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bmp4 (mouse) elisa kit  (Abnova)

 
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    Abnova bmp4 (mouse) elisa kit
    A Diagram of <t>BMP4-SEP</t> 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.
    Bmp4 (Mouse) Elisa Kit, supplied by Abnova, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bmp4 (mouse) elisa kit - by Bioz Stars, 2026-03
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    Images

    1) Product Images from "Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-024-53642-2

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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 .
    Figure Legend 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 .

    Techniques Used: RNA Sequencing, Expressing



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    A Diagram of <t>BMP4-SEP</t> 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.
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    A Diagram of <t>BMP4-SEP</t> 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.
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    Image Search Results


    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.

    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

    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.

    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

    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.

    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

    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 .

    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

    MSR1-activated macrophage PI3K/AKT/GSK3β/β-catenin signaling promotes osteogenic differentiation of BMSCs. (A) Heat map of several genes encoding molecules involved in BMSC osteogenic differentiation was performed based on the results of RNA sequencing (MSR1 KO vs. WT). Blue and yellow colors represent low and high expression values, respectively.(B) The amount of secreted BMP4 in 24-h in the serum-free medium by MSR1 WT and MSR1 KO macrophages after co-culture, or MSR1 WT macrophages treated with LY294002 or ARQ 092 before co-culture was assessed by ELISA. Values are expressed as mean ± SD, ***p < 0.001. (C) The amount of secreted BMP4 in 24-h serum-free MSR1 BL, Vec, and OE RAW264.7 cells after co-culture, or MSR1 OE RAW264.7 cells treated with LY294002, ARQ 092 before co-culture was determined by ELISA. Values are expressed as mean ± SD, ***p < 0.001. (D-F) In the co-culture system, knockout of MSR1 or inhibition of PI3K/AKT/GSK3β/β-catenin signaling in macrophages impaired pro-osteogenic differentiation of BMSCs as observed by AR staining (D). Quantitative evaluation of AR staining results (E) and ALP activities (F) on day 7 and 14 was performed. BMSC without co-culture was used as the Con group. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01. (G) mRNA expression levels of osteogenic biomarkers (Col1, ALP, Ocn and Runx2) in osteogenic differentiated BMSCs on day 14 were detected by qPCR in different groups. β-actin was used as an internal control. Values are expressed as mean ± SD, **p < 0.01, ***p < 0.001. (H-J) Inhibition of PI3K/AKT/GSK3β/β-catenin signaling in MSR1 OE RAW264.7 cells in the co-culture system decreased osteogenic differentiation of BMSCs as observed by AR staining (H). Quantitative evaluation of AR staining results (I) and ALP activities (J) on day 7 and 14 was performed. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance. (K) mRNA expression levels of Col1, ALP, Ocn and Runx2 in osteogenic differentiated BMSCs on day 14 detected by qPCR in the indicated groups. β-actin was used as an internal control. Values are expressed as mean ± SD, **p < 0. 01, ***p < 0.001, ns indicates no significance.

    Journal: Theranostics

    Article Title: Macrophage MSR1 promotes BMSC osteogenic differentiation and M2-like polarization by activating PI3K/AKT/GSK3β/β-catenin pathway

    doi: 10.7150/thno.36930

    Figure Lengend Snippet: MSR1-activated macrophage PI3K/AKT/GSK3β/β-catenin signaling promotes osteogenic differentiation of BMSCs. (A) Heat map of several genes encoding molecules involved in BMSC osteogenic differentiation was performed based on the results of RNA sequencing (MSR1 KO vs. WT). Blue and yellow colors represent low and high expression values, respectively.(B) The amount of secreted BMP4 in 24-h in the serum-free medium by MSR1 WT and MSR1 KO macrophages after co-culture, or MSR1 WT macrophages treated with LY294002 or ARQ 092 before co-culture was assessed by ELISA. Values are expressed as mean ± SD, ***p < 0.001. (C) The amount of secreted BMP4 in 24-h serum-free MSR1 BL, Vec, and OE RAW264.7 cells after co-culture, or MSR1 OE RAW264.7 cells treated with LY294002, ARQ 092 before co-culture was determined by ELISA. Values are expressed as mean ± SD, ***p < 0.001. (D-F) In the co-culture system, knockout of MSR1 or inhibition of PI3K/AKT/GSK3β/β-catenin signaling in macrophages impaired pro-osteogenic differentiation of BMSCs as observed by AR staining (D). Quantitative evaluation of AR staining results (E) and ALP activities (F) on day 7 and 14 was performed. BMSC without co-culture was used as the Con group. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01. (G) mRNA expression levels of osteogenic biomarkers (Col1, ALP, Ocn and Runx2) in osteogenic differentiated BMSCs on day 14 were detected by qPCR in different groups. β-actin was used as an internal control. Values are expressed as mean ± SD, **p < 0.01, ***p < 0.001. (H-J) Inhibition of PI3K/AKT/GSK3β/β-catenin signaling in MSR1 OE RAW264.7 cells in the co-culture system decreased osteogenic differentiation of BMSCs as observed by AR staining (H). Quantitative evaluation of AR staining results (I) and ALP activities (J) on day 7 and 14 was performed. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance. (K) mRNA expression levels of Col1, ALP, Ocn and Runx2 in osteogenic differentiated BMSCs on day 14 detected by qPCR in the indicated groups. β-actin was used as an internal control. Values are expressed as mean ± SD, **p < 0. 01, ***p < 0.001, ns indicates no significance.

