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: 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: The Journal of Biological Chemistry

Article Title: Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16 INK4a Protein and Confers Resistance to Oxidative Stress *

doi: 10.1074/jbc.M112.447821

Figure Lengend Snippet: Up-regulation of survivin during immortalization confers resistance to tBOOH. A, immunoblot analysis of survivin and p16INK4a in parental MRC5 cells and MRC5hTERT-1 cells at the indicated PDs (left panel) as well as precrisis MRC5hTERT cells transduced with MIGR1-survivin (SURV) or MIGR1 control vector (GFP) (right panel). HeLa cells were included as a positive control for detection of survivin and p16INK4a. B, MRC5hTERT-1 cells transduced with SURV or GFP retroviral vectors were treated with tBOOH for 48 h. Cell expansion was determined by trypan blue exclusion. C, quantification of apoptotic cells by annexin V-APC/PI staining. Values in B and C are presented as means ± S.E. calculated from four to five independent experiments. p values indicate the overall effect of retroviral transduction as determined by two-way ANOVA. *, p < 0.05 in Bonferroni post-test comparisons with normal GFP cells.

Article Snippet: The antibodies used were rabbit anti-survivin NB500-201 (Novus Biologicals, Littleton, CO), rabbit anti-actin (Sigma-Aldrich), mouse anti-p16INK4a (BD Biosciences), mouse anti-p53 clone D0–1 (Santa Cruz Biotechnology, Inc.), mouse anti-p21 cip1 (BD Biosciences).

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

Journal: The Journal of Biological Chemistry

Article Title: Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16 INK4a Protein and Confers Resistance to Oxidative Stress *

doi: 10.1074/jbc.M112.447821

Figure Lengend Snippet: Repression of p16INK4a enhances survival and proliferation of tBOOH-treated MRC5hTERT-1 cells. EP MRC5hTERT-1 cells were transduced with retroviral vectors carrying shRNA targeting p16INK4a (p16 shRNA) or an irrelevant gene (murine p63) as a control. A, qRT-PCR was performed to confirm down-regulation of p16INK4a mRNA in p16 shRNA-transduced cells relative to control vector-transduced cells. Values were calculated relative to p16INK4a expression in HeLa cells and are presented as means + S.E. from three assays, each with duplicate samples. B and C, transduced cells were treated for tBOOH for 48 h, counted using trypan blue (B), and assayed for apoptosis (C) by annexin V/PI staining. Values are expressed as means + S.E. calculated from four to five independent experiments. D, EP MRC5hTERT-1 cells transduced with p16 shRNA or control shRNA were maintained in replicate cultures and treated (or not treated) with tBOOH. On days 7 and 14, replicate cultures were stained with x-gal at pH 6.0 to detect senescence-associated β-galactosidase activity in senescent cells. Values are expressed as means + S.E. calculated from seven independent experiments. p values are from two-way ANOVA analysis of the effect of shRNA. *, p < 0.05; ***, p < 0.001 in Bonferroni post-test comparisons of p16 shRNA and control shRNA at the indicated tBOOH concentrations.

Article Snippet: The antibodies used were rabbit anti-survivin NB500-201 (Novus Biologicals, Littleton, CO), rabbit anti-actin (Sigma-Aldrich), mouse anti-p16INK4a (BD Biosciences), mouse anti-p53 clone D0–1 (Santa Cruz Biotechnology, Inc.), mouse anti-p21 cip1 (BD Biosciences).

Techniques: Transduction, shRNA, Quantitative RT-PCR, Plasmid Preparation, Expressing, Staining, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16 INK4a Protein and Confers Resistance to Oxidative Stress *

doi: 10.1074/jbc.M112.447821

Figure Lengend Snippet: Repression of p16INK4a increases expression of survivin and promotes immortalization. MRC5hTERT-1 cells transduced with p16 shRNA or control shRNA were maintained in long-term culture and sampled intermittently to determine expression of p16INK4a and survivin. A, qRT-PCR analysis of p16INK4a (top panel) and survivin (bottom panel) gene expression in transduced cells. Population doublings counted from the initiation of the original culture are indicated beneath the bottom of the graph. The arrow highlights the spontaneous down-regulation of p16INK4a expression in the control cells. Values were normalized to expression in HeLa cells and presented as means + S.E. B, immunoblot analysis of p16INK4a and survivin in shRNA-transduced MRC5hTERT-1 cells. Actin was used as a loading control. C, graphical representation of the long-term proliferation of MRC5hTERT-1 cells transduced with p16 shRNA or control shRNA. D, qRT-PCR analysis of p16INK4a and survivin gene expression in early-passage and immortal hTERT-transduced WI-38 cells. Results are shown for two independently transduced and immortalized cell lines. Values were normalized to expression levels in HeLa cells and presented as means + S.E. from four assays, each performed with duplicates.

Article Snippet: The antibodies used were rabbit anti-survivin NB500-201 (Novus Biologicals, Littleton, CO), rabbit anti-actin (Sigma-Aldrich), mouse anti-p16INK4a (BD Biosciences), mouse anti-p53 clone D0–1 (Santa Cruz Biotechnology, Inc.), mouse anti-p21 cip1 (BD Biosciences).

