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

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: Raji cells co-transfected with TAGLN2-GFP and the actin probe LifeAct-RFP were stimulated with IgM+IgG, and the localization of each protein was then followed over the next 18 min using time lapse fluorescence microscopy. Top: TAGLN2-GFP (Green), middle: LifeAct-RFP (Magenta), bottom: merged image. The images marked as 0 were acquired before stimulation. Both the actin and TAGLN2 signals increased in intensity and thickness after IgM+IgG stimulation and were depleted from the central area of the cells and moved together to the periphery.

Article Snippet: Raji B-cells (RCB3673) were provided by the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) through the National Bio-Resource Project of the MEXT, Japan and were maintained in RPMI 1640 medium supplemented with 10% FBS (Gibco, Thermo Fisher Scientific).

Techniques: Transfection, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: (A) The total protein levels of PLCγ2, PI3K, ERK, and Akt and the levels of their phosphorylated forms after 10 min IgM+IgG stimulation of TAGLN2 -knockdown (KD) Raji cells transfected with TAGLN2 siRNA, or of control cells with scrambled siRNA (Scramble), were detected by immunoblotting. Actin was blotted as a loading control. Suppression of TAGLN2 protein expression and PLCγ2 phosphorylation was seen in the TAGLN2 -KD Raji cells compared with the scrambled siRNA transfected cells. (B) Normalized expression levels of each protein and each phosphorylated protein assessed using membrane densitometry. Three independent experiments were performed. *p<0.05. (C) Significant inhibition of CXCL13-dependent TAGLN2 KD Raji cell chemotaxis as compared with negative controls. *p<0.05. RFU, relative fluorescence units. Migratory cells were lysed and quantified using fluorescent dye. The relative quantification was used to determine the change between various samples. Data were normalized by designating one sample in negative controls as equal to 1. Then, the ratiometric results were used to scale all values relative to that sample. (D) The CXCR5 and IgM expression levels were assessed by flow cytometry in TAGLN2 KD and control Raji cells. TAGLN2 KD appeared to have no effect on the surface expression of CXCR5 and IgM. Gray shading indicates isotype control.

Article Snippet: Raji B-cells (RCB3673) were provided by the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) through the National Bio-Resource Project of the MEXT, Japan and were maintained in RPMI 1640 medium supplemented with 10% FBS (Gibco, Thermo Fisher Scientific).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Phospho-proteomics, Membrane, Inhibition, Chemotaxis Assay, Fluorescence, Quantitative Proteomics, Flow Cytometry

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: Pathway analysis of microarray data. Functional grouping of significant, differentially expressed down regulated genes in TAGLN2 siRNA Raji cells versus the control (p <0.01).

Article Snippet: Raji B-cells (RCB3673) were provided by the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) through the National Bio-Resource Project of the MEXT, Japan and were maintained in RPMI 1640 medium supplemented with 10% FBS (Gibco, Thermo Fisher Scientific).

Techniques: Microarray, Functional Assay, Control, Ligand Binding Assay, Gene Expression, Clinical Proteomics, Membrane, Ubiquitin Proteomics, Transduction

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: (A) Validation of the microarray data regarding the change in expression of specific genes in TAGLN2 -knockdown Raji cells compared to control cells. Expression of the indicated genes was assessed using qRT-PCR and is indicated as fold change relative to that in control Raji cells transfected with scrambled siRNA. Results are the mean ± SEM of triplicate experiments. *p<0.05. (B) CCR6 is colocalized with TAGLN2 + B-cells in GC areas of enlarged follicles in lymph nodes in SLE patients. Double immunofluorescence of CCR6 and TAGLN2 was performed. Nuclei were stained with DAPI. Co-localization of CCR6 and TAGLN2 is indicated in yellow in the merged image (original magnification, x400). GC, germinal center.

Article Snippet: Raji B-cells (RCB3673) were provided by the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) through the National Bio-Resource Project of the MEXT, Japan and were maintained in RPMI 1640 medium supplemented with 10% FBS (Gibco, Thermo Fisher Scientific).

Techniques: Biomarker Discovery, Microarray, Expressing, Knockdown, Control, Quantitative RT-PCR, Transfection, Immunofluorescence, Staining