Journal: Cellular Microbiology

Article Title: Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

doi: 10.1111/j.1462-5822.2006.00698.x

Figure Lengend Snippet: A. Distribution of p53 in mock‐infected cells. B. Confocal images of cells expressing IBV proteins (green) and showing the distribution of p53 (red) and merged at 24 h pi. C. Enlarged images of IBV‐infected cells with IBV proteins labelled in green and p53 in red at 24 h pi. Transmission phase contrast (TPC) of the lower cell is shown below. Nucleoli are indicated (No) and the size bar is 10 µm. D. Western blot analysis of p53 in mock and infected cells at 0 and 24 h pi. Protein concentrations were standardized and compared with GAPDH.

Article Snippet: To detect the tumour suppressor protein p53 we used a mouse anti‐p53 antibody (D01, Santa Cruz) and to detect GAPDH we used a mouse anti‐GAPDH antibody (6C5, AbCam), with appropriate secondary antibody.

Techniques: Infection, Expressing, Transmission Assay, Western Blot

Journal: Cellular Microbiology

Article Title: Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

doi: 10.1111/j.1462-5822.2006.00698.x

Figure Lengend Snippet: A. Confocal analysis to investigate the potential colocalization of p53 (red) in cells expressing EGFP‐IBV N protein (green). B and C. Confocal analysis of the distribution of IBV proteins (blue), EGFP‐IBV N protein (green) and p53 (red) in IBV‐infected cells at 24 h pi. Colocalization between EGFP‐IBV N protein plus p53, EGFP‐IBV N protein plus IBV proteins, p53 plus IBV proteins, and between the three complexes, appears yellow, cyan, purple and white respectively. The nucleolus is indicated (No). Size bar is 10 µm.

Article Snippet: To detect the tumour suppressor protein p53 we used a mouse anti‐p53 antibody (D01, Santa Cruz) and to detect GAPDH we used a mouse anti‐GAPDH antibody (6C5, AbCam), with appropriate secondary antibody.

Techniques: Expressing, Infection

Journal: Oncogene

Article Title: Control of cell cycle progression in human mesothelioma cells treated with gamma interferon.

doi: 10.1038/sj.onc.1204199

Figure Lengend Snippet: Figure 4 p21WAF1/CIP1 (a), p27KIP1 (b) and p53 (c) protein expression in response to r-hu-IFNg in HM cell lines. Cells were cultured in the absence (7) or presence (+) of r-hu-IFNg (500 IU/ml) for 24, 48 and 72 h. Forty mg of protein extracts were subjected to SDS ± PAGE (12%) electrophoresis, followed by immunoblot analysis with antibodies against the corresponding proteins. ECL detection. Top: typical Western blots of a sensitive (HB) and resistant (FR) cell line. Bottom: Densitometric analysis of p21WAF1/

Article Snippet: The following antibodies were used: Primary mouse monoclonal antibody against human p21WAF1/CIP1, p27Kip1 and cyclin B1 (Transduction Lab, Med Gene Science, Pantin, France), and p53 D01 (Santa Cruz, Tebu, Le Perray en Yvelines, France).

Techniques: Expressing, Cell Culture, SDS Page, Electrophoresis, Western Blot

Journal: bioRxiv

Article Title: BK polyomavirus (BKPyV) is a risk factor for bladder cancer through induction of APOBEC3-mediated genomic damage

doi: 10.1101/2021.05.13.443803

Figure Lengend Snippet: Schematic model of BKPyV hit-and-run carcinogenesis hypothesis. Immune-insufficiency leads to sloughing of actively infected renal “decoy” cells and BKPyV viruria. BKPyV infects the G0-arrested urothelium. BKPyV LT-Ag inhibits retinoblastoma (pRb) and p53 inhibition of the cell cycle. BKPyV cell cycle re-entry progresses to arrest at the G2/M checkpoint. BKPyV stimulates APOBEC3 enzyme activity and host genome damage that inactivates tumour suppressors. The immune system clears the virus but initiated cells persist and over a period of years expand to form a tumour.

Article Snippet: Primary antibodies, applied overnight at 4°C, were anti-SV40 LT-Ag (1:200, mouse monoclonal “Pab 108”, Santa Cruz Biotechnology, sc-148), anti-VP1 (1:100, mouse “pab597”, kind gift from Chris Buck at the National Institute of Health, Bethesda), anti-Rad51 (1:1,000, rabbit “EPR4030(3)”, Abcam #ab133534), anti-Ki67 (1:400, mouse, “MM1”, Leica, NCL-L-Ki67-MM1), anti-MCM2 (1:500, rabbit, “D7G11”, Cell Signalling #3619), anti-Phospho-pRb Serine 608/807/811 (1:500, rabbit, Cell Signalling #9308), anti-p53 (1:40, mouse, “D01”, Cell Signalling #18032), anti-ZO3 (1:800, rabbit, Cell Signalling #3704) and anti-APOBEC3A/B/G (1:100 rabbit monoclonal, clone 5210-87-13 ).

