Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: Expression of BMP4 in human onychofibroblasts. (A) Major cell types in the human nail unit revealed by scRNA-seq. KC, keratinocyte; FIB, fibroblast; MFLC, myofibroblast-like cell; VEC, vascular endothelial cell; LEC, lymphatic endothelial cell; IMM, immune cell; NC, neural cell; MEL, melanocyte. (B) UMAP visualization of the human OF population. (C) Venn diagram illustrating the number of overlapping DEGs upregulated in OFs between scRNA-seq and bulk RNA-seq. (D) Relative mRNA expression of BMP4 and FGF10 in DF and OF groups. (E) Immunofluorescence staining and (F) MFI of BMP4 in two groups. MFI, Mean Fluorescence Intensity. (G) Western blotting and (H) quantitative analysis of BMP4 in two groups. (I) ELISA of BMP4 in the supernatants of cell cultures from two groups. Scale bars, 100 μm. The results presented are the mean ± SD of six biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: BMP4 derived from human onychofibroblasts regulates differentiation of nail stem cells. (A) Identity, marker genes, and cell proportions of human nail keratinocyte subtypes. (B) In vitro culture showing cell expansion and colony formation of human NSCs. (C) Flow cytometric analysis of KRT15 and KRT1 expression in human NSCs cultured in vitro . (D) Grouping of indirect co-cultures of OFs and NSCs. (E) Relative mRNA expression of stem and differentiation marker genes in keratinocytes under different co-culture conditions. (F) Immunofluorescence staining and (G) MFI of KRT15 and IGFBP7 in three groups. MFI, Mean Fluorescence Intensity. KD , knockdown . Scale bars, 200 μm. The results presented are the mean ± SD of three biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Derivative Assay, Marker, In Vitro, Expressing, Cell Culture, Co-Culture Assay, Immunofluorescence, Staining, Fluorescence, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: BMP4 induces in vitro differentiation of human NSCs through the TGF-beta signaling pathway. (A) QuSAGE analysis of human nail keratinocyte populations. (B) Effect of recombinant human BMP4 on NSC proliferation. (C) Relative mRNA expression of stem and differentiation marker genes in keratinocytes under different treatments. (D) Western blotting and (E) quantitative analysis of p-SMADs in three groups. The results presented are the mean ± SD of nine biologically independent replicates. (F) Immunofluorescence staining and (G, H) MFI of SMAD4 and p-SMADs in three groups. MFI, Mean Fluorescence Intensity. Scale bars, 100 μm. Unless otherwise stated, the results presented are the mean ± SD of three biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: In Vitro, Recombinant, Expressing, Marker, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: Proposed model: Onychofibroblast-mediated nail stem cell differentiation links amputation level with digit regeneration capacity. Under homeostatic conditions, BMP4 secreted by onychofibroblasts (OFs) activates the TGF-beta signaling pathway in nail stem cells (NSCs) and induces cell differentiation. It is known that NSCs and the mechanisms governing NSC differentiation are directly related to their ability to orchestrate digit regeneration. In instances of distal amputation, NSCs and OFs remain largely unaffected; regenerating nail epithelial cells can cover the wound site, preserving nail differentiation, and facilitating complete digit regeneration. Conversely, proximal amputation leads to decreased NSCs and OFs, along with diminished differentiation signals, resulting in impaired nail and digit regeneration.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Cell Differentiation, Preserving

