Journal: PLoS ONE

Article Title: A Novel BAT3 Sequence Generated by Alternative RNA Splicing of Exon 11B Displays Cell Type-Specific Expression and Impacts on Subcellular Localization

doi: 10.1371/journal.pone.0035972

Figure Lengend Snippet: HeLa cells (Adenocarcinoma), the human melanoma cell line MelJuSo, the B lymphoma Raji and monocyte-derived dendritic cells (moDCs) were plated on coverslips and stained for BAT3 using a polyclonal serum against a C-terminal peptide (middle lane). Cell nuclei (left lane) were visualized with DAPI (A) or 7AAD (B). Merged images are shown in the right lane. A. Immunofluorescence staining was evaluated with a standard fluorescence microscope and B. by confocal microscopy. Scale bars = 10 µm. C. Nuclear and cytosolic staining of endogenous BAT3 in Raji cells was evaluated in 10 single cells using ImageJ. MFI, mean fluorescence intensity per region of interest D. Western blot analysis of subcellular fractions from Raji and HeLa cells. Nuclei (N) and cytoplasm (C) were separated by SDS-PAGE and immunoblotted for BAT3, GADPH (cytosolic marker) and histone H3 (nuclear marker).

Article Snippet: The B lymphoma cell line Raji was cultured in RPMI and dendritic cells were grown in VLE-RPMI supplemented with antibiotics and glutamine (Biochrom AG, Berlin, Germany).

Techniques: Derivative Assay, Staining, Immunofluorescence, Fluorescence, Microscopy, Confocal Microscopy, Western Blot, SDS Page, Marker

Journal: PLoS ONE

Article Title: A Novel BAT3 Sequence Generated by Alternative RNA Splicing of Exon 11B Displays Cell Type-Specific Expression and Impacts on Subcellular Localization

doi: 10.1371/journal.pone.0035972

Figure Lengend Snippet: A. HeLa cells transfected with BAT3 Δ11B,24 were cultured on coverslips in the presence (lower panel) or absence (upper panel) of leptomycin B (LMB) for 2 h. Cells were subsequently stained with V5 mAb and inspected with standard immunofluorescence microscopy (second panel). Left panel shows DAPI stained nuclei, third panel merging of images and right panel displays phase contrast images. Scale bars = 10 µm. B. Raji cells were cultured for 2 h in the presence (lower panel) or absence (upper panel) of LMB and then plated on coverslips. Cells were subsequently stained with the polyclonal anti-BAT3 serum and with ISCR3 mAb (HLA-DR) for evaluation by immunofluorescence microscopy. Left panel shows DAPI staining, second panel staining for BAT3, third panel staining for HLA-DR and images were merged in the right panel. Scale bars = 5 µm.

Article Snippet: The B lymphoma cell line Raji was cultured in RPMI and dendritic cells were grown in VLE-RPMI supplemented with antibiotics and glutamine (Biochrom AG, Berlin, Germany).

Techniques: Transfection, Cell Culture, Staining, Immunofluorescence, Microscopy

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: V δ 2 + γδ T cells release granulysin in response to tumour. (a) The expression of exhaustion markers PD‐1 and Lag‐3 on, and the secretion of granulysin by, V δ 2 + γδ T cells during the 9‐day expansion process. (b) The percentage of V δ 2 + γδ T cells to express early activation marker CD69 following 24, 48 or 72 hr of culture with Daudi cells, Raji cells or Raji cells pre‐treated for 24 hr with 5 μ m zoledronic acid (ZA), as determined by flow cytometry. (c) The concentration of interferon‐ γ (IFN‐ γ ) found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (d) The percentage of V δ 2 + γδ T cells to express degranulation marker CD107a following 24, 48 or 72 hr of culture with Daudi cells, Raji cells or Raji cells pre‐treated for 24 hr with 5 μ m ZA, as determined by flow cytometry. (e) The concentration of granulysin found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (f) The concentration of granzyme B found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (g) Percentage killing of tumour cells by V δ 2 + γδ T cells following 24, 48 and 72 hr of culture, as determined by flow cytometry. Data shown are from six independent experiments, using V δ 2 + γδ T cells from six individual donors, with error bars (SD). Differences between groups were assessed by two‐way analysis of variance comparing negative control (V δ 2 + γδ T cells alone) with all other groups. * P < 0·05. ** P < 0·01. *** P < 0·001. **** P < 0·0001.

Article Snippet: Daudi and Raji B‐cell lymphoma lines (European Collection of Authenticated Cell Cultures, Salisbury, UK) were used in experiments as γδ T‐cell‐susceptible and ‐resistant target cells, respectively.

Techniques: Expressing, Activation Assay, Marker, Flow Cytometry, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Negative Control