Journal: British Journal of Cancer

Article Title: Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

doi: 10.1038/sj.bjc.6601549

Figure Lengend Snippet: Characteristics of AT heterozygote cell lines

Article Snippet: The p53-ser15 phosphorylation was measured using a specific p53-phosphoserine 15 antibody (#9284, Cell Signaling Technology, 1/1000 dilution), and the Chk2 phosphorylation was detected by the mobility shift of the protein using a specific Chk2 antibody (477, Abcam, 1/1500 dilution).

Techniques: Mutagenesis

Journal: British Journal of Cancer

Article Title: Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

doi: 10.1038/sj.bjc.6601549

Figure Lengend Snippet: Radiation-induced Chk2 and p53-ser15 phosphorylation. Protein extracts were prepared from the normal (IARC 2145), AT (AT11) and AT heterozygote (10–573, 1–15 308, 1–17 815, 1–17 053, 10–572 and 1–25 065) LCLs 2 and 4 h after exposure to 5 Gy, and from untreated cells. Ku80 immunodetection was performed on each gel to correct for variations in protein loading. Phosphorylation of Chk2 was assessed by the mobility shift of the protein band, while the intensity of the p53-ser15 bands was analysed by densitometry.

Article Snippet: The p53-ser15 phosphorylation was measured using a specific p53-phosphoserine 15 antibody (#9284, Cell Signaling Technology, 1/1000 dilution), and the Chk2 phosphorylation was detected by the mobility shift of the protein using a specific Chk2 antibody (477, Abcam, 1/1500 dilution).

Techniques: Phospho-proteomics, Immunodetection, Mobility Shift

Journal: British Journal of Cancer

Article Title: Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

doi: 10.1038/sj.bjc.6601549

Figure Lengend Snippet: Quantification of the p53-ser15 phosphorylation, presented as the p53-ser15/ku80 ratio to correct for variation in protein loading, 2 and 4 h after exposure to 5 Gy IR. The average ratio±s.d. was calculated for each group of cell lines, based on at least two independent experiments for each line ( n =number of lines).

Article Snippet: The p53-ser15 phosphorylation was measured using a specific p53-phosphoserine 15 antibody (#9284, Cell Signaling Technology, 1/1000 dilution), and the Chk2 phosphorylation was detected by the mobility shift of the protein using a specific Chk2 antibody (477, Abcam, 1/1500 dilution).

Techniques: Phospho-proteomics

Journal: Cancer Research

Article Title: Cancer-Specific Functions of SIRT1 Enable Human Epithelial Cancer Cell Growth and Survival

doi: 10.1158/0008-5472.can-05-1923

Figure Lengend Snippet: Figure 1. Selective silencing of SIRT1 in HCT116 cells. A, effects of SIRT1 siRNA, BCR-ABL siRNA, and lamin A/C siRNA on relative levels of mRNAs encoding SIRT1, lamin A/C, and GAPDH as indicated. Cells were harvested 48 hours after siRNA transfections and mRNA levels were determined by quantitative PCR (see Materials and Methods). B, immunoblots to show protein levels for SIRT1 and lamin A/C in cells harvested 48 hours posttransfection with siRNAs as indicated. C, immunoblot for acetylated p53 48 hours posttransfection with SIRT1 siRNA compared with mock-transfected controls (as indicated). Actin was used as internal reference for gel loading and protein levels were quantitated by densitometry (see Materials and Methods and text).

Article Snippet: Antibodies were anti-SIRT1 (H-300, Santa Cruz, Santa Cruz, CA); anti-p53 (DO-1, Santa Cruz); anti-lamin A/C (636, Santa Cruz); anti-cleaved caspase-3, caspase-6, caspase-7, caspase8, and caspase-9 (Cell Signalling); anti-actin (MAB1501, Chemicon, Temecula, CA); anti-phosphoserine-15 p53 [p-p53 (Ser15)-R, Santa Cruz]; anti-acetylated-p53 (Lys382, Cell Signalling); and anti-HDM2 (antibody 4B2; ref. 24).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer Research

Article Title: Cancer-Specific Functions of SIRT1 Enable Human Epithelial Cancer Cell Growth and Survival

doi: 10.1158/0008-5472.can-05-1923

Figure Lengend Snippet: Figure 2. Induction of RNAi per se does not initiate a p53 stress response. A, immunoblots to show lack of activation of endogenous p53 (as indicated by no detectable serine 15 phosphorylation nor up-regulation of the p53 target gene HDM2) in HCT116 cells mock transfected or transfected with control BCR-ABL or lamin A/C siRNAs. B, positive control showing activation of p53 in response to genotoxic stress in HCT116 cells treated for 24 hours with 375 Amol/L 5-fluorouracil.