Journal: Nature Communications

Article Title: Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells

doi: 10.1038/s41467-024-53642-2

Figure Lengend Snippet: A Diagram of BMP4-SEP fusion protein. A pH-sensitive GFP variant, super ecliptic pHluorin (SEP), was inserted into the linker domain of BMP4 between Asn307 and Cys308 and cloned into a plasmid under control of a CMV promoter. B Diagram of iMEPM neutralization by ammonium chloride (NH 4 Cl). The acidic environment of a vesicle (red, pH ~5.5) restricts the fluorescence of SEP. When NH 4 Cl is applied to a cell, the environment becomes neutralized (pH = 7.4), unquenching SEP and increasing fluorescence visualization. C A violin plot shows a significant increase in fluorescence amplitude between iMEPM cells expressing BMP4-SEP compared to a pCIG-GFP control after neutralization by the addition of 5 mM NH 4 Cl (** P = 0.003 by unpaired two-tailed t test). D Diagram of BMP4-SEP release in response to cellular depolarization induced by the addition of KCl. E ViolinPlot showing quantification of SEP fluorescence in iMEPM cells transfected with pcDNA-SEP (blue), TfR-SEP (pink) or BMP4-SEP (green). Changes in fluorescence amplitude in regions of interest (ROIs) taken in live imaging videos (see methods) were averaged and compared. BMP4-SEP and TfR-SEP transfected cells had significantly higher changes in fluorescence than pcDNA-SEP (vs BMP4-SEP P = 0.017; vs TfR-SEP P = 0.0003) but were not found to be different from one another ( P = 0.24). F – H Representative images (from seven plates of cells, two separate trials) show pcDNA-SEP, TFR-SEP, and BMP4-SEP fluorescence in iMEPM cells pre- and post-depolarization by 50 mM KCl. Red boxes denote the location of the magnified blue insets within each respective image. F’ – H’ Representative fluorescence traces from iMEPM cells transfected with pcDNA-SEP, TFR-SEP, or BMP4-SEP construct. The addition of 50 mM KCl is denoted with the red arrow (** P < 0.05 ns=not significant by two-way ANOVA). Source data are provided as a Source Data file.

Article Snippet: Fractions were prepared and assayed according to the standard protocol from the Abnova “BMP4 (Mouse) ELISA Kit” (catalog #: KA5051).

Techniques: Variant Assay, Clone Assay, Plasmid Preparation, Control, Neutralization, Fluorescence, Expressing, Two Tailed Test, Transfection, Imaging, Construct

Journal: Nature Communications

Article Title: Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells

doi: 10.1038/s41467-024-53642-2

Figure Lengend Snippet: A A schematic created in BioRender shows the method used to quantify the amount of BMP4 in conditioned media before and after depolarization of BMP4-transfected iMEPM cells. iMEPM cells were cultured for 24 h before conditioned media was collected and frozen. Cells were depolarized with KCl solution. Immediately following depolarization, conditioned media was collected and frozen. An ELISA was conducted with paired conditioned media samples collected before and after depolarization. B A paired box-and-whisker plot shows a significant increase in the BMP4 concentration after depolarization by KCl (*** P = 0.0003 by two-tailed paired t test, n = 20 plates of cells). Error bars represent minimum and maximum BMP concentration, horizontal line represents the median BMP concentration values, and the bounds of the box represent the 25th and 75th percentile. Source data are provided as a Source Data file.

Article Snippet: Fractions were prepared and assayed according to the standard protocol from the Abnova “BMP4 (Mouse) ELISA Kit” (catalog #: KA5051).

Techniques: Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Whisker Assay, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells

doi: 10.1038/s41467-024-53642-2

Figure Lengend Snippet: A Representative images show that depolarization by the addition of KCl at 20 s increases the fluorescence of GCaMP6 expressed in dissociated primary cultured E13.5 palate mesenchymal cells (blue stars), and cells have subsequent calcium events following depolarization (red arrows). Replicates=3 plates depolarized with KCl, transient changes in GCaMP fluorescence measured in 36 cells. B Representative fluorescence profile of one cell over time out of 36 cells with transient increases in GCaMP fluorescence. C Representative profile of fluorescence over time for a cell that undergoes a calcium transient with depolarization at 20 s followed by two endogenous transients. D Depolarization induces significant increases in fluorescence compared to background changes in fluorescence N = 3 plates of primary culture MEPMs imaged before (control) and after depolarized with KCl (experimental), violin plot represents 38 ROIs from the three plates before depolarization and 36 ROIs measured with depolarization (**** P = 5.7 × 10 −12 by two-tailed unpaired t test). E Mean fluorescence/F 0 traces of BMP4-SEP release averaged between cells treated with or without BAPTA-AM. Yellow represents DMSO controls ( n = 6 from separate plates), and blue represents BAPTA-AM treated cells ( n = 10 cells from separate plates). The red arrow denotes the addition of isosmotic KCl Tyrode’s media to induce depolarization. SEM is shown with shaded areas. F A box-and-whisker plot quantifying change in BMP4-SEP fluorescence amplitude over F 0 between DMSO controls and BAPTA-AM treated iMEPM cells. The error bars represent minimum and maximum fluorescence values, and the center line represents the median value (* P = 0.0002 by unpaired two-tailed t test). Source data are provided as a Source Data file.

