRNA and then treated with 3 mM ST for 48 h. (A) Cells were subjected to immunoblot analysis for p-p53 (Ser15), p53, p21, and (C) the regulators related to G2 arrest. NC: cells transfected with the same concentration of negative control siRNA. b-actin was used as the loading control. (B, D) Intensities of the immunoreactive bands in ‘‘A’’ and ‘‘C’’ were quantified by densitometric scanning and compared with those of the control (considered ‘‘1’’). (E) The cell cycle phases of the cells were analyzed by FCM. The values shown represent the means 6 SD, *P,0.05 compared with the solvent-treated control group. mP,0.05 compared with the ST-treated groups. #P,0.05 compared with the p53 siRNA-treated groups. doi:10.1371/journal.pone.0065044.g006

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse antihuman phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Concentration Assay, Negative Control, Control, Solvent

Journal: Molecular Biology Reports

Article Title: Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol

doi: 10.1007/s11033-013-2602-7

Figure Lengend Snippet: a Expression of p53 and proteins encoded by p53-regulated genes (p21 and MDM2). Whole-cell lysates of untreated cells (U) and of cells treated for the indicated number of hours with 50 μM resveratrol were analyzed. HSC70 is a loading control. b Changes in the levels of mRNAs encoding MDM2 or p21, measured by semi-quantitative real-time PCR in RNA samples isolated from untreated (U) cells and from cells treated with resveratrol for 24 or 96 h. The results represent the means and standard deviations from two independent experiments

Article Snippet: The following antibodies were from Cell Signaling Technology: anti-phospho-Ser1981 ATM (D6H9), anti-ATM (D2E2), anti-acetyl-Lys382 p53, anti-phospho-Ser15 p53 (rabbit polyclonal antibody), anti-phospho-Ser20 p53, anti-phospho-Ser37 p53, anti-phospho-Ser392 p53, anti-CHK2 (rabbit polyclonal antibody), anti-phospho-Thr68 CHK2, anti-phospho-Ser807/811 RB, and anti-PLK1 (208G4).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Isolation

Journal: Molecular Biology Reports

Article Title: Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol

doi: 10.1007/s11033-013-2602-7

Figure Lengend Snippet: Expression of p53 and its post-translationally modified forms in whole-cell lysates from untreated cells (U) or from cells treated with resveratrol for the indicated number of hours in a time-course experiment. Phosphorylation of serines 15, 20, 37, and 392 and acetylation of lysine 382 were examined

Article Snippet: The following antibodies were from Cell Signaling Technology: anti-phospho-Ser1981 ATM (D6H9), anti-ATM (D2E2), anti-acetyl-Lys382 p53, anti-phospho-Ser15 p53 (rabbit polyclonal antibody), anti-phospho-Ser20 p53, anti-phospho-Ser37 p53, anti-phospho-Ser392 p53, anti-CHK2 (rabbit polyclonal antibody), anti-phospho-Thr68 CHK2, anti-phospho-Ser807/811 RB, and anti-PLK1 (208G4).

Techniques: Expressing, Modification, Phospho-proteomics

Journal: Molecular Biology Reports

Article Title: Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol

doi: 10.1007/s11033-013-2602-7

Figure Lengend Snippet: Expression of DNA-damage signaling proteins (ATM, CHK2), their phosphorylated forms, p53, and post-translationally modified p53, as well as the major cell-cycle regulators (p21, CDC2, RB phosphorylated on Ser807/811), examined in whole-cell lysates from untreated U-2 OS cells (U) or from cells treated with resveratrol for the indicated number of hours in a time-course experiment. The pairs represent control cells (−) and WIP1-knockdown cells (+); knockdowns were performed using lentivirus-delivered shRNA molecules. The efficiency of knockdown is shown by immunodetection of WIP1 protein

Article Snippet: The following antibodies were from Cell Signaling Technology: anti-phospho-Ser1981 ATM (D6H9), anti-ATM (D2E2), anti-acetyl-Lys382 p53, anti-phospho-Ser15 p53 (rabbit polyclonal antibody), anti-phospho-Ser20 p53, anti-phospho-Ser37 p53, anti-phospho-Ser392 p53, anti-CHK2 (rabbit polyclonal antibody), anti-phospho-Thr68 CHK2, anti-phospho-Ser807/811 RB, and anti-PLK1 (208G4).

