Journal: PLoS ONE

Article Title: Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax

doi: 10.1371/journal.pone.0148217

Figure Lengend Snippet: (A) Growing Kit 225 cells were transfected with RelA-specific siRNA and cultured for 72 h. RelA expression was examined by RT-PCR. GAPDH was used as an internal control. (B) Growing Kit 225 cells were transfected with RelA-specific siRNA 24 h before adenovirus infection (Ad-Tax1 or Ad-Con), and harvested 48 h and 72 h post infection for western blotting. RelA, p100, p52 and Tax1 expression was monitored by immunoblotting with anti-RelA, anti-p52 and anti-Tax1 antibodies. β-Tubulin was used as an internal control. (C) siRNA-treated growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 to Ad-Con are shown. *, p <0.05. (D) siRNA-treated growing Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for 72 h. Tax1 expression and DNA fragmentation were measured by flow cytometry. (E) Percentage average number of cells undergoing apoptosis was calculated from three independent experiments. Values are shown as the means ± SE. *, p < 0.05.

Article Snippet: Anti-RelA (sc-372), anti-p52 (sc-3786), anti-p38α (sc-535), anti-JNK (sc-571), anti-nucleolin (sc-13057) and β-tubulin (sc-9104) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Infection, Western Blot, Activity Assay, MTT Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax

doi: 10.1371/journal.pone.0148217

Figure Lengend Snippet: (A) Kit 225 cells were transfected with p100-specific siRNA and cultured for 48 h. p100 expression was detected by western blotting. β-Tubulin was used as an internal control. (B) Growing Kit 225 cells were transfected with p100-specific siRNA and cultured for 24 h, and infected with Ad-Con or Ad-Tax1, followed by harvest at indicated times. Mitochondrial activity was measured by MTT assay. Relative values of Ad-Tax1 to Ad-Con are shown. *, p < 0.05. (C) Expression of adenovirus-derived Tax1 and its mutant Tax225–232 proteins was measured by immunoblotting with anti-Tax1 antibody (Taxy-7). p100 and p52 levels were detected by anti-p52 antibody, which recognizes both p100 and p52. β-Tubulin was used as an internal control. (D) Growing Kit 225 cells were infected with Ad-Tax1, its mutant Ad-Tax225–232 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 or Ad-Tax225–232 samples to Ad-Con samples are shown. *, p < 0.05. (E) Growing Kit 225 cells were infected with Ad-Tax1 or Ad-Tax225–232, and cultured for 72 h. DNA fragmentation was measured by flow cytometry. The thick line and gray area indicate Ad-Tax1- or Ad-Tax225–232-treated cells and Ad-Con-treated cells, respectively.

Article Snippet: Anti-RelA (sc-372), anti-p52 (sc-3786), anti-p38α (sc-535), anti-JNK (sc-571), anti-nucleolin (sc-13057) and β-tubulin (sc-9104) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Expressing, Western Blot, Control, Infection, Activity Assay, MTT Assay, Derivative Assay, Mutagenesis, Flow Cytometry

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: Primary Antibodies Used in This Study

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Concentration Assay

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: Inhibition of the junctional protein VE-cadherin does not alter the expression level of IL-33 and knockdown of IL-33 does not alter the cellular distribution or expression level of junctional proteins CD31 or VE-cadherin. Immunocytochemical double staining for IL-33 (IL-33Nter, red) and VE-cadherin (mouse IgG1, green) in HUVECs preincubated with vehicle (PBS, A) or anti-VE-cadherin (IgG2a, B) from point of seeding until fixation. Pictures were obtained using identical exposure times and image-enhancement parameters. Arrowheads point to areas of intercellular contact. Note that cells in B with redistribution or lowered expression level of VE-cadherin maintained expression of IL-33. C and D: Immunocytochemical double staining for IL-33 (IL-33Nter, red) and CD31 (green) in HUVECs transfected with mock (C) or specific siRNA (D). E and F: Immunocytochemical double staining for IL-33 (red) and VE-cadherin (green) in HUVECs transfected with mock (E) or specific siRNA (F). Pictures were obtained using identical exposure times and image enhancement parameters. G: Western blot of IL-33 in cell lysates of HUVEC monolayers transfected with either scrambled siRNA controls (scr1 and scr2) or concentration-matched IL-33 targeting siRNA (spec1 and spec2). Shown are lanes from the same experiment, which was independently repeated twice with similar results. Scale bars = 10 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Inhibition, Expressing, Double Staining, Transfection, Western Blot, Concentration Assay