Article Snippet: Fractions were prepared and assayed according to the standard protocol from the Abnova “BMP4 (Mouse) ELISA Kit” (catalog #: KA5051).

Techniques: Fluorescence, Cell Culture, Control, Two Tailed Test, Whisker Assay

Journal: Nature Communications

Article Title: Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells

doi: 10.1038/s41467-024-53642-2

Figure Lengend Snippet: A UMAP detailing cluster identities adapted from Ozekin et al. . FeaturePlots represent data from a single-cell RNA sequencing of the E13.5 mouse anterior palate showing expression of ion channels and connexins in green with non-expressing cells in gray: B Cacna1c (Cav1.2, L-type calcium channel). C Cacna1d (Cav1.3, L-type calcium channel). D Cacna1a (Cav1.2, L-type calcium channel). E Cacna1g (T-type calcium channel). F Kcnj2 (Kir2.1, inwardly rectifying potassium channel). G Kcnb1 (Kv2.1, voltage-gated potassium channel subfamily B). H Kcnb2 (Kv2.2), I Kcnc3 (Kv3.3, voltage-gated potassium channel subfamily C), J Kcnn2 (KCa2.2, Potassium Calcium-activated channel subfamily N), K ATP2a2 (SERCA2/Atpase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2), L Stim1 , M Stim2 , N Scn3a (Nav1.3, voltage-gated sodium channel, O Scn8a (Nav1.6 Voltage-gated sodium channel), P Gja1 (Gap junction protein alpha, Connexin 43), Q Gjc1 (Gap Junction protein gamma 1, Connexin 45), R – T FeaturePlots of Bmp4 ( Bone morphogenetic protein 4) (green), Kcnj2 (orange), and overlapped FeaturePlot of Bmp4 and Kcnj2 . Cells with high coexpression of both features will appear on a gradient to yellow. U – W FeaturePlots of Gjc1 (green), Gja1 (orange), and overlapped FeaturePlot of Gjc1 and Gja1 . Cells with high coexpression of both features will appear on a gradient to yellow. RNA sequencing data is available at Raw and processed data has been made available via a NCBI GEO Submission (accession code GSE222205). Code is accessible via GitHub https://github.com/yunusozekin/WT_E13.5_AntPalate_scRNAseq_Ozekin.git .

Article Snippet: Fractions were prepared and assayed according to the standard protocol from the Abnova “BMP4 (Mouse) ELISA Kit” (catalog #: KA5051).

