Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is induced by BMP6. (A) Hep3B cells were treated with 5, 25, and 50 ng/mL of human BMP6 for 16 hours and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 3 to 8 (depending of the dose) independent experiments is presented. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock. (B-C) Hep3B cells were transfected with siRNA control (5nM), siRNA TMPRSS6 (5nM), or TMPRSS6-FLAG (8 μg), and treated with 25 ng/mL of BMP6 for 48 hours. Cells were analyzed for matriptase-2 level relative to pan-cadherin protein by Western blot (B) followed by chemiluminescence quantification (C). (B) *A shorter exposure of lane 5 to better distinguish the 2 bands. (C) The mean of 3 experiments is presented, and results are reported as the mean ± SEM. (D) A total of 15 μg of protein from conditioned media of Hep3B cells transfected with siRNA control (5nM) and siRNA TMPRSS6 (5nM) and treated with BMP6 (25 ng/mL) for 48 hours were incubated with 666μM of N-(tert-butoxycarbonyl)-Gln-Ala-Arg-p-nitroanilide. Activity of matriptase-2 was assessed by measurement of the release of the dye p-nitroaniline during up to 20 minutes at a wavelength of 405 nm at 37°C using a spectrophotometer. The resulting activities (1 U corresponds to a release rate of 1 mmol of p-nitroaniline per minute) were measured in duplicate in 3 independent experiments. Results are reported as the mean ± SEM. (A,C-D) Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: 35 For BMP6 antibody experiments, 8-week-old males received an intraperitoneal injection of monoclonal anti-BMP6 antibody (R&D Systems) 15 mg/kg daily for 1 week.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Incubation, Activity Assay, Spectrophotometry

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Regulation of TMPRSS6 mRNA expression in response to BMP6. Hep3B cells were treated with 25 ng/mL of BMP6 for several time points between 1 and 48 hours (A), and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. (B-C) Hep3B cells received 10 μg/mL of cycloheximide (B) or 60nM of LDN-193189 (C) before BMP6 (25 ng/mL) treatment and were analyzed for gene expression relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 6 (A-B) and 3 (C) independent experiments is presented. (B-C) Results are reported as the mean ± SEM for the fold change from mock (just before adding BMP6) and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: 35 For BMP6 antibody experiments, 8-week-old males received an intraperitoneal injection of monoclonal anti-BMP6 antibody (R&D Systems) 15 mg/kg daily for 1 week.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is controlled by ID1 in response to BMP6. Hep3B cells transfected with 10nM of siRNA control, siRNA SMAD7 (A-B), or siRNA ID1 (C-D), and treated in the absence or presence of 25 ng/mL of BMP6 for 24 hours were analyzed for TMPRSS6 (B,D) SMAD7 (A), and ID1 (C) mRNA expression relative to RPL19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock (siRNA control alone). Significant changes are as follows: *P < .05.

Article Snippet: 35 For BMP6 antibody experiments, 8-week-old males received an intraperitoneal injection of monoclonal anti-BMP6 antibody (R&D Systems) 15 mg/kg daily for 1 week.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is up-regulated in vivo by BMP6 and chronic iron treatment. (A) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of BMP6 750 μg/kg animal weight (BMP6, black bars) or vehicle alone (mock, gray bars; n = 3 per group) for 6 and 12 hours. (B) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of neutralizing BMP6 antibody 15 mg/kg once a day for 1 week (n = 5 per group). (C) Seven-week-old male C57BL/6 mice were killed at time zero (baseline) or after initiation of a 2% carbonyl iron diet for 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks (n = 6 per group). Tissues were analyzed for hepatic hepcidin and Tmprss6 relative to Rpl19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; and ***P < .005.

Article Snippet: 35 For BMP6 antibody experiments, 8-week-old males received an intraperitoneal injection of monoclonal anti-BMP6 antibody (R&D Systems) 15 mg/kg daily for 1 week.

