Journal: Stem cell reviews and reports

Article Title: MSC secreted extracellular vesicles carrying TGF-beta upregulate Smad 6 expression and promote the regrowth of neurons in spinal cord injured rats.

doi: 10.1007/s12015-021-10219-6

Figure Lengend Snippet: Fig. 2 BMSC-EVs upregulated Smad 6 expression in NSCs via the secretion of TGF-β. A. To confirm whether IL-6, BMP4, and TGF-β existed in the BMSC-EVs, the expression of these factors was determined by PCR (n = 3, Student’s t-test). B. ELISA from five individual samples confirmed the presence of TGF-β and IL-6 in BMSC- EVs. C, D. To evaluate whether TGF-β or IL-6 played a key role in mediating the expression of Smad 6 in NSCs, we added SB431542 and JSH-23, respec- tively, to NSCs in the presence of BMSC-EVs. This revealed that the BMSC-EVs-induced upregulation of Smad 6 could be abolished by the addition of SB431542 (C, n = 5; the data were revealed as fold changes to control NSCs, Student’s t-test). Conversely, the addition of JSH- 23 did not alter the expression of Smad 6 in NSCs (D, n = 5; the data were presented as fold changes to control BMSC-EV treated NSCs, Student’s t-test) E. We added TGF-β to NSCs to assess the effect of TGF-β on mediating Smad 6 expression; this resulted in an increase of Smad 6 expression in NSCs. This TGF-β-induced upregula- tion of Smad 6 expression could be significantly abolished by the addition of SB431542 (n = 5; the data were presented as fold changes to control NSCs, Stu- dent’s t-test). (Smad 6 mRNA expression was normalized to GAPDH mRNA; the data were presented as mean ± S.D; *p < 0.05, **p < 0.01, ***p < 0.001, #p > 0.05.)

Article Snippet: Passage 2 NSCs or the Smad 6-knockdown NSCs were dissociated and reseeded on glass coverslips in 5% FBSDMEM/F12 for 24 h. The medium was then switched to DMEM/F12 supplemented with one of the following: BMSC- EVs or 10 ng/mL TGF-β (R&D Systems); BMSCEVs + 10 μM SB431542 [the TGF-β type I receptor kinase inhibitor (Sigma)]; BMSC-EVs + 20 ng BMP4; 10 ng/ mL TGF-β + 10 μM SB431542; 10 ng/mL TGF-β + 20 ng BMP4 (R&D Systems); 20 ng/mL IL-6 (Sigma), with or without 30 μM JSH-23 (NF-κB inhibitor, MCE); 20 ng/mL IL-6 + BMSC-EVs, with or without SB 431,542; 20 ng/mL BMP4, with or without 200 ng/mL Noggin [BMP- antagonist (Sigma)]; 20 ng/mL BMP4 + BMSC-EVs, with or without SB 431,542; 40 ng/mL IL-6 and 40 ng/mL BMP4, with 1 3 or without BMSC-EVs.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

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: Toxicological Sciences

Article Title: Bone morphogenetic protein 4 is involved in cadmium-associated bone damage

doi: 10.1093/toxsci/kfac121

Figure Lengend Snippet: Effects of CdCl 2 on BMP/SMAD pathway. The hBMSCs were exposed to 0, 2.5, or 5.0 μM CdCl 2 and subjected to osteogenic differentiation for 14 days. A and C, Western blots were performed, and (B and D) relative protein levels of BMP-2, BMP-4, BMP-6, BMP-7, SMAD1, p-SMAD1, SMAD4, SMAD5, and p-SMAD1/5/9 were determined. E and F, Expression levels of BMP-2 and BMP-4 were detected by ELISA. These are the results from 3 independent biological replicates experiments (mean ± SD, n = 3). * p < .05, ** p < .01, different from hMSCs in the absence of CdCl 2 . Abbreviations: BMP, bone morphogenetic protein; hBMSC, human bone marrow mesenchymal stem cell.

Article Snippet: For BMP-4 groups, the medium included 50 ng/ml of recombinant BMP-4 (MedChemexpress).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: Bone morphogenetic protein 4 is involved in cadmium-associated bone damage

doi: 10.1093/toxsci/kfac121

Figure Lengend Snippet: Association among Cd, BMP-4, and osteoporosis. The mediating role of plasma BMP-4 ligand in the relationship between U-Cd and occurrence of osteoporosis. Causal mediation analysis estimated the indirect effect of U-Cd on osteoporosis through plasma BMP-4 ligand. Abbreviations: BMP, bone morphogenetic protein; CI, confidence interval; DE, direct effect; IE, indirect effect; PM, proportion mediated; U-Cd, urinary cadmium.

Article Snippet: For BMP-4 groups, the medium included 50 ng/ml of recombinant BMP-4 (MedChemexpress).

Techniques: Clinical Proteomics

Journal: Toxicological Sciences

Article Title: Bone morphogenetic protein 4 is involved in cadmium-associated bone damage

doi: 10.1093/toxsci/kfac121

Figure Lengend Snippet: The osteogenic effect of BMP-4 on hBMSCs. The hBMSCs were treated with BMP-4 (50 ng/ml) and/or CdCl 2 (5.0 μM) for 14 days. A, The BMP-4 levels detected by ELISA. B, Mineralization was measured using Alizarin Red S staining. C, Western blots were performed. D, Relative protein levels of Runx2, OSX, SMAD4, and p-SMAD1/5/9 complex were determined. These are the results from 3 independent biological replicates experiments (mean ± SD, n = 3). ∗ p < .05, ** p < .01. Abbreviations: BMP, bone morphogenetic protein; hBMSC, human bone marrow mesenchymal stem cell.

