Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: (A) BrdU incorporation after 4 days of TCR stimulation with the indicated treatments. Results are represented as absorbance relative to control (horizontal bar). Means ± SD of two independent experiments run in duplicates are shown (* p≤0.05; by t test). Control refers to no treatment for BMP2 and BMP4, unspecific immunoglobulins for BMPRIA-Fc and DMSO for DMH1. (B-D) After 4 days of TCR stimulation in the presence of DMSO or DMH1, the proliferation rate, measured by CFSE loss (B), Hoechst staining (C) and number of cells recovered (D), and the percentage of apoptotic/necrotic cells (E) were analyzed by flow cytometry. Bars represent means ± SD of five independent experiments (* p≤0.05; by t test). Cell counts were performed in duplicates and the means ± SD of at least 4 independent experiments are shown. Histograms in (C) and dot plots in (E) correspond to one representative experiment. (F) Cells were harvested at the indicated time points and stained for CD25, phosphorylated Smad-1/5/8 (pBR-Smad) and Hoechst. Cell populations were defined according to the expression of CD25 and pBR-Smad and the percentage of Hoechst positive cells was determined for each subset. Means ± SD of three independent experiments are shown (* p≤0.05; ** p≤0.01; by t test).

Article Snippet: Inhibition of BMP signaling was performed by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera (R&D Systems) or the inhibitor molecule DMH1 (Tocris) at the indicated doses, while unspecific mouse immunoglobulins or vehicle (DMSO), respectively, were used as controls.

Techniques: BrdU Incorporation Assay, Control, Staining, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: TCR-induced IL-2 production by T cells in the presence of DMSO or DMH1 at day 4 (A) and 2 (B, left graph). (B, right graph) mRNA expression for IL2 after 2 days of activation. GNB2L1 was used as endogenous control. Means ± SD of three to five independent experiments performed in duplicates are shown (* p≤0.05; ** p≤0.01; by t test). (C) Proliferation rate measured by CFSE loss in T cells after 4 days of TCR stimulation with DMSO, DMH1 alone or DMH1 supplemented with the indicated doses of rhIL-2. Bars represent the means ± SD of three independent experiments (* p≤0.05; by t test).

Article Snippet: Inhibition of BMP signaling was performed by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera (R&D Systems) or the inhibitor molecule DMH1 (Tocris) at the indicated doses, while unspecific mouse immunoglobulins or vehicle (DMSO), respectively, were used as controls.

Techniques: Expressing, Activation Assay, Control

Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: (A) Expression of BMP2/4 and BMP6 was determined by flow cytometry at the indicated time points in T cells cultured in media alone (grey histograms) or in the presence of IL-7 (5 ng/ml) (black histograms). Grey filled histograms represent isotype control stainings. A representative experiment out of four is shown. (B) Differential expression of phosphorylated Smad-1/5/8 (pBR-Smad) analyzed by flow cytometry in naive CD4 + T cells after 11 days of culture in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM). Percentages represent the increment relative to cultures in media alone. One representative of three independent experiments is shown.

Article Snippet: Inhibition of BMP signaling was performed by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera (R&D Systems) or the inhibitor molecule DMH1 (Tocris) at the indicated doses, while unspecific mouse immunoglobulins or vehicle (DMSO), respectively, were used as controls.

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Quantitative Proteomics

Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: (A) Naive CD4 + T cells cultured in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM) were harvested and counted at the indicated time points. Cell counts were performed in duplicates. Results represent the mean ± SD of four to twelve samples pooled from at least two independent experiments (** p≤0.01; *** p≤0.005; by t test. IL-7/DMSO vs IL-7/DMH1). (B) Differential expression of CD127 analyzed by flow cytometry after 36 hours of culture under the indicated conditions. Similar stainings were obtained in two independent experiments. (C) Proliferation rate measured by CFSE loss along 20 days in T cells cultured in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM). Means ± SD of four independent experiments are shown (* p≤0.05; by t test. IL-7/DMSO vs IL-7/DMH1). (D) Cell viability calculated as percentage of PI - /Annexin-V - cells throughout the culture. Means ± SD of four independent experiments are shown (** p≤0.01; *** p≤0.005; by t test. IL-7/DMSO vs IL-7/DMH1). (E) Bcl-2 levels determined by flow cytometry after 6 days of culture. White filled histograms represent media alone; grey-filled IL-7/DMSO; black-filled IL-7/DMH1. The mean fluorescence intensity is indicated in each histogram. Similar stainings were obtained in two independent experiments.

Article Snippet: Inhibition of BMP signaling was performed by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera (R&D Systems) or the inhibitor molecule DMH1 (Tocris) at the indicated doses, while unspecific mouse immunoglobulins or vehicle (DMSO), respectively, were used as controls.

Techniques: Cell Culture, Quantitative Proteomics, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: T cells were cultured in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM) and the expression of several homing receptors (A), CXCR4 (B) and CCR9 (C) was analyzed by flow cytometry after 36 hours of culture. Bars represent the mean ± SD of two independent experiments (* p≤0.05; by t test. IL-7/DMSO vs IL-7/DMH1).

Article Snippet: Inhibition of BMP signaling was performed by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera (R&D Systems) or the inhibitor molecule DMH1 (Tocris) at the indicated doses, while unspecific mouse immunoglobulins or vehicle (DMSO), respectively, were used as controls.

Techniques: Cell Culture, Expressing, Flow Cytometry