Techniques: Expressing, In Vivo, Injection, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Schematic representation showing proposed role of TMPRSS6 regulation by the BMP6-SMAD signaling pathway and iron via ID1. We propose that, in addition to being stimulated by several signals that inhibit hepcidin, such as iron deficiency, erythropoeitic drive, and hypoxia, TMPRSS6 expression is also stimulated indirectly by the hepcidin activators BMP6 and iron. Stimulation by BMP6 and/or iron induces an increase of the BMP6-HJV-SMAD pathway activity, possibly through a mechanism involving HFE and transferrin receptor 2 (TFR2), leading to binding of SMAD complexes to BMP-responsive elements (BMP-REs) on the hepcidin promoter and up-regulation of hepcidin transcription. In parallel, BMP6-SMAD pathway directly up-regulates SMAD7 and ID1 transcription. ID1 induction leads to the up-regulation of TMPRSS6 expression. TMPRSS6 then serves as a negative feedback inhibitor of BMP-SMAD pathway activity and hepcidin expression by cleaving the BMP coreceptor HJV. Inhibitory SMAD7 can also act as a negative feedback inhibitor by blocking SMAD activation. By acting as negative feedback inhibitors, TMPRSS6 and SMAD7 are important to prevent excessive hepcidin increases in response to BMP6 and iron, thereby maintaining tight control of iron homeostasis. Abbreviations: BMPR indicates BMP receptor; TF-Fe, holotransferrin; sHJV, soluble hemojuvelin; and BMP-RE, BMP responsive element.

Article Snippet: 35 For BMP6 antibody experiments, 8-week-old males received an intraperitoneal injection of monoclonal anti-BMP6 antibody (R&D Systems) 15 mg/kg daily for 1 week.

Techniques: Expressing, Activity Assay, Binding Assay, Blocking Assay, Activation Assay, Control

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: BMP-6 inhibits proliferation of human B cells . A) CD19 + B cells were isolated from peripheral blood and stimulated in triplicates with anti-IgM (37.5 μg/ml) or anti-IgM and CD40 ligand (CD40L; 10 ng/ml) in the presence or absence of BMP-6 for 72 hours. DNA synthesis was measured by [3H]-thymidine incorporation for the last 18 hours. Data are given as relative proliferation obtained by normalizing the mean counts per minute (cpm) for each stimulation to the mean cpm obtained for anti-IgM stimulated cells ± SEM. (mean cpm = 25 352 for anti-IgM stimulated cells, *p ≤ 0.0002 (n = 8), **p ≤ 0.023 (n = 6). B) Dose dependent inhibition of BMP-6 of anti-IgM induced DNA-synthesis of CD19 + B cells (relative proliferation ± SEM, n = 3) and C) the Burkitt lymphoma cell line Ramos (relative proliferation ± SEM, n = 3). Ramos cells were cultured for 72 hours and [3H]-thymidine were added for the last 4 hours. D) Noggin (5 μg/ml) and BMP-6 (0,25 or 1 μg/ml) were preincubated for 1 h at 37°C and then added to the CD19 + B cells in the presence of anti-IgM(37.5 μg/ml). Cells were cultured for 72 h and DNA synthesis was measured by 3 H-thymidine incorporation. One representative of three separate experiments is shown (mean cpm ± SD of triplicates). E) Highly purified CD19 + CD27 - or CD19 + CD27 + cells were obtained by cell sorting of CD19 + cells and stimulated with anti-IgM in the presence or absence of BMP-6, as indicated for 72 hours. DNA synthesis was measuredby [3H]-thymidine incorporation. Data are given as relative proliferation obtained by normalizing the mean cpm for each stimulation to the mean cpm obtained for anti-IgM stimulated cells (mean cpm = 18 221 for CD19 + CD27 - naïve B cells, mean cpm = 8 930 for CD19 + CD27 + memory B cells, n = 5; * p ≤ 0,004, **p ≤ 0.001).