    Article Snippet: The BMP4 ELISA kit was obtained from CUSABIO (CSB-E04512m, WuHan, China).

    Techniques: RNA Sequencing, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Knock-Out, Inhibition, Staining, Control

    Detection of key soluble morphogens in the system of sweat gland morphogenesis and the impact of BMP receptor inhibitor and EGF receptor inhibitor on sweat gland development in vitro. BMP4 and EGF demonstrated sharped difference in the medium of the sweat gland organogenesis system. BMP receptor inhibitor could block the formation of sweat gland in this system, while EGF receptor inhibitor significantly reduced the expression of K18. (a) The variation tendency of BMP4 and EGF in the medium of system. The error bar meant the standard error of BMP4 and EGF concentrations in different systems at different time points. (b) The impact of BMP receptor inhibitor and EFG receptor inhibitor on sweat gland morphogenesis. In comparison with the control group, EGF receptor inhibitor significantly reduced the expression of K18, but glandular structure was still observed. BMP receptor inhibitor completely blocks the expression of K18, and no glandular structure was observed. All the nuclei were counterstained with DAPI (DAPI: blue; K18: red; bars = 200 μ m and 50 μ m; K18: cytokeratin 18; IM: light microscope; H&E: hematoxylin-eosin staining; IF: immunofluorescence staining).

    Journal: Stem Cells International

    Article Title: Developing a Novel and Convenient Model for Investigating Sweat Gland Morphogenesis from Epidermal Stem Cells

    doi: 10.1155/2019/4254759

    Figure Lengend Snippet: Detection of key soluble morphogens in the system of sweat gland morphogenesis and the impact of BMP receptor inhibitor and EGF receptor inhibitor on sweat gland development in vitro. BMP4 and EGF demonstrated sharped difference in the medium of the sweat gland organogenesis system. BMP receptor inhibitor could block the formation of sweat gland in this system, while EGF receptor inhibitor significantly reduced the expression of K18. (a) The variation tendency of BMP4 and EGF in the medium of system. The error bar meant the standard error of BMP4 and EGF concentrations in different systems at different time points. (b) The impact of BMP receptor inhibitor and EFG receptor inhibitor on sweat gland morphogenesis. In comparison with the control group, EGF receptor inhibitor significantly reduced the expression of K18, but glandular structure was still observed. BMP receptor inhibitor completely blocks the expression of K18, and no glandular structure was observed. All the nuclei were counterstained with DAPI (DAPI: blue; K18: red; bars = 200 μ m and 50 μ m; K18: cytokeratin 18; IM: light microscope; H&E: hematoxylin-eosin staining; IF: immunofluorescence staining).

    Article Snippet: BMP4 and EGF concentrations in supernatant were measured using a mouse BMP4 (CSB-E04512m, Cusabio, Wuhan, Hubei, China) and EGF ELISA kit (EM014-96, ExCell Bio, Shanghai, China).

    Techniques: In Vitro, Blocking Assay, Expressing, Comparison, Control, Light Microscopy, Staining, Immunofluorescence

    a Total RNAs from UPJ tissues of control and obstructed kidneys were collected from C57BL/6 and IL33KO mice for qRT-PCR analyses ( n = 5 from each group). PC1 shows the log2 fold change in gene expression between UUO UPJ tissues and control (Ctrl) UPJ tissues from wild-type C57BL/6 mice. PC2 shows the log2 fold change in gene expression between IL33KO UUO UPJ tissues and C57BL/6 UUO UPJ tissues. The heatmap represents the expression of urothelial differentiation genes in UPJ tissues. b Western blot analysis of the SHH protein in UPJ tissues. c Immunofluorescent staining of SHH and IL-33 in UPJ tissues. d Levels of SHH, BMP4, and BMP5 in control and hydronephrotic urine samples were analyzed using ELISA ( n = 5 in each group). e Proposed working model for the mechanism by which the upregulation of IL-33 following obstructive renal injury induces type 2 immune responses and UPJ urothelium hyperplasia

    Journal: Experimental & Molecular Medicine

    Article Title: IL-33/ST2 axis mediates hyperplasia of intrarenal urothelium in obstructive renal injury

    doi: 10.1038/s12276-018-0047-8

    Figure Lengend Snippet: a Total RNAs from UPJ tissues of control and obstructed kidneys were collected from C57BL/6 and IL33KO mice for qRT-PCR analyses ( n = 5 from each group). PC1 shows the log2 fold change in gene expression between UUO UPJ tissues and control (Ctrl) UPJ tissues from wild-type C57BL/6 mice. PC2 shows the log2 fold change in gene expression between IL33KO UUO UPJ tissues and C57BL/6 UUO UPJ tissues. The heatmap represents the expression of urothelial differentiation genes in UPJ tissues. b Western blot analysis of the SHH protein in UPJ tissues. c Immunofluorescent staining of SHH and IL-33 in UPJ tissues. d Levels of SHH, BMP4, and BMP5 in control and hydronephrotic urine samples were analyzed using ELISA ( n = 5 in each group). e Proposed working model for the mechanism by which the upregulation of IL-33 following obstructive renal injury induces type 2 immune responses and UPJ urothelium hyperplasia

    Article Snippet: Mouse serum or urine levels of IL-33 (DY413, R&D System), IL-5 (DY405, R&D System), IL-13 (DY413, R&D System), SHH (DY461, R&D System), BMP4 (EK0316, Boster), and BMP5 (LS-F20454, LSBio) were analyzed using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions.

    Techniques: Control, Quantitative RT-PCR, Gene Expression, Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay