Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of the Zebrafish Cell Landscape at Single-Cell Resolution

doi: 10.3389/fcell.2021.743421

Figure Lengend Snippet: Genetic regulation during tissue regeneration. (A) Virtual inference of protein-activity by enriched regulon analysis in caudal fin Replicate 1. Red represents activated transcription factors; blue indicates repressed transcription factors. Act, activation. (B) A gene-gene correlation network of regeneration module. Red corresponds to high activation transcription factors; blue corresponds to low activation transcription factors; orange corresponds to co-factor genes; green corresponds to targeted genes. (C) Gene set enrichment analysis of caudal fin regeneration module in Replicate 1. (D) Gene set enrichment analysis between caudal fin regeneration module and non-regeneration module in Replicate 1. (E) Histogram showing BMP signaling pathway-related gene expression of each subgroup in caudal fin regeneration from Replicate 1. (F) Scheme of DMH1 and dorsomorphin treatment from –12 h to 3 dpa. dpa, days post-amputation. (G,H) DMH1 and dorsomorphin (Dor) treatment both significantly inhibited fin regeneration from –12 h to 3 dpa (pre-blastema formation, blastema formation, and regenerative outgrowth stages), when compared to DMSO treatment. Red dashed lines indicate the amputation planes. ** p < 0.01 by Student’s t -test. Error bars represent the standard error of 4 independent experiments. dpa, days post-amputation; scale bars, 500 μm in (G) .

Article Snippet: 10 μM DMH1 (Target Mol), and 10 μM Dorsomorphin (Target Mol) were used as specific BMP inhibitors.

Techniques: Activity Assay, Activation Assay, Gene Expression

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: Toxins

Article Title: Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays

doi: 10.3390/toxins12050276

Figure Lengend Snippet: Cell culture supplements.

Article Snippet: On the next day, the medium was exchanged with neural medium containing 3 μM CHIR99021 (CHIR, Axon Medchem, Groningen, Netherlands), 2 μM Dorsomorphin homolog 1 (DMH1) (Bertin Pharma, Montigny le Bretonneux, France), and 2 μM SB431542 (SB, Stemcell, Cologne, Germany).

Techniques: Cell Culture

Journal: Development (Cambridge, England)

Article Title: Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells

doi: 10.1242/dev.112979

Figure Lengend Snippet: Elongation process. (A) Examples of aggregates from Sox1::GFP mESCs aggregated for 2 days in N2B27 before further treatment with N2B27, which may include a 24 h pulse on day 3 with Chiron, 3 days of Chiron, or Chiron and the MEK inhibitor PD03, or 3 days with the BMP inhibitor DMH1. Aggregates were imaged on day 5 by wide-field epifluorescence microscopy. The phase-contrast and fluorescence images shown are representative examples. Aggregates exposed to a 24 h pulse of Chiron are able to show a single large extension containing Sox1::GFP-expressing cells with a region at the tip that is negative for the fluorescence reporter. (B) Wnt reporter TLC2 mESC aggregates differentiated in N2B27 with a 24 h pulse of Chiron on day 3 and imaged on day 5. (C) Aggregate as in B fixed and immunostained for brachyury (white) and Sox2 (green). The fluorescent reporter is expressed predominately in the aggregate extension corresponding to a Sox2 + Bra + region. The white arrowhead indicates the region of highest Sox2 expression. Hoechst was used to label the nuclei. Scale bars: 500 µm in A; 200 µm in B,C.

Article Snippet: Using Sox1::GFP mESCs and exposing them to continuous differentiation in N2B27, we observe the emergence of Sox1::GFP-positive cells throughout the aggregate ( A) and a similar pattern is observed if, on day 3, the aggregates are exposed to DMH1, a BMP inhibitor ( A; 2-5 DMH1), SB43, an activin/nodal inhibitor (data not shown) or PD03 ( A; 2-5 Chi+PD03).

Techniques: Epifluorescence Microscopy, Fluorescence, Expressing