Techniques: Expressing, Modification, Control, Knockdown, shRNA, Immunodetection

Journal: EMBO molecular medicine

Article Title: PHD1 regulates p53-mediated colorectal cancer chemoresistance.

doi: 10.15252/emmm.201505492

Figure Lengend Snippet: Figure 4. PHD1 hydroxylase is required for p53 phosphorylation upon chemotherapy treatment.

Article Snippet: The following antibodies were used for the detection of the proteins by immunoblotting: rabbit anti-PHD1 (Novus, NB100-310, 1/250 in 5% milk), mouse anti-p53 (Santa Cruz Biotechnology, DO1, 1/4,000 in 5% milk), rabbit anti-p53 (Santa Cruz Biotechnology, FL-393, 1/1,000 in 5% milk), mouse anti-vinculin (Sigma-Aldrich, V9131, 1/5,000 in 5% milk), rabbit anti-caspase-3 (Cell Signaling, #9665, 1/500 in 5% BSA), rabbit anti-Flag (Sigma-Aldrich, F7425, 1/1,000 in 5% milk), rabbit anti-phospho-serine 15 p53 (Cell Signaling, #9284, 1/1,000 in 5% BSA), rabbit anti-pH2AX (Cell Signaling, #2577, 1/1,000 in 5% BSA), rabbit anti-cleaved parp (Cell Signaling, #5625, 1/1,000 in 5% BSA), rabbit anti-p38 (Cell Signaling, #9212, 1/1,000 in 5% BSA), and rabbit anti-XPB (Santa Cruz Biotechnology, sc-293, 1/500 in 5% milk).

Techniques: Phospho-proteomics

Journal: EMBO molecular medicine

Article Title: PHD1 regulates p53-mediated colorectal cancer chemoresistance.

doi: 10.15252/emmm.201505492

Figure Lengend Snippet: Figure 5. PHD1 hydroxylase favors p38a-mediated p53 phosphorylation.

Article Snippet: The following antibodies were used for the detection of the proteins by immunoblotting: rabbit anti-PHD1 (Novus, NB100-310, 1/250 in 5% milk), mouse anti-p53 (Santa Cruz Biotechnology, DO1, 1/4,000 in 5% milk), rabbit anti-p53 (Santa Cruz Biotechnology, FL-393, 1/1,000 in 5% milk), mouse anti-vinculin (Sigma-Aldrich, V9131, 1/5,000 in 5% milk), rabbit anti-caspase-3 (Cell Signaling, #9665, 1/500 in 5% BSA), rabbit anti-Flag (Sigma-Aldrich, F7425, 1/1,000 in 5% milk), rabbit anti-phospho-serine 15 p53 (Cell Signaling, #9284, 1/1,000 in 5% BSA), rabbit anti-pH2AX (Cell Signaling, #2577, 1/1,000 in 5% BSA), rabbit anti-cleaved parp (Cell Signaling, #5625, 1/1,000 in 5% BSA), rabbit anti-p38 (Cell Signaling, #9212, 1/1,000 in 5% BSA), and rabbit anti-XPB (Santa Cruz Biotechnology, sc-293, 1/500 in 5% milk).

Techniques: Phospho-proteomics

Journal: Mechanisms of ageing and development

Article Title: Cellular senescence in vascular wall mesenchymal stromal cells, a possible contribution to the development of aortic aneurysm.

doi: 10.1016/j.mad.2021.111515

Figure Lengend Snippet: Fig. 5. (A and B) representative images of immunofluorescence labeling of p – H2AX in h – MSCs and AAA – MSCs isolated from two different donors. A FITC conjugated secondary antibody was used to detect the nuclear localization of p – H2AX. AAA – MSCs nuclei showed a stronger green fluorescent signal compared to h – MSCs nuclei (magnification 600X; bar: 100 nm); (C) representative western blot images showing total p53 and P-p53 protein expression in h – MSCs and AAA – MSCs. Results from two different donors were shown. (D) Relative amounts of total p53 and P-p53 protein expression were normalized to the intensity of actin and represented as fold increase relative to h – MSCs of each donor. Western blotting was performed in duplicate and the relative quantification was expressed as mean value ± SD. * represents a significant difference compared to h - MSCs, p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The membranes were blocked with dry milk (blocking reagent) (Invitrogen, Thermo Fisher Scientific, Monza, Italy) for 30 min at room temperature and were then incubated with the following primary antibodies: mouse anti-human p53 antibody (Cell Signaling Technologies, Euroclone, Milan, Pero); mouse anti-human phospho - p53 (Ser 15) (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human p21CIP antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti human p16INK4A antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human Beclin (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human LC3 (Cell Signaling Technologies, Euroclone, Milan, Italy); mouse anti-human tubulin antibody (Sigma- Aldrich, St Louis, Missouri, USA) and mouse anti-human actin antibody (Millipore Merck, Darmstadt, Germany).