Techniques: RNA Sequencing, Expressing

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: ( a ) Gating strategy for flow cytometric characterization of MYH6-specific cardiac T cells expressing the Vα2 and Vβ8 TCR chains. ( b ) Gating strategy for flow cytometric characterization of heart-infiltrating myeloid cells (upper panel) with colored frames indicating specific monocyte/macrophage populations analyzed in TCRM and transgene-negative littermate control mice at the age of 4 and 8 weeks (N = 7 mice per group from at 2 independent experiments). ( c ) Representative microscopy images from hematoxylin&eosin-stained sections from 8-week-old Ctrl and TCRM mice. ( d ) Quantification of picrosirius red-stained collagen networks on heart sections (N = 6 mice per group from 2 independent experiments). ( e ) UMAP representation of cardiac cell populations derived from sc- and snRNA-seq (upper panel) and of 4- and 8-week-old mice (bottom panel). ( f ) Heatmap showing the expression of marker genes used to assign cell identity to cardiac cells from Ctrl and TCRM mice detected by sn- and scRNA-seq analysis. ( g ) Bmp2 and Bmp4 mRNA expression as determined by RT-PCR in the indicated FACS-sorted cardiac cells from 8-week-old Ctrl or TCRM mice; FB, CD45 – PDPN + CD31 – fibroblast; EC, CD45 – PDPN – CD31 + endothelial cells; CD45, CD45 + immune cells. Bars show mean ± SEM (N = 6 mice per group from 3 independent experiments). ( h ) mRNA expression of the indicated BMPs from cardiac fibroblasts measured by sc/snRNAseq (N = 5 from 2 independent experiments). ( i ) Concentrations of the corresponding BMPs from cardiac homogenates measured by ELISA (N = 6 mice per group from two independent experiments). ( j ) GREM1 and GREM2 concentrations measured by ELISA from cardiac homogenates from 8-week-old littermate controls (Ctrl) or TCRM mice; N = 6, mice per group data from 2 independent experiments. Box and whiskers show min to max, mean ± interquartile range. Statistical analysis was performed using Student’s t test (g-j); one-way ANOVA with Dunnett’s multiple comparison test (b and d) with *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Expressing, Control, Microscopy, Staining, Derivative Assay, Marker, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: a – c , Enumeration of heart-infiltrating CD45 + cells ( a ), MYH6-specific (Vα2 + Vβ8 + CD4 + ) T cells ( b ) and CD11b + myeloid cells ( c ) from 4-week-old and 8-week-old TCRM or control (Ctrl) mice. d , Histopathological disease severity in control and TCRM mice. e , Representative confocal microscopy images showing CD4 + and CD11b + cells and COL1 deposition in hearts from 8-week-old control and TCRM mice. Boxed areas in left panels denote magnified area in right panels ( n = 4). f – k , scRNA-seq and snRNA-seq analysis from total cardiac cells from age-matched and sex-matched control and TCRM mice. f , UMAP representation showing 16 cell populations in the heart of control and TCRM mice. g , Significantly enriched pathways according to GO enrichment analysis based on differentially expressed genes between total cardiac cells from control and TCRM mice. h , Scatter plot indicating incoming and outgoing interaction strengths of individual cell subsets based on network centrality measures on the aggregated cell–cell communication network. Dot size reflects interaction count. i , Top significantly enriched ligand–receptor pairs grouped according to functional similarity. Numbers in brackets indicate the quantity of receptor pairs within a functional group. j , UMAP projection showing the expression of the indicated genes. k , Dot plot depicting the average expression of the indicated BMP pathway-related genes in cardiac cells. l , Quantification of BMP4 protein in heart homogenates by ELISA. scRNA-seq/snRNA-seq data represent a total of 28,913 cells per nuclei from control ( n = 6) and TCRM ( n = 6) mice. Pooled data from 2–3 independent experiments with n = 7, 8 and 8 mice per group ( a – c ), n = 8, 8 and 9 mice per group ( d ) or n = 6 mice per group ( l ). Representative sections from one out of five control or TCRM mice ( c ). Dots represent values from individual mice; box and whiskers show minimum to maximum, mean ± interquartile range ( a – d and l ). CM, cardiomyocytes; EC, endothelial cells; FB, fibroblasts; Mono, monocytes; Mph, macrophages; NK cells, natural killer cells; PVC, perivascular cells; Ts, T cells; wk, weeks.