Techniques: Expressing, Transduction, shRNA, Quantitative RT-PCR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16 INK4a Protein and Confers Resistance to Oxidative Stress *

doi: 10.1074/jbc.M112.447821

Figure Lengend Snippet: Repression of p16INK4a is intrinsically linked to survivin by E2F1 binding at the BIRC5 promoter and resistance to apoptotic cell death. A, EP MRC5hTERT-1 cells transduced with p16 shRNA or control shRNA were transfected with Sc or Survivin siRNA and then treated with tBOOH for 48 h. Apoptotic cells were quantified by annexin V-APC/PI staining. Values are presented as means ± S.E. from three independent experiments. The p value is from ANOVA analysis of the effect of shRNA. *, p < 0.05; ***, p < 0.001 in Bonferroni post-test comparisons of p16 shRNA and control shRNA at the indicated tBOOH concentrations. B, E2F1 and IgG (control) antibodies were used in ChIP assays of immortal MRC5hTERT-1 (279 PD) and precrisis MRC5hTERT-1 cells transduced with p16 shRNA or control shRNA. Values obtained with the E2F1 antibody are presented relative to IgG values. The results are means + S.E. from three assays, with each performed in duplicate. **, p < 0.01 in Student's t test comparisons of control and p16 shRNA-transduced cells.

Article Snippet: The antibodies used were rabbit anti-survivin NB500-201 (Novus Biologicals, Littleton, CO), rabbit anti-actin (Sigma-Aldrich), mouse anti-p16INK4a (BD Biosciences), mouse anti-p53 clone D0–1 (Santa Cruz Biotechnology, Inc.), mouse anti-p21 cip1 (BD Biosciences).

Techniques: Binding Assay, Transduction, shRNA, Transfection, Staining

Journal: Journal of Virology

Article Title: Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

doi: 10.1128/jvi.01442-10

Figure Lengend Snippet: FIG. 1. E1B-55K induces SUMO1 modification of p53 in Ad5-infected cells. (a) A549 cells (p53) were infected with Ad5 mutants (indicated at the top) with inactivating mutations in the indicated viral genes (). (Top) Anti-p53 Western blot (WB) assay of total cell extract 20 hpi; the molecular mass marker is 55 kDa. (Bottom) Extracts were immunoprecipitated with anti-SUMO1 or control rabbit IgG, as indicated, and the immunoprecipitate (IP) was Western blotted with anti-p53. The red dot in panels a and b indicates the position of SUMO1-p53. HC is IgG heavy chain. The images come from the same exposure of Western blot assays of D0-1 and control IgG immunoprecipitates prepared at the same time. (b and c) The same extracts of infected p53 A549 cells were immunoprecipitated with anti-p53 MAb D0-1, and the immunoprecipitates were Western blotted with antibody to SUMO1 (b) or SUMO2 and SUMO3 (c). The vertical red line in panel c denotes specific bands detected with the anti-SUMO2/3 MAb. (d) H1299 (p53) cells were transfected with the indicated expression vectors, and whole-cell extract was prepared after 24 h and Western blotted with anti-p53 (lanes 1 to 6). In lanes 7 to 10, extracts were immunoprecipitated with anti-p53 MAb and the IP was Western blotted with anti-SUMO1 (7, 8) or anti-Flag (9, 10). (e) H1299 cells were transfected with expression vectors for E1B-55K and p53, whole-cell extract was prepared at 24 h posttransfection, 2-fold dilutions were run in neighboring lanes of an SDS gel, and the gel was subjected to Western blotting with anti-p53 MAb. Arrowheads indicate bands of p53-SUMO1 and p53 of comparable intensities.

Article Snippet: Alternatively, the diluted SDS lysis buffer was subjected to preclearing with Santa Cruz mouse IgG conjugated to agarose (sc-2343), followed by immunoprecipitation as described above with anti-p53 MAb D0-1 coupled to agarose (sc-126 AC).

Techniques: Infection, Western Blot, Marker, Immunoprecipitation, Control, Transfection, Expressing, SDS-Gel

Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: Mutant p53 and p63 bind to the let-7i promoter to regulate let-7i expression. (a) Schematic representation of let-7i promoter region containing the putative mutant p53-binding site. (b) H1299 cells stably expressing EV or p53R273H were cotransfected with the luciferase promoter plasmid carrying the let-7i promoter (pGL4-let-7i) and pRL-TK (internal control). Luciferase activity was measured after 36 h (left panel). Repression of let-7i promoter by mutant p53 was also analyzed by cotransfecting H1299-p53R273H cells with control (CTL) or p53 siRNAs and pGL4-let-7i for 48 h and measuring luciferase activity (right panel). (c) Parental H1299 cells were cotransfected with the let-7i promoter reporter (pGL4-let-7i), EV or p53R273H plasmids and pRL-TK (internal control). Luciferase activity was measured after 48 h. The effect of stable knockdown of mutant p53 in MDA-MB-231 and MIA-PaCa-2 cells on mature let-7i (d) and primary let-7i (e) levels was assessed by qRT–PCR normalized to U6 or UBC. (f) Mutant p53 co-immunoprecipitates with p63 and p73 in whole-cell lysates prepared from MDA-MB-231 cells. (g) Chromatin immunoprecipitation (ChIP) assays were performed to pulldown mutant p53 or p63 from MDA-MB-231 cells. Fold enrichment of the let-7i promoter region in the mutant p53 or p63 ChIP was determined by qPCR. (h) MDA-MB-231 cells were transfected with CTL or p63 siRNAs for 48 h and the levels of primary let-7i transcript (left panel) or mature let-7i (right panel) was assessed by qRT–PCR normalized to UBC or U6.

Article Snippet: 55 For each IP, 2.0 μg of anti-p53 (D0–1, Santa Cruz, Santa Cruz, CA, USA), anti-p63 (H-137, Santa Cruz), anti-p73 (Bethyl, Montgomery, TX, USA), anti-Ago2 (Mouse 2A8), rabbit IgG, mouse IgG and anti-HA beads was used.

Techniques: Mutagenesis, Expressing, Binding Assay, Stable Transfection, Luciferase, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Transfection