Techniques: Immunofluorescence, Labeling, Isolation, Western Blot, Expressing, Quantitative Proteomics

Journal: Oncogene

Article Title: Proteomic identification of p53-dependent protein phosphorylation.

doi: 10.1038/onc.2008.124

Figure Lengend Snippet: Figure 1 Induction and phosphorylation of p53 and apoptosis in mitomycin C (MMC)-treated HCT116 cells. (a) HCT116 p53 þ / þ and p53/ cells were treated with MMC for 16 and 24 h. Whole-cell lysates were analysed for p53, p21, MDM2 and phosphorylation of p53 at Ser15, Ser20 and Ser392 by western blotting. b-Actin expression was used as loading control. (b) Immunostaining of HCT116 p53 þ / þ and p53/ cells after 16 h treatment with MMC. Phosphorylation of p53 was detected by antibodies specific for phosphorylated Ser15 and Ser37. (c). Apoptosis was assessed by fluorescence-activated cell sorting (FACS) analysis of caspase activity in HCT116 p53 þ / þ and p53/ cells treated with MMC for 16 and 24 h. HCT116 p53 þ / þ and p53/ colon carcinoma cells (provided by Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, USA) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum, glutamin and gentamycin in 5% CO2 at 37 1C. HCT116 cells were grown with or without 10 mg/ml mitomycin C to activate endogenous p53 (Sigma, St Louis, MI, USA). Western blotting was performed according to standard procedure using the following primary antibodies: mouse monoclonal anti-p53 (DO-1, Eurogenetics, Tessenderlo, Belgium), mouse monoclonal anti- MDM2 (2A10, provided by Sonia Lain, University of Dundee), mouse monoclonal anti-p21 (BD Pharmingen, San Diego, CA, USA), mouse monoclonal anti-b-actin (Sigma, St Louis, MI, USA). For detection of phosphorylation of p53, rabbit polyclonal anti-p53 Ser15, Ser20 and Ser392 (Cell Signaling Technology, Danvers, MA, USA) were used. Immunofluorescence staining was performed as described (Rahman et al., 2005). Caspase activity was measured using CaspaTag Pan caspase kit (Chemicon, Temecula, LA, USA).

Article Snippet: For detection of phosphorylation of p53, rabbit polyclonal anti-p53 Ser15, Ser20 and Ser392 (Cell Signaling Technology, Danvers, MA, USA) were used.

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Immunostaining, FACS, Activity Assay, Staining

Journal: Molecular Biology of the Cell

Article Title: Failure of cell cleavage induces senescence in tetraploid primary cells

doi: 10.1091/mbc.E14-03-0844

Figure Lengend Snippet: Tetraploidy arrest does not involve DNA damage. (A) Both REF52 and TAG cells were exposed to 10 μM DCB for 24 h and then released for 24 h and stained for γ-H2AX, a marker for cell response to DNA damage ( Paull et al. , 2000 ). The counterstain for DNA is DAPI. Binucleate cells of either cell type show no intense DNA damage response. γ-Irradiation (4Gy) of REF52 and TAG cells serves as a positive control. All images were taken from the same experiment, using identical image capture settings. (B) Quantitation of the number of γ-H2AX foci present in nuclei, visualized with microscopy, using procedures detailed in Materials and Methods . All foci that were above a set intensity threshold were automatically counted. The intensity of foci in irradiated nuclei was substantially greater than in other conditions, as seen in A. Results are expressed as mean ± SD. Foci within at least 100 nuclei were counted in each condition. (C) Western blot of DNA-damage markers in HFF cells, phospho-Ser-15 p53, and phospho-Thr-68 Chk2, demonstrating that these markers are not elevated after tetraploidy induction by two rounds of 10 μM DCB treatment compared with control random cycling cells. Adriamycin induction of DNA damage 72 h after drug treatment is the positive control for DNA damage response (Adr). Actin is a loading control.

