Journal: PLoS ONE

Article Title: Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

doi: 10.1371/journal.pone.0059223

Figure Lengend Snippet: ( A ) Confocal microscopy images of MOLT3 cells infected with HHV-6B for 24, 48 or 72 hrs. The cells were analyzed with antibodies against p53 (Alexa488 green), p41 (Alexa546 red) and DAPI (blue). ( B ) Confocal microscopy images of HCT116 cells treated with or without doxorubicine or infected with HHV-6B for 48 hrs, followed by analysis with antibodies against p53 (red), p41 (green) and DAPI (blue). ( C ) Heat-map showing hierarchical cluster analysis on 128 p53-target genes. Red indicates high expression and blue indicates low expression. The symbol [denotes a cluster (c1 and c2) that responds to γ radiation in the wt cells, but with no or impaired response in the HHV-6B-infected cells and p53 −/− cells. ( D ) Functional distribution of genes from c1 and c2 in the heat-map analysis in (C). Cell death, 24.1%; Proliferation, 13.1%; Arrest/Repair, 31.1%; Other, 31.1%.

Article Snippet: HCT 116 cells were cultured in McCoy’s 5A medium, the human embryonic kidney cell line 293T (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), and the human leukemia T-cell line MOLT3 (a gift from Z. Berneman) was cultured in RPMI medium.

Techniques: Confocal Microscopy, Infection, Expressing, Functional Assay

Journal: PLoS ONE

Article Title: Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

doi: 10.1371/journal.pone.0059223

Figure Lengend Snippet: ( A & B ) Western blot analysis of PARP cleavage in cells either mock-treated or infected with HHV-6B for 48 hrs. The cells were subsequently treated with mock, leupeptin (10 µM), UV radiation (10 min exposure followed by 4 hrs of incubation), doxorubicin (0.2 µg/ml for 24 hrs), γ radiation (30 Gy followed by 24 hrs of incubation), or MG132 (10 µM). 7C7 was used as infection control and GAPDH was used as loading control. ( C & D ) Western blot analyses with antibodies against PARP, GAPDH, and 7C7 (infection control) on lysates from HCT116 (C) or MOLT3 (D) cells treated with varying doses of UV radiation (10, 20, 240, 600 J/cm 2 followed by 4 hrs of incubation). ( E ) ATP cell viability assay on MOLT3 cells with or without HHV-6B infection and treated with varying amounts of UV (0, 2.5, 5, 10, 15, 20, 40 and 60 J/cm 2 followed by 4 hrs of incubation) or γ radiation (0 and 30 Gy followed by 24 hrs of incubation). ( F ) Western blot analyses with antibodies against PARP, p53, PUMA, p21 or GAPDH (loading control) on lysates from HCT116 cells either mock-treated or infected with HHV-6B for 24 hrs followed by γ radiation (30 Gy) and 24 hrs of incubation. ( G ) Confocal microscopy images of HCT116 cells either mock-treated or infected with HHV-6B for 24 hrs, γ-irradiated (30 Gy) followed by 24 hrs of incubation. The cells were analyzed for cleaved caspase-3 (Alexa 488 green), DR6 (Alexa 546 red), and DAPI (blue).

Article Snippet: HCT 116 cells were cultured in McCoy’s 5A medium, the human embryonic kidney cell line 293T (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), and the human leukemia T-cell line MOLT3 (a gift from Z. Berneman) was cultured in RPMI medium.

Techniques: Western Blot, Infection, Incubation, Viability Assay, Confocal Microscopy, Irradiation

Journal: PLoS ONE

Article Title: Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

doi: 10.1371/journal.pone.0059223

Figure Lengend Snippet: ( A ) Western blot analyses of HCT116 cells infected for 24, 48, or 72 hrs and analyzed with antibodies against p53 p-Ser15, p53 p-Ser46, p53, 7C7 (infection control), or GAPDH (loading control). ( B ) Western blot analyses of nuclear (NF) and cytoplasmic (CF) fractions from mock-treated or HHV-6B-infected HCT116 cells (48 hpi). The membranes were stained with antibodies against p53, p53 p-Ser46, 7C7 (infection control), RCC1 (nuclear control), or GAPDH (cytoplasmic control). ( C ) Western blot analyses of HCT116 cells mock-treated or HHV-6B-infected for 24 hrs followed by γ irradiation (30 Gy) and additional incubation for 24 hrs. The membrane was probed with antibodies against p53, p53 p-Ser15, or GAPDH (loading control). Numbers at the bottom of the figure indicate fold induction of Ser15 phosphorylation relative to GAPDH. ( D & E ) Confocal microscopy on MOLT3 cells infected with HHV-6B for 24 hrs followed by staining with antibodies against the viral protein p41 (green), p53 (red) DAPI (blue). The p53 antibodies were either the monoclonal DO-7 (D) or a polyclonal antibody (FL393) (E). Arrows point to nuclear p53 staining. ( F ) Confocal microscopy on MOLT3 cells infected with HHV-6B for 24 hrs followed by staining with antibodies against p53 phospho-Ser20 (green), 7C7 (red) and DAPI (blue). ( G ) Confocal microscopy on MOLT3 cells infected with HHV-6B for 24 hrs, γ-irradiated (30 Gy) for 24 hrs followed by staining with antibodies against the viral protein DR6 (green), p53 (DO-7) (red) DAPI (blue).