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Isolation, DNA Synthesis, Inhibition, Cell Culture, Purification, FACS

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: Ramos cells were cultured in the presence or absense of BMP-6 (1 μg/ml) for 48 h before analysis of cell death by PI staining. Data are shown as mean percentage PI + cells (± SEM, n = 3, p ≤ 0,001).

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Cell Culture, Staining

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: BMP-6 induces cell death in B cells . CD19 + CD27 - naïve B cells or CD19 + CD27 + memory B cells were cultured for 48 h with BMP-6 (1 μg/ml) with or without anti-IgM (37.5 μg/ml). Cell death (PI + cells) was then measured by flow cytometry analysis. Data are shown as mean percentage PI + cells from five independent donors (± SEM; p ≤ 0,003).

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Cell Culture, Flow Cytometry

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: BMP-6-induced phosphorylation of Smad1/5/8 in CD19 + B cells and Ramos, but not in HL60 cells . CD19 + B cells, or the cell lines Ramos or HL60

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques:

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: were cultured in X-vivo 15 over night before treatment with BMP-6 for 30 minutes, or for the indicated time points before total cell lysates were prepared. The amount of phosphorylated Smad 1/5/8 was determined by western-blot analysis. The membranes were reprobed for Smad1. One representative experiment of three is shown.

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Cell Culture, Western Blot

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: BMP-6 induced upregulation of Id1 at the mRNA and protein level . CD19 + B cells were cultured in X-vivo 15 over night before treatment with BMP-6 for the indicated time points. Total RNA was extracted and Id1 , Id2 or Id3 -expression was analysed by real-time RT-PCR; values are normalised to the expression level of PGK1 mRNA and expressed as relative quantification (2 -average ΔΔC T – relative to Id1 , Id2 or Id3 expression in the cell-line Ramos). One representative of three independent experiments is shown.

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: Id protein expression was determined by western-blot analysis. CD19 + B cells were cultured in X-vivo 15 over night before treatment with BMP-6 for the indicated time points and cell lysates were prepared. One representative experiment of three is shown. HeLa cells were used as a positive control for Id1 protein detection.

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Expressing, Western Blot, Cell Culture, Positive Control

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: Anti-IgM rapidly upregulates BMP-6 mRNA expression in human B-cells . CD19-positive B cells were cultured in X-vivo 15 over night and stimulated with anti-IgM for the indicated time periods, before total RNA was extracted. The expression of BMP-6 mRNA by realtime RT-PCR; values are normalised to the expression level of PGK1 mRNA and expressed as relative quantification (2 -average ΔΔC T – relative to BMP-6 expression in the cell-line Ramos. One representative of five independent experiments is shown.

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Immunology

Article Title: BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

doi: 10.1186/1471-2172-6-9

Figure Lengend Snippet: Fetal calf serum and human AB serum upregulates BMP-6 mRNA expression in human B-cells . CD19-positive B cells were cultured in X-vivo 15 over night and with fetal calf serum or human AB serum at the indicated dilutions for four hours, before total RNA was extracted. The expression of BMP-6 mRNA by realtime RT-PCR; values are normalised to the expression level of PGK1 mRNA and expressed as relative quantification (2 -average ΔΔC T – relative to BMP-6 expression in the cell-line Ramos. One representative of three independent experiments is shown.

Article Snippet: Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Chinese Medicine

Article Title: BMP6, a potential biomarker of inflammatory fibrosis and promising protective factor for dilated cardiomyopathy

doi: 10.1186/s13020-025-01062-9

Figure Lengend Snippet: Significant gene and assessment of biomarker sensitivity. A Heatmap of differential expression of immune-related genes between DCM and NC. B Heatmap of differential expression of senescene-related genes between DCM and NC. C Intersection of ImmDEGS, SenDEGS and gene sets in MEyellow. D Sensitivity and specificity analysis of ROC curves between BMP6 and DCM. E Correlation between BMP6 and 22 types of immune cells

Article Snippet: Additionally, the expression levels of proteins, including BMP6 (M06924-1, Boster, USA), SMAD3 (25494-1-AP, ProteinTech, USA), SMAD6 (sc-25321, Santa Cruz, USA), IL-1β (16806-1-AP, ProteinTech, USA), IL-18 (60070-1-Ig, ProteinTech, USA), IL-6 (21865-1-AP, ProteinTech, USA), TNF-α (17590-1-AP, ProteinTech, USA), and COL1A1 (sc-293182, Santa Cruz, USA), were detected using western blotting.