Article Snippet: Antibodies were anti-SIRT1 (H-300, Santa Cruz, Santa Cruz, CA); anti-p53 (DO-1, Santa Cruz); anti-lamin A/C (636, Santa Cruz); anti-cleaved caspase-3, caspase-6, caspase-7, caspase8, and caspase-9 (Cell Signalling); anti-actin (MAB1501, Chemicon, Temecula, CA); anti-phosphoserine-15 p53 [p-p53 (Ser15)-R, Santa Cruz]; anti-acetylated-p53 (Lys382, Cell Signalling); and anti-HDM2 (antibody 4B2; ref. 24).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Transfection, Control, Positive Control

Journal: Cancer Research

Article Title: Cancer-Specific Functions of SIRT1 Enable Human Epithelial Cancer Cell Growth and Survival

doi: 10.1158/0008-5472.can-05-1923

Figure Lengend Snippet: Figure 6. FoxO4 cosilencing rescues HCT116 p53+/+ cells from SIRT1 siRNA-induced apoptosis and also from Bcl-2 siRNA-induced apoptosis. A, phase contrast images of HCT116 p53+/+ cells 48 hours posttransfection with either FoxO3 siRNA, FoxO4 siRNA, or SIRT1 siRNA, and cotransfection of FoxO3 siRNA with SIRT1 siRNA, or FoxO4 siRNA with SIRT1 siRNA. B, FoxO4 mRNA levels in HCT116 p53+/+ cells 48 hours posttransfection with BCR-ABL siRNA, FoxO4 siRNA sequence 1, or FoxO4 siRNA sequence 2 (see Materials and Methods). C, Annexin V–positive, apoptotic HCT116 p53+/+ cells were determined 48 hours posttransfection with siRNAs as indicated.

Article Snippet: Antibodies were anti-SIRT1 (H-300, Santa Cruz, Santa Cruz, CA); anti-p53 (DO-1, Santa Cruz); anti-lamin A/C (636, Santa Cruz); anti-cleaved caspase-3, caspase-6, caspase-7, caspase8, and caspase-9 (Cell Signalling); anti-actin (MAB1501, Chemicon, Temecula, CA); anti-phosphoserine-15 p53 [p-p53 (Ser15)-R, Santa Cruz]; anti-acetylated-p53 (Lys382, Cell Signalling); and anti-HDM2 (antibody 4B2; ref. 24).

Techniques: Cotransfection, Sequencing

Journal:

Article Title: Induction and Bypass of p53 during Productive Infection by Polyomavirus

doi: 10.1128/JVI.76.18.9526-9532.2002

Figure Lengend Snippet: Induction of a p53 response in vivo. Newborn C3H/BiDa pups were inoculated with a lethal dose of LID. After 8 days, pups were sacrificed and kidney homogenates were prepared for immunoblot analysis. p53 was detected with anti-p53 (CM5 from Novocastra) and p21 with C-19 (Santa Cruz). Immunoblot analysis for phosphoserine-18 (Pser18) p53 was performed in a separate experiment using anti-phosphoserine-15 p53 from Cell Signaling.

Article Snippet: To block immunoreactivity, 2 μl of a 1:10 dilution of the phosphoserine-15 p53 antibody and 1 μl of blocking peptide (Cell Signaling) in 97 μl of Tris-buffered saline containing 0.1% Tween and 5% bovine serum albumin were incubated at room temperature for 1 h before being added to slides.

Techniques: In Vivo, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Expression of Arl2 is associated with p53 localization and chemosensitivity in a breast cancer cell line.

doi: 10.4161/cc.7.19.6777

Figure Lengend Snippet: Figure 3. p53 staining of MA-, MP and MA+ cells after incubation to cytotoxic compounds shows differences in nuclear translocation. (A) Representative images of each subline exposed to DMSO (vehicle), to 1 μM gemcitabine (Gem) or to 10 nM vinorelbine after p53 (DO7 clone) (yellow/green) and DNA (DAPI, blue) staining. (B) mean percentages of cells with p53 positive nuclei exposed to cytotoxic compounds, evaluated by confocal microscopy. Scale bar represents 30 μm. Bars represent standard deviations. Statistical significance was determined using Student’s t test (+: p < 0.05 between subline of interest and other sublines).

Article Snippet: Antibodies against p53 (DO7-clone) were purchased from Dako (Denmark) and specific antibodies directed against phospho-serine forms of p53 (phosphoser6, phospho-ser9, phospho-ser15 (monoclonal: 16G8-clone), phospho-ser20, phospho-ser37 and phospho-ser392) were from Cell Signaling Technology (USA).

