Journal: Viral Immunology

Article Title: Membrane Vesicles Released by Intestinal Epithelial Cells Infected with Rotavirus Inhibit T-Cell Function

doi: 10.1089/vim.2009.0113

Figure Lengend Snippet: RV infection induces release of larger quantities of proteins associated with MV. (A) Supernatants collected from RV-infected cells (MOI 5) or mock-infected Caco-2 cells for 24 h were filtered with 0.22-μm filters (F1), and ultracentrifuged at 100,000 × g for 90 min. The supernatant (F2) and pellet (F3) were recovered after ultracentrifugation. HSC70, HSP70, VP6, the ER protein calnexin, the exosome marker CD63, and lactadherin (MFG-E8, showing a 30-kDa intracellular protein form, and one 46 kDa in size associated with MV fractions) were evaluated in all fractions by Western blot. As a positive control we used 20 μg of cell lysate (Cx+), and equal volumes of each fraction were loaded in the gels. A representative Western blot of five independent experiments with similar results is shown. Western blots for CD63 identification were done in non-reducing conditions, producing a smeared band. (B) TGF-β1 was measured in five independent F3 preparations by ELISA. In three of these preparations AChE was simultaneously measured. The quantity of TGF-β1 is reported relative to the quantity of producing cells (p = 0.062 by Wilcoxon test for the comparison between mock- and RRV-infected F3), or to the AChE activity. The bars represent medians. (C) AChE activity was measured at 0, 6, 16, 24 and 48 h post-infection in apical and basolateral supernatants of RRV- and mock-infected Caco-2 cells, and the means from three independent experiments are shown.

Article Snippet: In some experiments, the CD4 + T cells were treated with 10 μM of the TGF-β receptor inhibitor ALK5i (SB431542; Sigma-Aldrich) for 30 min at 37°C before stimulation.

Techniques: Infection, Marker, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Viral Immunology

Article Title: Membrane Vesicles Released by Intestinal Epithelial Cells Infected with Rotavirus Inhibit T-Cell Function

doi: 10.1089/vim.2009.0113

Figure Lengend Snippet: The TGF-β-receptor inhibitor ALK5i restores viability and proliferation of T cells. CD4+ T cells purified by magnetic beads were stained with CFSE and treated with ALK5i before exposure to the polyclonal stimulus (SEB/α-CD49d/α-CD28), and the F3 of mock-infected or RRV-infected cells or media. After 5 d the cells were stained with violet viability dye and 1 × 105 cells were acquired and analyzed with a FACS Aria. (A) Percentages of viable CD4+ T cells are shown. (B) Percentages of proliferating CD4+ T cells (CFSElow) are shown. Bars represent medians of three independent experiments.

Article Snippet: In some experiments, the CD4 + T cells were treated with 10 μM of the TGF-β receptor inhibitor ALK5i (SB431542; Sigma-Aldrich) for 30 min at 37°C before stimulation.

Techniques: Purification, Magnetic Beads, Staining, Infection

Journal: Oncotarget

Article Title: Gremlin-1 is a key regulator of the invasive cell phenotype in mesothelioma

doi: 10.18632/oncotarget.21550

Figure Lengend Snippet: ( A ) Invasive growth of H2052 and JP5/grem1 cells was analyzed in 3D collagen 1 matrix in the presence of DMSO or TGF-β/activin receptor inhibitors (SB431542 or SB505124 at 10 μM concentration). Graphs show quantification as relative spheroid surface area. The error bars represent SD ( n = 3). * p < 0.05. ( B ) Expression of PAI1 , SNAI2 , MMP2, MMP14 and ITGAV in SB431542 treated mesothelioma cells (JL-1 and JP5/grem1). The results are expressed relative to each control (DMSO treated cells), which was set to 1. The error bars represent SD ( n = 3). * p < 0.05.

Article Snippet: TGF-βR inhibitors SB431542 and SB505124 were from Sigma-Aldrich and used at 10 μM concentration.

Techniques: Concentration Assay, Expressing

Journal: Oncotarget

Article Title: Gremlin-1 is a key regulator of the invasive cell phenotype in mesothelioma

doi: 10.18632/oncotarget.21550

Figure Lengend Snippet: ( A ) Tumor blood vessels imaged by photography. ( B ) Staining of tumor blood vessels using mouse CD31 specific antibody. The graph shows CD31 positive staining area (%) in JP5/ctrl and JP5/grem1 tumors. The error bars represent SD ( n = 6). * p < 0.05. ( C ) Expression of VEGFA gene in gremlin-1 silenced (grem1_siRNA) H2052 or JL-1 cells and in JP5/grem1 cells. The results are expressed relative to each control (control siRNA transfected or JP5/ctrl), which was set to 1. The error bars represent SD ( n = 3). * p < 0.05. ( D ) Expression of VEGFA in SB431542 treated mesothelioma cells (JL-1 and JP5/grem1). The results are expressed relative to each control (DMSO treated cells), which was set to 1. The error bars represent SD ( n = 3). * p < 0.05.

Article Snippet: TGF-βR inhibitors SB431542 and SB505124 were from Sigma-Aldrich and used at 10 μM concentration.

Techniques: Staining, Expressing, Transfection