Article Snippet: HCT 116 cells were cultured in McCoy’s 5A medium, the human embryonic kidney cell line 293T (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), and the human leukemia T-cell line MOLT3 (a gift from Z. Berneman) was cultured in RPMI medium.

Techniques: Western Blot, Infection, Staining, Irradiation, Incubation, Confocal Microscopy

Journal: PLoS ONE

Article Title: GFI1 Is Repressed by p53 and Inhibits DNA Damage-Induced Apoptosis

doi: 10.1371/journal.pone.0073542

Figure Lengend Snippet: (A) MO7e and Molt3 cells were infected with the lentivirus containing a p53 shRNA. The expression of GFI1 and p53 proteins was examined by Western blot analysis. GFI1 mRNA levels were examined by RT-PCR. Subsequently, control (Ctr) and p53 knocked down (KD) MO7e (B) and Molt3 (D) cells were treated with Doxo (25 ng/ml) for times as indicated and examined for expression of p53 and GFI1. The results shown in B and D were quantitated using ImageJ and normalized based on the levels of β-actin protein and GAPDH mRNA for MO7e (C) and Molt3 (E) cells. (F) Human umbilical cord blood CD34+ cells were treated with Doxo prior to evaluation of the expression of p53 and GFI1.

Article Snippet: Human Burkitt lymphoma cell line Ramos (American Type Culture Collection), human T lymphoblastic cell line Molt3 , and human myeloid leukemic cell lines U937 and HL-60 were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% P/S solution.

Techniques: Infection, shRNA, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: GFI1 Is Repressed by p53 and Inhibits DNA Damage-Induced Apoptosis

doi: 10.1371/journal.pone.0073542

Figure Lengend Snippet: (A) p53 −/− HCT116 cells were transfected with the GFI1 promoter (−1933/+468 bp) luciferase reporter construct along with the wild type (WT) or W248 mutant p53. Luciferase activities were measured 36 hrs later and normalized for β-Gal activities. Data are shown as mean ± SD. (B) p53 +/+ and p53 −/− HCT116 cells were transfected with GFI1 promoter luciferase reporter construct and treated with Doxo (400 ng/ml) 8 hrs later. Luciferase activities were measured 16 hrs after Doxo treatment. (C) p53 −/− HCT116 cells were transfected with pGL3-basic plasmid containing GFI1 promoter fragment (−4840/+184 bp) either alone or together with p53. ChIP assays were carried out using the anti human p53 or an irrelevant species-matched antibody. The indicated regions of GFI1 promoter were amplified by PCR. (D) ChIP experiment was carried out on Molt3 cells using the anti human p53 and control antibodies.

Article Snippet: Human Burkitt lymphoma cell line Ramos (American Type Culture Collection), human T lymphoblastic cell line Molt3 , and human myeloid leukemic cell lines U937 and HL-60 were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% P/S solution.

Techniques: Transfection, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Amplification

Journal: Viruses

Article Title: Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation

doi: 10.3390/v10120730

Figure Lengend Snippet: Assessment of specificity of primers in a human DNA background. MELT curves from each Sybr Green-based PCR detected the indicated virus. A single peak of dissociation (red arrow) was observed from each individual reaction ran in the presence of human genomic DNA from the MOLT3 cell line. Agarose gel electrophoresis (bottom right) showed that a unique PCR product of the expected size was amplified in each individual Sybr green-based PCR. MM: 100 bp (base pairs) molecular weight marker. Black arrowheads point at bands corresponding to 100 bp and 500 bp.

Article Snippet: Every standard also contained 100 ng of DNA from the human cell line MOLT3 (ATCC-CRL-1552) as background DNA.

Techniques: SYBR Green Assay, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker

Journal: Viruses

Article Title: Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation

doi: 10.3390/v10120730

Figure Lengend Snippet: Assessment of specificity of primers in a human DNA background. MELT curves from each Sybr Green-based PCR detected the indicated virus. A single peak of dissociation (red arrow) was observed from each individual reaction ran in the presence of human genomic DNA from the MOLT3 cell line. Agarose gel electrophoresis (bottom right) showed that a unique PCR product of the expected size was amplified in each individual Sybr green-based PCR. MM: 100 bp (base pairs) molecular weight marker. Black arrowheads point at bands corresponding to 100 bp and 500 bp.

Article Snippet: We first tested specificity of the primers in a background of human genomic DNA using DNA from the EBV, HCMV, HHV6, HHV7 and KSHV negative human cell line MOLT3 (ATCC-CRL-1552).

Techniques: SYBR Green Assay, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker