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

Journal: Nature Communications

Article Title: A tunable human intestinal organoid system achieves controlled balance between self-renewal and differentiation

doi: 10.1038/s41467-024-55567-2

Figure Lengend Snippet: a Schematic of screening strategy to optimize cultures of human intestinal organoids. b Schematic of the targeting strategy to generate LGR5-mNeonGreen reporter system. c Medium composition comparison among ES, IF, IL, and TpC culture systems. 1 Noggin or BMP pathway inhibitor DMH1, 2 R-Spondin1 conditioned media, 3 WNT3a protein, WNT3a conditioned media or WNT surrogate. d Representative brightfield and fluorescence images of LGR5-mNeonGreen organoids cultured in IL patterning condition, IF condition or TpC condition. Representative images from three independent experiments. Scale bars, 200 μm. Quantification of LGR5-mNeonGreen proportion ( e ) and relative LGR5-mNeonGreen intensity ( f ) in IF, IL patterning and TpC organoids cultured for 4 weeks. n = 3 samples. Quantification of colony forming efficiency ( g ) and cell proliferation as indicated by the number of cells ( h ) in IF, IL patterning and TpC organoids cultured for ten days from single cells (8000 cells per well seeding). n = 4 samples. One-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. i Proportion of secretory lineage cells in IF and TpC organoids quantified by positive staining of LYZ, MUC2, and CHGA, respectively. Two-tailed unpaired t -test; data are presented as mean ± SD; n = 3 samples. j Representative images of enterocytes (ALPI), goblet cells (MUC2), enteroendocrine (CHGA), and Paneth cells (LYZ) in TpC organoids. Representative images from five independent experiments. k Representative confocal images of LGR5-mNeonGreen and DEFA5 positive Paneth cells in TpC organoid. White arrowheads indicate Paneth cells adjacent to LGR5 intestinal stem cells, red arrowhead points out a double-positive cell. Representative images from four independent samples. l The growth and mNeonGreen expression of a single LGR5-mNeonGreen + cell cultured in TpC condition over 13 days. Also showing Paneth (LYZ), goblet (MUC2), and enteroendocrine (CHGA) cells in the 13-day colonies detected by immunofluorescence staining. Representative images from three biological replicates. Scale bars, ( j – l ), 50 μm. Arrowheads indicate the emergence of mNeonGreen expression at the same location. Source data for this Figure are provided as a Source Data file.

Article Snippet: TpC-hSIO culture medium was made by supplementing the basal medium with 50 ng/mL EGF(Peprotech), 100 ng/mL human IGF-I (Peprotech), 50 ng/mL FGF-2 (Peprotech), 2 μM DMH1 (ApexBio), 10% R-spondin1 conditioned medium, 10 nM Gastrin I (MCE), 0.5 μM A83-01 (ApexBio), 4 μM CHIR (ApexBio), 20 nM TSA (ApexBio), 100 μg/mL pVc (Sigma) and 0.5 μM CP673451 (TargetMol).

Techniques: Comparison, Fluorescence, Cell Culture, Staining, Two Tailed Test, Expressing, Immunofluorescence