Techniques: Infection, Inhibition, Activity Assay, Virus

Journal: bioRxiv

Article Title: BK polyomavirus (BKPyV) is a risk factor for bladder cancer through induction of APOBEC3-mediated genomic damage

doi: 10.1101/2021.05.13.443803

Figure Lengend Snippet: Homologous recombination was induced by BKPyV infection of human urothelium. (A) Western blotting for p53 protein showed significant stabilisation (no increase in transcription was observed) during BKPyV infection. (B) However, there was no increased expression of verified p53 target genes . RAD51 and (D) RAD51AP1 transcripts were significantly induced by BKPyV infection. (E) Western blotting confirmed significant Rad51 protein induction and phosphorylation was observed as a slight increase in molecular weight when Rad51 was induced by BKPyV. (F) Analysis of indirect immunofluorescence for Rad51 found nuclear speckles were significantly increased in BKPyV infection. In some cells, indirect immunofluorescence revealed LT-Ag and Rad51 protein colocalisation to large granular deposits in the nuclei of BKPyV-infected urothelial cells. Scale bar denotes 10 μm, immunofluorescence performed on n=3 independent donors with representative images shown. Full blots and further immunofluorescence images for p53 and Rad 51 in Extended Data Fig. 17 & 18, respectively.

Article Snippet: Primary antibodies, applied overnight at 4°C, were anti-SV40 LT-Ag (1:200, mouse monoclonal “Pab 108”, Santa Cruz Biotechnology, sc-148), anti-VP1 (1:100, mouse “pab597”, kind gift from Chris Buck at the National Institute of Health, Bethesda), anti-Rad51 (1:1,000, rabbit “EPR4030(3)”, Abcam #ab133534), anti-Ki67 (1:400, mouse, “MM1”, Leica, NCL-L-Ki67-MM1), anti-MCM2 (1:500, rabbit, “D7G11”, Cell Signalling #3619), anti-Phospho-pRb Serine 608/807/811 (1:500, rabbit, Cell Signalling #9308), anti-p53 (1:40, mouse, “D01”, Cell Signalling #18032), anti-ZO3 (1:800, rabbit, Cell Signalling #3704) and anti-APOBEC3A/B/G (1:100 rabbit monoclonal, clone 5210-87-13 ).

Techniques: Homologous Recombination, Infection, Western Blot, Expressing, Phospho-proteomics, Molecular Weight, Immunofluorescence

Journal: bioRxiv

Article Title: BK polyomavirus (BKPyV) is a risk factor for bladder cancer through induction of APOBEC3-mediated genomic damage

doi: 10.1101/2021.05.13.443803

Figure Lengend Snippet: Schematic of hypothetical model of urothelial DNA damage at displacement loops at the G2 checkpoint during BKPyV infection. LT-Ag interactions might derive from (A) its DNA-binding helicase activity or (B) one of the other dimerization domains present in the LT-Ag protein. (C) LT-Ag inhibition of p53 is likely important for this process as p53 would normally prevent Rad51 oligomerisation by direct binding . The evidence from this study is most robust for BKPyV-induced APOBEC3B activity but APOBEC3A protein was consistently more abundant during infection and its role requires further validation.

Article Snippet: Primary antibodies, applied overnight at 4°C, were anti-SV40 LT-Ag (1:200, mouse monoclonal “Pab 108”, Santa Cruz Biotechnology, sc-148), anti-VP1 (1:100, mouse “pab597”, kind gift from Chris Buck at the National Institute of Health, Bethesda), anti-Rad51 (1:1,000, rabbit “EPR4030(3)”, Abcam #ab133534), anti-Ki67 (1:400, mouse, “MM1”, Leica, NCL-L-Ki67-MM1), anti-MCM2 (1:500, rabbit, “D7G11”, Cell Signalling #3619), anti-Phospho-pRb Serine 608/807/811 (1:500, rabbit, Cell Signalling #9308), anti-p53 (1:40, mouse, “D01”, Cell Signalling #18032), anti-ZO3 (1:800, rabbit, Cell Signalling #3704) and anti-APOBEC3A/B/G (1:100 rabbit monoclonal, clone 5210-87-13 ).