Techniques: Staining, Incubation, Translocation Assay, Confocal Microscopy

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Expression of Arl2 is associated with p53 localization and chemosensitivity in a breast cancer cell line.

doi: 10.4161/cc.7.19.6777

Figure Lengend Snippet: Figure 4. Exposure to gemcitabine enhances p53 transcriptional activity which is correlated with Arl2 content. (A) effect of gemcitabine on expression levels of p21/waf1 (p21), PIG3 and PUMA mRNA in MA-, MP and MA+, expressed as ratios of mRNA levels in gemcitabine exposed-cells to vehicle- exposed cells, determined by quantitative real time RT-PCR. (B) representative images of each subline exposed with 1 μM gemcitabine (Gem) after p21 (yellow/green) and DNA (DAPI, blue) staining and mean percentages of cells with p21 positive nuclei in cells exposed to 1 μM gemcitabine, evaluated by confocal microscopy. Scale bars represent 10 μm. Bars represent standard deviations. Statistical significance was determined using Student’s t test (+: p < 0.05 between exposed and control conditions and * p < 0.05 between interest exposed cells and exposed MP cells).

Article Snippet: Antibodies against p53 (DO7-clone) were purchased from Dako (Denmark) and specific antibodies directed against phospho-serine forms of p53 (phosphoser6, phospho-ser9, phospho-ser15 (monoclonal: 16G8-clone), phospho-ser20, phospho-ser37 and phospho-ser392) were from Cell Signaling Technology (USA).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Staining, Confocal Microscopy, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Expression of Arl2 is associated with p53 localization and chemosensitivity in a breast cancer cell line.

doi: 10.4161/cc.7.19.6777

Figure Lengend Snippet: Figure 5. p53 and p53 phosphorylated content in microtubule and soluble tubulin fractions of MA-, MP and MA+ cells. (A) Left: representative Western blots ofp53 in soluble and microtubule (MT) fractions, standardized on β-tubulin content. Right: representative blot of p53 in total extracts from DMSO (vehicle) or doxorubicin-exposed (Doxo, 150 nM) MP cells and from soluble and microtubule (MT) MP cells fractions under basal conditions. The histograph represents percentage of p53 content of each fraction as a ratio of total p53 content. Bars represent standard deviations. Statistical significance was determined using Student’s t test (+: p < 0.05 between subline of interest and other sublines). (B) Representative Western blots ofphospho-ser15p53 (pser15-p53), phospho- ser37-p53 (pser37-p53), standardized on β-tubulin content, in soluble and microtubule (MT) and total extracts; and ratios of phospho-ser15-p53 levels to total p53 content. (C) Representative images of each subline after phospho-ser15-p53 (Red) and DNA (DAPI, blue) staining in different phases of the cell cycle (upper: mitotic cells; lower: interphasic cells). Bars represent standard deviations. Statistical significance was determined using Student’s t test (+: p < 0.05 between subline of interest and other sublines).

Article Snippet: Antibodies against p53 (DO7-clone) were purchased from Dako (Denmark) and specific antibodies directed against phospho-serine forms of p53 (phosphoser6, phospho-ser9, phospho-ser15 (monoclonal: 16G8-clone), phospho-ser20, phospho-ser37 and phospho-ser392) were from Cell Signaling Technology (USA).

Techniques: Western Blot, Staining

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Expression of Arl2 is associated with p53 localization and chemosensitivity in a breast cancer cell line.

doi: 10.4161/cc.7.19.6777

Figure Lengend Snippet: Figure 6. PP2A implication in p53 localization and resistance phenotype. (A) Upper: representative Western blots of p53 in soluble and microtubule (MT) extracts of the MA+ cell line exposed to DMSO (vehicle) or to 10 nM okadaic acid (OA), standardized on β-tubulin content; and ratio of phospho-p53 to total p53 content. (B) Upper left: mean IC50 values of MA+ cells preincubated either during 2 hours with DMSO (vehicle) or okadaic acid (OA), or during 24 hours with DMSO or cantharidic acid (CA) then incubated for six days in the presence of taxol (Tx) or vinorelbine (Vnb). Upper right: representative images, after DAPI-staining, of MA+ cells preincubated during 24 hours with either DMSO or cantharidic acid then exposed for three days to 10 nM vinore- lbine. Bright blue condensed disks correspond to apoptotic nuclei (white arrows). Lower left: Survival curves of MA+ cells exposed to cisplatin (CisP), after preincubation for 24 hours with vehicle (DMSO/CisP), or with 50 nM, 150 nM or 400 nM cantharidic acid (50 nM, 150 nM, 400 nM CA/CisP). (C) Ratio of IC50 values, after three days of incubation in the presence of taxol (Tx), of MA-, MP and MA+ cells, after preincubation with cantharidic acid (IC50-CA) or not (IC50-vehicle). Statistical significance was determined using Student’s t test (+: p < 0.05 between subline of interest and other sublines, *p < 0.05 between IC50-CA and IC50-vehicle for each subline).

Article Snippet: Antibodies against p53 (DO7-clone) were purchased from Dako (Denmark) and specific antibodies directed against phospho-serine forms of p53 (phosphoser6, phospho-ser9, phospho-ser15 (monoclonal: 16G8-clone), phospho-ser20, phospho-ser37 and phospho-ser392) were from Cell Signaling Technology (USA).

Techniques: Western Blot, Incubation, Staining