Techniques: Biomarker Discovery, Quantitative Proteomics

Journal: Chinese Medicine

Article Title: BMP6, a potential biomarker of inflammatory fibrosis and promising protective factor for dilated cardiomyopathy

doi: 10.1186/s13020-025-01062-9

Figure Lengend Snippet: BMP6 expression in different cellular subpopulations. A Differential expression of BMP6 in various cell types of the heart. B Differential gene expression in fibroblasts between DCM and NH. C Differential gene expression in endothelial cells between DCM and NH. D Elbow diagram obtained using the harmony method and we selected 0–15 as the number of dimensions. E Specific markers and expression levels in fibroblasts and myofibroblasts. F – I subdividing and annotating fibroblast subpopulations by TSNE and Umap methods. J BMP6 expression between conventional fibroblasts and myofibroblasts. K differential expression of BMP6 in fibroblasts between DCM and NH. L Specific markers and expression levels of macrophage subpopulations. M – P Subdividing and annotating macrophage subpopulations by TSNE and Umap methods. Q Differences in the percentage of macrophage subpopulations abundance between DCM and NH

Article Snippet: Additionally, the expression levels of proteins, including BMP6 (M06924-1, Boster, USA), SMAD3 (25494-1-AP, ProteinTech, USA), SMAD6 (sc-25321, Santa Cruz, USA), IL-1β (16806-1-AP, ProteinTech, USA), IL-18 (60070-1-Ig, ProteinTech, USA), IL-6 (21865-1-AP, ProteinTech, USA), TNF-α (17590-1-AP, ProteinTech, USA), and COL1A1 (sc-293182, Santa Cruz, USA), were detected using western blotting.

Techniques: Expressing, Quantitative Proteomics, Gene Expression

Journal: Chinese Medicine

Article Title: BMP6, a potential biomarker of inflammatory fibrosis and promising protective factor for dilated cardiomyopathy

doi: 10.1186/s13020-025-01062-9

Figure Lengend Snippet: Mendelian randomization analysis between BMP6 and DCM. A Scatter trend plot of effective SNPs. B The black line represents the nature of each SNP, and the red lines represent the combined nature of the SNPs analyzed by the IVW and MR Egger methods, respectively. C SNPs distribution map. D Leave-one-out analysis. The red line shows the results of the combined analysis

Article Snippet: Additionally, the expression levels of proteins, including BMP6 (M06924-1, Boster, USA), SMAD3 (25494-1-AP, ProteinTech, USA), SMAD6 (sc-25321, Santa Cruz, USA), IL-1β (16806-1-AP, ProteinTech, USA), IL-18 (60070-1-Ig, ProteinTech, USA), IL-6 (21865-1-AP, ProteinTech, USA), TNF-α (17590-1-AP, ProteinTech, USA), and COL1A1 (sc-293182, Santa Cruz, USA), were detected using western blotting.

