csb e17298h  (Cusabio)

Journal: Oncology Reports

Article Title: POSTN + fibroblast-secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling

doi: 10.3892/or.2026.9067

Figure Lengend Snippet: BMP4 activates BMPR2/Smad signaling to induce macrophage M2 polarization. (A) PCA analysis of macrophages were treated with sEV-derived CAF-S6 transfected with shNC and shPOSTN in three replicate times. (B) Venn-diagram of DEGs between shPOSTN sEV vs. CTRL and shPOSTN sEVs vs. shNC sEVs. (C) Heatmap showing the top DEGs in each group. (D) Volcano plot showing BMP4 downregulation in macrophages treated with shPOSTN sEVs vs. shNC sEVs. (E) GSEA analysis of hallmark pathways in macrophages treated with sEV-derived CAF-S6 transfected with either shPOSTN or shNC. (F) GO enrichment analysis was performed to identify pathways associated with the representative DEGs. (G) The bubble plot displays the top 20 markedly enriched KEGG pathways for the DEGs. (H) The mRNA expression of BMP4 in macrophages induced sEVs derived from CAF-S5/-S6 transfected with shNC and shPOSTN were analyzed by RT-qPCR. (I) The mRNA expression of TNF-α, IL-6, TGF-β and IL-10 in macrophages stimulated with BMP4 at concentrations of 0, 50 and 100 ng/ml. (J) The expression of CD163 and CD206 in macrophages treated with BMP4 at concentrations of 0, 100 ng/ml examined by flow cytometry. (K) The level of BMPR2, pSmad1/5/9, Smad 5 and Smad 9 in macrophages treated with BMP4 (100 ng/ml) for 48 h were examined by western blotting (n=3 per group). (L) The level of pSmad1/5/9, Smad5, Smad9, CD163 and CD206 in macrophages treatment with or without LDN193189 inhibitor (n=3 per group). Statistical significance was determined using a one-way ANOVA test, ns, not significant *P<0.05; **P<0.01; ****P<0.0001. Error bars represent the mean ± SEM. Source data for blotting assays, see . PCA, principal component analysis; sEV, small extracellular vesicles; sh, short hairpin; NC, negative control; DEGs, differentially expressed genes; POSTN, perostin; sEV, small extracellular vesicles; GSEA, gene set enrichment analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR; pSmad, phosphorylated Smad.

Article Snippet: To assess the content of BMP4 on the surface of sEV-derived CAF-S5/-S6 transfected with shNC and shPOSTN, Human BMP4 ELISA kits was purchased (cat. no. CSB-E17298h; Cusabio Technology, LLC).

Techniques: Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

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: 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, 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: Cell communication and signaling : CCS

Article Title: Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

doi: 10.1186/s12964-024-01559-0

Figure Lengend Snippet: Fig. 1 Modification of MDA-MB-231-HM (231-HM) breast cancer cells to investigate canonical and non-canonical BMP4 signalling. a Schematic diagram of canonical and non-canonical BMP4 signalling. b Western blotting validation of enforced BMP4 expression, SMAD1/5/8 signalling and knockdown of SMAD4 using two different short hairpin RNA constructs in 231-HM cells. c Quantitation of secreted BMP4 protein levels in 24-h conditioned medium. n = 3/group, mean ± SEM. d RT-qPCR analysis of the expression of canonical target genes in 231-HM cells with modified levels of BMP4 and/or SMAD4. n = 3/group, mean ± SEM. e Effect of enforced BMP4 expression and/or SMAD4 knockdown on the proliferation of cultured 231-HM cells. 500 cells were seeded on day 0 and proliferation was tracked for 5 days. n = 6/group, mean ± SEM. Statistical analysis was completed using the exponential growth curve equation function in Prism. ns, not significant. f Effect of enforced BMP4 expression and/or SMAD4 knockdown on colony formation of 231-HM cells. 60 cells were seeded on day 0 and colonies were counted on day 22. n = 3/group, mean ± SEM. For bar plots, statistical analysis was completed by Student’s t test. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. See also Fig. S1

Article Snippet: The concentration of secreted BMP4 protein was determined using a human BMP4 DuoSet ELISA kit (R&D systems #DY314) and normalized to the protein concentration in each well.