Techniques: Infection, Binding Assay, Activity Assay, Inhibition, Biomarker Discovery

Journal:

Article Title:

doi:

Figure Lengend Snippet: A. Distribution of p53 in mock‐infected cells. B. Confocal images of cells expressing IBV proteins (green) and showing the distribution of p53 (red) and merged at 24 h pi. C. Enlarged images of IBV‐infected cells with IBV proteins labelled in green and p53 in red at 24 h pi. Transmission phase contrast (TPC) of the lower cell is shown below. Nucleoli are indicated (No) and the size bar is 10 µm. D. Western blot analysis of p53 in mock and infected cells at 0 and 24 h pi. Protein concentrations were standardized and compared with GAPDH.

Article Snippet: To detect the tumour suppressor protein p53 we used a mouse anti‐p53 antibody (D01, Santa Cruz) and to detect GAPDH we used a mouse anti‐GAPDH antibody (6C5, AbCam), with appropriate secondary antibody.

Techniques: Infection, Expressing, Transmission Assay, Western Blot

Journal: Anticancer research

Article Title: Inhibition of Proteasome Activity by Bortezomib in Renal Cancer Cells Is p53 Dependent and VHL Independent

doi:

Figure Lengend Snippet: Baseline expression under normoxic conditions of VHL and HIF1α protein by Western blot analysis using anti-pVHL- and anti-pHIF1α specific antibodies in cell extracts of 786-0, RC-28, RC-26B, RC-13 and CAKI-1 cell lines. CAKI-1 expression of HIF1α was activated following 4 hours of continuous treatment with cobalt chloride at 50, 100 and 200 μM. Determination of actin expression served as a control for equal loading.

Article Snippet: The primary antibodies were survivin (polyclonal; Santa Cruz Biotech., Santa Cruz, CA, USA), actin (polyclonal), p21 (monoclonal), p53 (D01, monoclonal), VHL (11E12, mouse monoclonal, RB), and HIF1α (mouse monoclonal; BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Western Blot

Journal: Cancer Research

Article Title: Cells with Defective p53-p21-pRb Pathway Are Susceptible to Apoptosis Induced by p84N5 via Caspase-6

doi: 10.1158/0008-5472.can-07-0334

Figure Lengend Snippet: Figure 1. A, analyses of uninfected cells (control) and cells infected with AAV at various times after infection. Cellular DNA was stained with propidium iodide and analyzed by FACS. B, Western blot analysis of activated p53 (p53 Ser15P) and p21 from lysates of uninfected U2OS cells and AAV-infected cells at various days post infection. C, Western blot analyses of lysates of Saos-2 cells (c) that were transduced with either empty vector (v) or vector that carried a p21 gene (+p21). D, FACS analyses of DNA content and methylene blue staining of uninfected cells and AAV-infected untransduced and transduced cells.

Article Snippet: Equal amounts of protein were loaded onto SDSpolyacrylamide gels and transferred onto nitrocellulose membranes and analyzed with antibodies to p53 (Novocastra, NCL-p53-D01), phospho-Ser15 p53 (Cell Signaling), p21 (Santa Cruz Biotechnology), pRb (BD PharMingen), actin (Santa Cruz Biotechnology), caspase-3 and caspase-6 (Cell Signaling), or p84N5 (BD Transduction).

Techniques: Control, Infection, Staining, Western Blot, Transduction, Plasmid Preparation

Journal: Cancer Research

Article Title: Cells with Defective p53-p21-pRb Pathway Are Susceptible to Apoptosis Induced by p84N5 via Caspase-6

doi: 10.1158/0008-5472.can-07-0334

Figure Lengend Snippet: Figure 3. A, Western blot analysis of lysates from cells that were transduced with retroviruses encoding either scrambled shRNA (shC) or pRb-specific shRNA (shRb). Antibodies against pRb and p53 were used. On extensive exposure of the blot, a residual pRb protein band is observed in the shRb lane, indicating noncomplete abolition of the pRb protein by the shRNA. B, methylene blue staining of AAV-infected cells transduced with scrambled shRNA or pRb-specific shRNA.

Article Snippet: Equal amounts of protein were loaded onto SDSpolyacrylamide gels and transferred onto nitrocellulose membranes and analyzed with antibodies to p53 (Novocastra, NCL-p53-D01), phospho-Ser15 p53 (Cell Signaling), p21 (Santa Cruz Biotechnology), pRb (BD PharMingen), actin (Santa Cruz Biotechnology), caspase-3 and caspase-6 (Cell Signaling), or p84N5 (BD Transduction).

Techniques: Western Blot, Transduction, shRNA, Staining, Infection