Article Snippet: Antibodies used were p21waf1 (C-19; Santa Cruz Biotechnology, Dallas, TX), p27kip1 (610241; BD Transduction Lab, San Jose, CA), α-tubulin (B511; Sigma-Aldrich), anti-ERK1/2 (9102; Cell Signaling), anti–phospho-ERK1/2 (4377; Cell Signaling), anti–γ-H2AX (4411-PC; Trevigen), anti–cyclin D1/2 (05-362; Upstate Biotechnology, Lake Placid, NY), anti-anillin (C. Field, Harvard Medical School, Boston, MA; Oegema et al. , 2000 ), anti–phospho-Ser-15 p53 (12571; Cell Signaling), anti–phospho-Thr-68 Chk2 (2197; Cell Signaling), anti-p16INK4a (mouse mAb 11104; Immuno-Biological Laboratories, Minneapolis, MN), and PRC1 antibody described previously ( Mollinari et al. , 2002 ).

Techniques: Staining, Marker, Irradiation, Positive Control, Quantitation Assay, Microscopy, Western Blot, Control

Journal: Molecular Biology of the Cell

Article Title: Failure of cell cleavage induces senescence in tetraploid primary cells

doi: 10.1091/mbc.E14-03-0844

Figure Lengend Snippet: Treatment with DCB does not arrest HCT116 p53 +/+ cell proliferation. (A) HCT116 p53 +/+ cells were treated with 10 μM DCB for 24 h and then released for the indicated times. The HCT116 cells proceed from tetraploidy to aneuploidy during 72 h of release. (B) HCT116 p53 +/+ and HCT116 p53 − / − were exposed to 10 μM DCB or 2 μg/ml Adriamycin ( Skoufias et al. , 2004 ) for 24 h and then released. Cell counts were then performed at the indicated times. Cells continue to proliferate when tetraploid and aneuploid, regardless of p53 status, but do not recover from Adriamycin.

Article Snippet: Antibodies used were p21waf1 (C-19; Santa Cruz Biotechnology, Dallas, TX), p27kip1 (610241; BD Transduction Lab, San Jose, CA), α-tubulin (B511; Sigma-Aldrich), anti-ERK1/2 (9102; Cell Signaling), anti–phospho-ERK1/2 (4377; Cell Signaling), anti–γ-H2AX (4411-PC; Trevigen), anti–cyclin D1/2 (05-362; Upstate Biotechnology, Lake Placid, NY), anti-anillin (C. Field, Harvard Medical School, Boston, MA; Oegema et al. , 2000 ), anti–phospho-Ser-15 p53 (12571; Cell Signaling), anti–phospho-Thr-68 Chk2 (2197; Cell Signaling), anti-p16INK4a (mouse mAb 11104; Immuno-Biological Laboratories, Minneapolis, MN), and PRC1 antibody described previously ( Mollinari et al. , 2002 ).

Techniques:

Journal: Developmental biology

Article Title: Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

doi: 10.1016/j.ydbio.2019.01.011

Figure Lengend Snippet: Fluorescent Immunohistochemistry

Article Snippet: Mouse monoclonal anti-p53 (phospho Ser15) 16G8 , Cell Signaling , Cat#9286; RRID:AB_331741.

Techniques: Transduction, Recombinant, Imaging, Binding Assay, Software

Journal: Developmental biology

Article Title: Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

doi: 10.1016/j.ydbio.2019.01.011

Figure Lengend Snippet: (A) RT-PCR for Cdkn1a mRNA in E18.5 control and cDnmt1KO urethras. (B-C) E18.5 urethra sections were labeled with antibodies against Gamma-H2AX (in red, DNA damage marker) and LacZ (in green, labels Shh lineage epithelium). (D-E) E18.5 urethra sections were labeled with antibodies against Phospho-p53 Ser15 (in red, marks active p53). (F-G) E18.5 urethra sections were labeled with antibodies against Cleaved caspase 3 (in red, marks apoptotic cells) and EYFP (in green, labels Shh lineage epithelium). Green channel is excluded for ease of visualization. (H) Percentage of cleaved Caspase 3 labeled cells in the Shh lineage urethral epithelium. White arrowheads indicated cleaved Caspase 3 positive apoptotic cells. White dotted lines indicate epithelial-mesenchymal interface. DAPI staining is shown in blue. Scale bar is 50 microns. Graphical results are the mean + SEM of at least three mice per group. p-values indicate significant differences (* p<0.05, ** p<0.01) between groups based on Student’s unpaired t-test.

Article Snippet: Mouse monoclonal anti-p53 (phospho Ser15) 16G8 , Cell Signaling , Cat#9286; RRID:AB_331741.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Labeling, Marker, Staining