Techniques:

Journal: Chinese Medicine

Article Title: BMP6, a potential biomarker of inflammatory fibrosis and promising protective factor for dilated cardiomyopathy

doi: 10.1186/s13020-025-01062-9

Figure Lengend Snippet: Immunofluorescence staining and cardiac tissue protein assay and BMP6 knockdown. A Immunofluorescence staining of markers specific to macrophages and type 2 macrophages. B and C Western blot was used to detect the changes in protein levels in the cardiac tissues between DCM and NC (n = 3). D and E The knockdown of BMP6 and the expression levels of SMAD6 and COL1A1 (n = 3). The siRNA group represents the knockdown group, si-NC represents the empty vector group, and blank represents the control group. The significance of * P in the above graph: * P < 0.05; ** P < 0.01. F Potential interventional herbs based on BMP6 inverse prediction

Article Snippet: Additionally, the expression levels of proteins, including BMP6 (M06924-1, Boster, USA), SMAD3 (25494-1-AP, ProteinTech, USA), SMAD6 (sc-25321, Santa Cruz, USA), IL-1β (16806-1-AP, ProteinTech, USA), IL-18 (60070-1-Ig, ProteinTech, USA), IL-6 (21865-1-AP, ProteinTech, USA), TNF-α (17590-1-AP, ProteinTech, USA), and COL1A1 (sc-293182, Santa Cruz, USA), were detected using western blotting.

Techniques: Immunofluorescence, Staining, Knockdown, Western Blot, Expressing, Plasmid Preparation, 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: Freshly isolated human peripheral blood naive CD4 + T cells were stimulated with anti-CD3/CD28 mAb. (A) Transcripts for several components of the canonical BMP signaling pathway were determined by real-time PCR ex vivo (0h) or after 6 days of stimulation (6d). GNB2L1 was used as endogenous control. Means ± SD of at least three independent experiments run in duplicates are shown. Note the logarithmic scale on y-axis. (B) Percentage of BMPRIA + cells detected by flow cytometry throughout the culture. Bars represent the mean ± SD of two to five independent experiments. (C) Expression of BMPRIA and CD25 in T cells (upper dot plots) and differential expression of BMPRIA in the CD25 - and CD25 + cell populations (lower histograms) during activation. A representative experiment out of four is shown. (D) Expression of BMPRIA in T cells cultured with different stimuli. Grey histograms represent isotype controls. Similar stainings were obtained in two to three independent experiments. mDCs: mature dendritic cells; PHA: Phytohaemagglutinin; Ck: cytokine cocktail (rhIL-2, rhIL-6, rhTNF-α) (E) Determination of BMP2/4 and BMP6 production by flow cytometry in T cells cultured in media alone (grey histograms) or in the presence of anti-CD3/CD28 mAb (black histograms). Grey filled histograms represent isotype control stainings. A representative experiment out of three is shown. (F) Expression of CD25 and phosphorylated BR-Smad (pBR-Smads) during activation. For comparison, T cells were kept in culture media alone. Results are representative of three independent experiments.

Article Snippet: For the intracellular stainings, and according to the manufacturer’s instructions, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with Perm/Wash buffer (BD Biosciences), and stained with purified anti-human BMP2/4 mAb (100230), biotin-conjugated anti-human BMP6, purified anti-human BMPRIA (all from R&D Systems) or PE-conjugated anti-human Bcl-2 mAb (6C8) (BD Biosciences), followed when required by fluorochrome-conjugated, multiadsorbed F(ab’)2 fragments of donkey anti-goat, anti-rabbit or anti-mouse IgG or streptavidin (Jackson ImmunoResearch Laboratories), all diluted in Perm/Wash buffer.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Ex Vivo, Flow Cytometry, Expressing, Activation Assay, Cell Culture, Comparison

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: For the intracellular stainings, and according to the manufacturer’s instructions, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with Perm/Wash buffer (BD Biosciences), and stained with purified anti-human BMP2/4 mAb (100230), biotin-conjugated anti-human BMP6, purified anti-human BMPRIA (all from R&D Systems) or PE-conjugated anti-human Bcl-2 mAb (6C8) (BD Biosciences), followed when required by fluorochrome-conjugated, multiadsorbed F(ab’)2 fragments of donkey anti-goat, anti-rabbit or anti-mouse IgG or streptavidin (Jackson ImmunoResearch Laboratories), all diluted in Perm/Wash buffer.

Techniques: Expressing, Flow Cytometry, Cell Culture