Techniques: Modification, Western Blot, Biomarker Discovery, Expressing, Knockdown, shRNA, Construct, Quantitation Assay, Quantitative RT-PCR, Cell Culture

Journal: Cell communication and signaling : CCS

Article Title: Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

doi: 10.1186/s12964-024-01559-0

Figure Lengend Snippet: Fig. 2 Non-canonical BMP4 signalling, but not canonical signalling, promotes tumor growth. a Timeframe for tumor growth and onset of metastatic disease of the 231-HM tumor model. Cells (1,000,000) were injected into the mammary glands of NSG mice. Tumor growth was tracked before resection until approximately 400 mm3. Metastatic burden was assessed 15 days after resection. Created with BioRender.com. b Effect of enforced BMP4 expression on SMAD4-expressing (top) and SMAD4-knockdown (middle and lower panels) 231-HM tumors. n = 15/ group, mean ± SEM. c Immunohistochemical analysis of the levels of SMAD4 in resected 231-HM tumors, shown at both low and high magnification in different tumors

Article Snippet: The concentration of secreted BMP4 protein was determined using a human BMP4 DuoSet ELISA kit (R&D systems #DY314) and normalized to the protein concentration in each well.

Techniques: Injection, Expressing, Knockdown, Immunohistochemical staining

Journal: Cell communication and signaling : CCS

Article Title: Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

doi: 10.1186/s12964-024-01559-0

Figure Lengend Snippet: Fig. 3 BMP4 suppresses metastasis independent of tumor-intrinsic expression of SMAD4. a Visualisation of luciferase-tagged metastatic lesions in mice using the IVIS Spectrum imaging system. b Representative images of TurboGFP-tagged metastatic lesions in the lungs and livers visualized ex vivo using the Maestro imaging system. c Metastatic burden in the lungs (left panel), livers (middle panel) and spines (right panel) at endpoint, as quantitated by determining the levels of tumor-specific TurboGfp genomic DNA in each organ. n ≥ 9/group, mean ± SEM. Statistical analysis was completed by Student’s t test. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. See also Fig. S2

Article Snippet: The concentration of secreted BMP4 protein was determined using a human BMP4 DuoSet ELISA kit (R&D systems #DY314) and normalized to the protein concentration in each well.

Techniques: Expressing, Luciferase, Imaging, Ex Vivo

Journal: Cell communication and signaling : CCS

Article Title: Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

doi: 10.1186/s12964-024-01559-0

Figure Lengend Snippet: Fig. 6 Prognostic value of canonical and non-canonical BMP4 signalling in breast cancer patients included in the Metabric dataset. a Expression of genes included in the canonical signature in 231-HM tumors with modified expression of BMP4 and/or SMAD4. b Canonical signature score was calculated based on the upregulated genes minus the down-regulated genes from (a). Average signature scores for tumors of different grades in the Metabric dataset are visualized. c Correlation between canonical signature score and overall survival in breast cancer patients. d Expression of genes included in the non-canonical signature in 231-HM tumors with modified expression of BMP4 and/or SMAD4. e Non-canonical signature score was calculated based on the upregulated genes minus the down-regulated genes from (d). Average signature scores for tumors of different grades in the Metabric dataset are visualized. f Correlation between non-canonical signature score and overall survival in breast cancer patients. Statistical analysis was completed by the stat_compare_means function in R for (b) and (e). N.S., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Statistical analysis was completed by the coxph function in R for (c) and (f). HR, hazard ratio; CI, confidence interval; Cox, Cox proportional-hazards model

Article Snippet: The concentration of secreted BMP4 protein was determined using a human BMP4 DuoSet ELISA kit (R&D systems #DY314) and normalized to the protein concentration in each well.

Techniques: Expressing, Modification