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Control, Confocal Microscopy, Functional Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: a , b , i – l , sc-RNA-seq analysis of sorted CD45 − PDPN + CD31 − cardiac fibroblasts isolated from 4-week-old and 8-week-old control or TCRM mice. a , UMAP representation showing 10 cell clusters depicting cardiac fibroblast heterogeneity (top) and condition (bottom). b , Projection of the indicated gene expression on cardiac fibroblast UMAP plot from control and TCRM mice. c , d , Ccl2 ( c ) and Il6 ( d ) mRNA expression in sorted cardiac fibroblasts of 8-week-old control or TCRM mice. e – h , Multi-parametric flow cytometric analysis of surface expression of CD157 ( e ), NCAM ( f ), SCA-1 ( g ) and ICAM1 ( h ) by cardiac fibroblasts from control or TCRM mice. MFI of the indicated activation markers in CD45 − PDPN + CD31 − cardiac fibroblasts. Dots represent values from individual mice;box plots as in Fig. . i , Diffusion map representation based on gene expression in cardiac fibroblasts colored according to heterogeneity (top) and origin (bottom). j , k , Projection of the indicated gene signatures onto diffusion maps. l , Expression profile of Bmp4 in cardiac fibroblast subsets shown as violin plots. m , n , Representative confocal microscopy analysis of hearts from control ( m ) and TCRM ( n ) mice using the indicated markers. Single-cell transcriptomics data represent a total of 65,545 cardiac fibroblasts from control ( n = 4) and TCRM ( n = 5) mice. Pooled data from 2–3 independent experiments with n = 6 mice per group ( c ) or n = 9, 6 and 6 mice per group ( d – h ). Representative sections from one out of five control or TCRM mice ( m , n ). Statistical analysis was performed using two-tailed Student’s t -test ( c , d ) or one-way ANOVA with Tukey’s multiple comparisons test ( e – h ) with * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Isolation, Control, Expressing, Activation Assay, Diffusion-based Assay, Confocal Microscopy, Single-cell Transcriptomics, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: Cardiac fibroblasts were isolated from hearts of 8-week-old Ccl19 -Cre R26R-EYFP mice. a , Representative dot plots showing the gating strategy for FACS of PDPN + CD31 − , Bmp4 -deficient (EYFP + ) and Bmp4 -proficient (EYFP − ) cardic fibroblasts. b , Purity of Bmp4 -deficient (EYFP + ) and Bmp4 -proficient (EYFP − ) cardiac fibroblasts after 10 d of culture. Representative dot plots from three independent isolations. c , Production of the indicated BMPs by Bmp4 −/− (EYFP + ) or Bmp4 +/+ (EYFP − ) fibroblasts. d , e , Production of the cytokines IL-6 ( d ) and TNF ( e ) by Bmp4 −/− (EYFP + ) or Bmp4 +/+ (EYFP − ) fibroblasts after 24 h of culture ( n = 5; pooled data from independent wells from two independent experiments). f , g , Expression of ICAM1 by Bmp4 −/− (EYFP + ) or Bmp4 +/+ (EYFP − ) fibroblasts after exposure to medium or BMP4 (10 ng ml −1 ) ( f ) or IL-1β (1 ng ml −1 ) ( g ) as assessed by flow cytometry with MFI of ICAM1 in f and MFI fold change relative to ICAM1 expression by untreated Bmp4 +/+ fibroblasts in g . Dots represent values from individual wells; pooled data of two independent experiments ( n = 5). All box plots as in Fig. . Statistical analysis was performed using Student’s t -test ( c – e ) or one-way ANOVA with Dunnett’s multiple comparison test ( f , g ) with * P < 0.05, ** P < 0.01 and *** P < 0.001. FACS, fluorescence-activated cell sorting.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Isolation, Expressing, Flow Cytometry, Comparison, Fluorescence, FACS