Article Snippet: For BMP-4 groups, the medium included 50 ng/ml of recombinant BMP-4 (MedChemexpress).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot

Journal: Toxicological Sciences

Article Title: Bone morphogenetic protein 4 is involved in cadmium-associated bone damage

doi: 10.1093/toxsci/kfac121

Figure Lengend Snippet: The effects of BMP-4 knockdown on osteogenic differentiation and SMADs. After si-BMP-4 transfect the hBMSCs were osteogenic induction for 14 days. A, Mineralization was measured using Alizarin Red S staining. B, Western blots were performed. C, Relative protein levels of BMP-4, Runx2, OSX, SMAD4, and p-SMAD1/5/9 complex were determined. These are the results from 3 independent biological replicates experiments (mean ± SD, n = 3). ** p < .01.

Article Snippet: For BMP-4 groups, the medium included 50 ng/ml of recombinant BMP-4 (MedChemexpress).

Techniques: Knockdown, Staining, Western Blot

Journal: Respiratory research

Article Title: Levosimendan mediates the BMP/Smad axis through upregulation of circUSP34-targeted miR-1298 to alleviate pulmonary hypertension.

doi: 10.1186/s12931-024-02945-5

Figure Lengend Snippet: Fig. 5 Lev upregulates circUSP34-targeted miR-1298-mediated BMP/Smad axis to inhibit cell proliferation, migration and promote apoptosis in hypoxia- treated PASMCs. (A) The expression of BMPR2 in PASMCs was measured by WB. (B) The protein expression levels of BMPR2 in PASMCs under different treatment conditions were detected by WB. (C) The cell proliferation ability of PASMCs under different treatment conditions was examined by CCK-8. (D) The expression of Ki-67 in PASMCs was measured by IF. (E) Cell migration ability of PASMCs was tested by cell scratch assay. (F) Apoptosis of PASMCs was determined by flow cytometry. (G) WB was performed to test the protein expression levels of BMP4, p-Smad1/5/8, Id1 in PASMCs under different treat ment conditions. Data are displayed for three individual experiments with mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: After blocking with 5% skim milk for 1 h, the membranes were kept at 4 °C and incubated overnight with the following primary antibodies: BMPR2 (1:1000, Abcam, ab130206), BMP4 (1:1000, Cell Signaling Technology, 4680), Smad1/5/8 (1:1000, Abcam, ab13723), p-Smad1/5/8 (1:1000, Sigma-Aldrich, AB3848-I), Id1 (1:1000, Abcam, ab168256), β-actin (1:2000, Cell Signaling Technology, 4967).

Techniques: Migration, Expressing, CCK-8 Assay, Wound Healing Assay, Flow Cytometry

Journal: Respiratory research

Article Title: Levosimendan mediates the BMP/Smad axis through upregulation of circUSP34-targeted miR-1298 to alleviate pulmonary hypertension.

doi: 10.1186/s12931-024-02945-5

Figure Lengend Snippet: Fig. 6 Lev upregulates circUSP34-targeted miR-1298-mediated BMP/Smad axis to alleviate hypoxia combined with SU5416-induced PH. (A) Detection of RVSP in PH rat. (B) The mPAP levels were measured in PH rat. (C) Detection of RVI in PH rat. (D) Detection of LVSP in PH rat. (E) Examination of vascular wall changes in PH rat by HE staining. (F) The collagen deposition phenomenon in the lung interstitial tissue in PH rat was detected by Masson staining. (G) Detection of Ki-67 expression in pulmonary artery tissues in PH rat by IHC. (H) Apoptosis in the pulmonary artery tissue in PH rat was measured by TUNEL. (I) Levels of circUSP34 and miR-1298 were probed by qRT-PCR in PH rat. (J) WB was performed to measure the expression levels of BMPR2, BMP4, p-Smad1/5/8, and Id1 proteins in PH rat under different treatment conditions. n = 6, *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: After blocking with 5% skim milk for 1 h, the membranes were kept at 4 °C and incubated overnight with the following primary antibodies: BMPR2 (1:1000, Abcam, ab130206), BMP4 (1:1000, Cell Signaling Technology, 4680), Smad1/5/8 (1:1000, Abcam, ab13723), p-Smad1/5/8 (1:1000, Sigma-Aldrich, AB3848-I), Id1 (1:1000, Abcam, ab168256), β-actin (1:2000, Cell Signaling Technology, 4967).

Techniques: Staining, Expressing, TUNEL Assay, Quantitative RT-PCR

Journal: Methods in Molecular Biology

Article Title: Molecular Dermatology

doi: 10.1007/978-1-62703-227-8

Figure Lengend Snippet: Fig. 2. The stages of iPSC differentiation during keratinocyte derivation. iPSCs generated from an ICR mouse were differ- entiated into keratinocytes using differentiation Protocol I. ( a ) EBs treated with RA for 2 days in suspension culture. ( b ) EB outgrown on a ColIV-coated plate in the presence of BMP4 on day 10 of differentiation. ( c ) EB outgrown on a ColIV-coated plate on day 17 of differentiation before rapid attachment plating. ( d ) iPSC-derived keratinocytes at passage 2 post-rapid attachment plating (day 25 of differentiation). All images were taken with 10× objectives.

Article Snippet: 100 ng/ m l stock solution of mouse BMP4 (R&D Systems) reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin (BSA).

Techniques: Generated, Suspension, Derivative Assay