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: Figure 4: 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 μm and 50 μm; 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

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: miR‐1187 directly targets BMP4. (A) The predicted binding site between miR‐1187 and BMP4 by bioinformatics analysis. (B) The luciferase activity of the BMP4‐WT and BMP4‐MUT in MC3T3‐E1 cells treated with miR‐1187 mimics or NC. (C) mRNA expression of Bmp4 was significantly downregulated in the miR‐1187 mimics‐transfected group. (D) Western blot analysis for the expression of BMP4 protein in miR‐1187 mimics and inhibitor‐transfected MC3T3‐E1 cells on Pti. (E) ELISA assay analysis for the expression of supernatant BMP4 secreted protein in miR‐1187 mimics and inhibitor‐transfected MC3T3‐E1cells on Pti. All values represent means ± SD.( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: BMP4 positively regulates osteogenic differentiation in MC3T3‐E1 cells. (A) Level of BMP4 decreased after transfected with different siRNA‐BMP4, si‐BMP4‐785 was identified to have the highest transfection efficiency. (B) BMP4 transcript levels was determined by RT‐PCR after transfected with overexpression plasmids of BMP4. (C) qRT‐PCR analysis. Level of Col‐1 , Alp , Runx2 , and Ocn mRNA expression was significantly downregulated in BMP4 silencing. (D) qRT‐PCR analysis. Level of Col‐1 , Alp , Runx2 , and Ocn mRNA expression was significantly upregulated in BMP4 overexpression. (E) Western blotting. Expression of BMP4 protein and osteogenesis‐related proteins COL‐1, ALP, RUNX2 as well as statistical analysis after transfecting si‐BMP4. (F) Western blotting. Expression of BMP4 protein and osteogenesis‐related proteins COL‐1, ALP, RUNX2 as well as statistical analysis after transfecting pCDNA3.1‐BMP4. (G) ALP activity detection on day 7 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells. (H) ALP staining on day 10 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells (scale bar = 500 μm). (I) Alizarin red S staining and quantitative analysis of Alizarin Red S accumulation on day 21 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells (scale bar = 200 μm/100 μm), the black arrow indicate the magnified area, the magnified pictures were marked within the square frame in the image. All values represent means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Staining

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: LIPUS promotes osteogenic differentiation of MC3T3‐E1 through inhibiting miR‐1187 and upregulating BMP4. (A) qRT‐PCR analysis. Effect of daily LIPUS stimulation on mRNA expression of Bmp4. (B) ELISA. Supernatant BMP4 secreted by cultured MC3T3‐E1 cells was determined by ELISA assay. (C) Western blotting. BMP4 level was analyzed by western blot after 7 days LIPUS treatment. (D) Expression of osteogenesis‐related mRNA was analyzed by qRT‐PCR after si‐BMP4 or si‐bmp4 + LIPUS treatment. (E) Expression of osteogenesis‐related protein was analyzed by western blot after si‐BMP4 or si‐BMP4 + LIPUS treatment. (F) ALP activity detection on day 7 in si‐BMP4 and si‐BMP4 + LIPUS‐treated MC3T3‐E1 cells. (G) Bmp4 and osteogenesis‐related genes Col‐1, Alp, Runx2, and Ocn expression in MC3T3‐E1 cells on Pti as indicated treatment by qRT‐PCR. (H) ALP activity in MC3T3‐E1 cells on Pti on day 7 as indicated treatment. (I) Expression of BMP4 and osteogenesis‐related proteins including COL‐1, ALP, RUNX2 in MC3T3‐E1 cells on Pti as indicated treatment by Western blot analysis. All values represent means ± SD ( n = 3). Expression of osteogenesis‐related protein was analyzed by western blot (E1) and quantitative analysis (E2) after si‐BMP4, LIPUS or si‐BMP4+LIPUS treated. Expression of BMP4 and osteogenesis‐ related proteins including COL‐ 1, ALP, RUNX2 in MC3T3‐ E1 cells on Pti as indicated treatment by Western blot analysis(I)andquantitative analysis (J). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activity Assay

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: LIPUS regulates bone formation in vivo. (A) Study plan of the vitro study. (B) qRT‐PCR analysis of mRNA expression of Bmp4 as well as other osteogenesis‐related gene in the de novo bone of different groups. B1: MRNA expression of Bmp4; B2, B3: Osteogenesis‐related gene including Col‐1, Alp, Runx2, and Ocn at 4 weeks and 8 weeks respectively. (C) Micro‐CT analysis. C1: 3D reconstruction of the bone defect area at week 4 and 8. More bone ingrowth is observed as time increases in each group. The white part is the scaffold and the red part is the bone tissue. C2: The POF values for the Pti at 4 and 8 weeks. The POF values differ significantly between the LIPUS and control group, between the si‐BMP4 and control group, as well as between the si‐BMP4 and si‐BMP4 + LIPUS group. (D) D1: The representative merged images of fluorescent double labeling of calcein(green color) and xylenol orange(orange color). The red square indicate the magnified area, the magnified pictures were marked within the white square frame in the image. (scale bar = 200 μm/100 μm). D2: The MAR of the four groups at 4 and 8 weeks. All values represent means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Micro-CT, Control, Labeling

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: The schematic of LIPUS promotes osteogenesis of porous titanium alloys through inhibiting miR‐1187 and upregulating BMP4.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques:

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