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: ( a ) Computationally predicted murine BMP4-BMPR1a/BMPR2 interactions by cells in the homeostatic and inflamed cardiac microenvironment based on sc/snRNA-seq data using the CellChat tool. ( b ) Representative confocal images of Bmp4 -deficient (EYFP + ) and Bmp4 -proficient (EYFP − ) fibroblasts after 10 days of culture. ( c , d ) Production of BMP4 (c) and IL-6 (d) measured by ELISA from Bmp4 -proficient (EYFP − ) fibroblasts cardiac fibroblasts exposed IL-1β (1 ng/ml) or left untreated (medium) for 24 h. Box and whiskers show min to max, mean ± interquartile range. (N = 6; pooled data from two independent experiments). Statistical analysis was performed using Student’s t test (c-d) with ***, p < 0.001.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: ( a ) Heatmap representing binding of 3-A1-3, 20-D1-5 and 14-D10-2 mAbs to human GREM1 and GREM2 as determined by ELISA. ( b ) Dose-dependent binding of the indicated anti-GREM1/2 mAbs to human GREM1 and GREM2 as determined by ELISA. Area under the curve (AUC) was calculated as the area under the titration curve from 1:2 serial dilutions of each antibody (2.5 - 0.02 µg) and GREM1 and GREM2 (0.5–0.008 µg/ml). ( c–e ) Neutralization capacity of the indicated mAbs using GREM1 and GREM2 in BMP4-induced luciferase activity by SL-0051 cells. Hundred percent BMP4 activity was determined as relative light units in the absence of GREM1 (c) or GREM2 (d) (N = 3, mean ± SEM; representative data from 1 out of 3 independent experiments with similar results). (e) The concentration of the indicated mAb necessary to restore 50% of BMP4 activity (EC 50 ) was determined based on the values shown in (c) and (d); N = 6 independent replicates from 2 independent experiments. ( f–h ) Effect of anti-GREM1/2 mAb treatment in the adoptive T cell transfer myocarditis model. (f) Schematic representation of the experimental set up. (g) Quantification of heart-infiltrating CD45 + cells (h) and cytokine production of heart-infiltrating MYH6-specific CD4 + Vβ8 + T cells in Rag1 −/− mice at day 28 post T cell adoptive transfer. N = 8, 10, 8 or 7 mice per group from 3 independent experiments. Box and whiskers show min to max, mean ± interquartile range; dots indicate individual mice. ( i , j ) Intracellular phospho-SMAD1/5/9 expression by CD45 − CD31 − PDPN + CD56 + cardiac fibroblasts isolated from 8-week-old TCRM mice treated with IgG2b isotype control or GREM1/2-neutralizing antibody (14-D10-2) with representative histograms shown in (i). (j) Fold change of pSMAD1/5/9 mean fluorescence intensity (MFI) calculated relative to baseline pSMAD1/5/9 expression in CD56 − fibroblasts (N = 4 mice per group from 2 independent experiments) Dots indicate individual mice, mean ± SEM is displayed. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test (g-h) or two tailed Student’s t test (j) with *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Neutralization, Luciferase, Activity Assay, Concentration Assay, Adoptive Transfer Assay, Expressing, Isolation, Control, Fluorescence, Comparison, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: a , Schematic representation of the experimental design. b , Enumeration of CD45 + heart-infiltrating cells in 8-week-old TCRM mice treated with IgG2b isotype control or GREM1/2-neutralizing antibody (14-D10-2) using flow cytometry. c , Representative confocal microscopy images showing collagen deposition and immune infiltrates in the hearts of TCRM mice treated with isotype or 14-D10-2 antibodies. d , e , Flow cytometry-based enumeration ( d ) and functional characterization ( e ) of MYH6-specific, Vα2 + Vβ8 + CD4 + T cells with representative dot plots (left) and quantification of cytokine-producing MYH6-specific T cells (right). f – h , Flow cytometric enumeration of CD11b + Ly6C + CCR2 + inflammatory monocytes ( f ), CD11b + Ly6C int Ly6G + neutrophils ( g ) and CD11b + CD64 + MHCII hi activated macrophages ( h ). i , MFI of CD86 expression on CD11b + CD64 + MHCII hi activated macrophages. j , Tnf mRNA expression by sorted CD11b + Ly6G − myeloid cells. k , IL-1β protein concentration in cardiac homogenates. l , Fraction of CD157 + SCA1 + cells of CD45 − PDPN + CD31 − cardiac fibroblasts. m , MFI of the indicated activation markers by CD45 − PDPN + CD31 − cardiac fibroblasts. n , Representative confocal microscopy images showing BMP4 production by CD34 + fibroblasts in inflamed hearts of isotype antibody-treated TCRM mice and restoration of BMP4 production by 14-D10-2 antibody treatment. o , Quantification of BMP4 protein in heart homogenates by ELISA. Box plots as in Fig. ; pooled data from three independent experiments with n = 6 (control) or n = 9 (TCRM) mice per group ( b , d – m , o ). Representative sections from one out of five control or TCRM mice ( c , n ). Statistical analysis was performed using Student’s t -test with * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Control, Flow Cytometry, Confocal Microscopy, Functional Assay, Expressing, Protein Concentration, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: a – k , snRNA-seq from left or right ventricular EMBs from patients with acute myocarditis (AM) ( n = 5), inflammatory cardiomyopathy (ICM) ( n = 8), dilated cardiomyopathy (DCM) ( n = 3) or undergoing heart transplantation (HTx) ( n = 7). a , b , UMAP representation ( a ) and abundance of cardiac cell populations ( b ) in individual patients. c – e , Frequencies of T cells ( c ), inflammatory macrophages ( d ) and cardiomyocytes ( e ) in patients stratified according to high (>6%, T cell High , n = 7), intermediate (3–6%, T cell Int , n = 9) and low (<3%, T cell Low , n = 7) proportions of heart-infiltrating T cells. Dots indicate individual patients; bars indicate geometric means. f , Correlation between T cells and inflammatory macrophages (left) and T cells and resident macrophages (right). g , Heat maps showing average gene expression of the indicated, differentially expressed genes grouped by function in T cells, macrophages and cardiomyocytes. h , Significantly enriched pathways according to GO enrichment analysis based on differentially expressed genes between cardiac fibroblasts from T cell Low and T cell High cardiac biopsies. i , Average BMP4 gene expression in cardiac fibroblast. Dots indicate values of individual patients; box plots as in Fig. . j , ELISA-based quantification of BMP4 concentration in serum from healthy donors or patients with biopsy-confirmed and/or cardiac MRI-confirmed acute myocarditis. Dots indicate values of individual patients; data are mean ± s.e.m. k , ROC curve of BMP4 serum concentrations of patients with acute myocarditis and healthy controls. snRNA-seq data represent a total of 44,114 nuclei, two biopsies per patient ( n = 23 patients). Statistical analysis was performed using one-way ANOVA with Dunnett’s post test ( c – e ), Benjamini, Krieger and Yekutieli post test ( i ) or Mann–Whitney U -test ( k ) with ** P < 0.01 and *** P < 0.001. Simple linear regression test was used in f . CI, confidence interval; LEC, lymphatic endothelial cells; NC, neural cells; SMC, smooth muscle cells.

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Transplantation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: Network plots depicting significantly enriched gene sets in cardiac fibroblasts from ( a ) T cell Low and ( b ) T cell High groups according to GO enrichment analysis based on differentially expressed genes in snRNA-seq analysis of cardiac EMBs. ( c ) Correlation analysis BMP4 mRNA expression and T cell infiltration in EMBs. Colors indicate the proportion of infiltrating T cells. High, red dots (>6%, T cell High ); intermediate, green dots (3–6%, T cell Int ) and low, blue dots (<3%, T cell Low ) proportions of heart-infiltrating T cells. ( d , e ) Multiparametric correlation analysis between proportions of infiltrating cardiac cells and genes of the BMP family. (d) Pearson’s correlation comparisons of indicated cell type abundances and expression of indicated genes. (e) P values from the Pearson’s correlation comparisons in (d). Statistical analysis was performed using simple linear regression test (c).

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Expressing

Journal: Nature Cardiovascular Research

Article Title: Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

doi: 10.1038/s44161-024-00432-0

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: BMP4 levels in the samples were measured using a human BMP4 SimpleStep ELISA Kit (Abcam) following the manufacturer’s instructions.

Techniques: Concentration Assay

Journal: Redox biology

Article Title: Endothelial TET2 regulates the white adipose browning and metabolism via fatty acid oxidation in obesity.

doi: 10.1016/j.redox.2023.103013

Figure Lengend Snippet: Fig. 6. NRF2 recruits TET2 to regulate FAO. A, Binding sites of TET2 and NRF2 were predicted using Gramm-X. Blue, TET2; Cyan, NRF2; Red, binding sites. B and C, Lysate of HUVECs was immunoprecipitated with anti-TET2 antibody (B) or anti-NRF2 antibody (C) and then immunoblotted with the indicated antibodies. D, Schematic illustration of different plasmids of TET2 and NRF2. E and F, TET2-His and NRF2-Flag were ectopically expressed in HEK293T and the lysate of HEK293T was immunoprecipitated with anti-His (E) antibody or anti-Flag antibody (F). G, NRF2 was ectopically expressed with different domains of TET2 HEK293T and the lysate of HEK293T was immunoprecipitated with anti-Flag antibody. H, The 5-mC levels of HUVECs transfected with or without TET2 siRNA. n = 3/group. I, The methylation status of BMP4 and CPT1A promoter in HUVECs treated with AdNC or AdTET2. J, Predict binding sites of NRF2 in the promoters of CPT1A and BMP4 by JASPAR. K. qPCR analysis of the mRNA expression levels of CPT1A and BMP4 in HUVECs infected with TET2 adenovirus in presence or absence of NRF2 siRNA. n = 3/group. L. qPCR analysis of the mRNA expression levels of CPT1A and BMP4 in HUVECs treated with bardoxolone methyl in presence or absence of TET2 siRNA. n = 3/group. All data are mean ± SEM. E, Two-way ANOVA with Bonferroni post hoc test. *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Insulin, BMP4 and global DNA methylation levels were measured by murine INS ELISA kits (Cat#YCQZ-10385, YC Bio), murine BMP4 ELISA kits (Cat#EK0316, Boster), and Global DNA Methylation–LINE-1 Kit (Cat#55017, Active Motif) respectively.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Methylation, Expressing, Infection