Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Cardiomyocytes develop from anterior primitive streak cells induced by β-catenin activation and the blockage of BMP signaling in hESCs.

doi: 10.1111/j.1365-2443.2010.01455.x

Figure Lengend Snippet: Figure 2 Differences in the cardiac potential of posterior and anterior PS cells derived from hESCs. (A) The percentages of the aggregates containing contracting cells at day 16 of culture in cells treated with 4-hydroxy-tamoxifen (4OHT) only, 4OHT and BMP4, and 4OHT and Noggin (***P < 0.001). The error bars represent the means ± SEM of three independent experiments. (B) Immunostaining for a-actinin protein of aggregates for each condition on day 26–29 (green). Merged images are indicated, and nuclei were stained with DAPI. (C) Quantitative RT-PCR analysis for cardiac marker genes, TBX5, NKX2-5, MYH6 and ACTN2. The expression of TBX5 and the other genes was examined on days 8 and 16 of culture. The expression level of each gene in the 4OHT-only treated samples was defined as 1.0. Scale bars: 100 lm.

Article Snippet: From the next day (referred to as day 0 of culture), the cells were cultured in N2B27 medium with 100 nM 4OHT (SigmaAldrich, St Louis, MO, USA), 10 ng ⁄ mL recombinant human BMP4 (R&D Systems, Minneapolis, MN, USA) and ⁄ or 100 ng ⁄ ml recombinant human Noggin ⁄ Fc Chimera (R&D Systems) (Sumi et al. 2008).

Techniques: Derivative Assay, Immunostaining, Staining, Quantitative RT-PCR, Marker, Expressing

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 cell research & therapy

Article Title: Centrifugal gravity-induced BMP4 induces chondrogenic differentiation of adipose-derived stem cells via SOX9 upregulation.

doi: 10.1186/s13287-016-0445-6

Figure Lengend Snippet: Fig. 3 CG induced SOX9 upregulation via increased expression BMP4. a Expression of BMP2, BMP4, and TGF-β1 mRNAs in CG-stimulated ASCs. ASCs were loaded with different degrees of CG (0, 300, 600, 1200, and 2400 g) for 15 min. b The duration of BMP4 mRNA expression in CG-stimulated ASCs. c Expression of BMP4 protein in CG-stimulated ASCs. ASCs were centrifuged at 2400 g for 30 min and then harvested at the indicated time points. Expression of BMP4 mRNA and protein was examined by RT-PCR and western blotting, respectively. d BMP4-dependent SOX9 expression in CG- stimulated ASCs. ASCs were stimulated with CG (2400 g for 30 min) and treated with recombinant BMP4 (10 nM) or Dorsomorphin (25 nM). SOX9 protein expression was examined using western blotting. The intensities of the bands in western blots were determined using ImageJ 1.40. BMP bone morphogenetic protein, CG centrifugal gravity, SOX SRY (sex-determining region Y)-box, TGF transforming growth factor

Article Snippet: Protein lysates (30 μg) and concentrated supernatants were resolved by 10–12% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then probed with primary antibodies against SOX9 (EMD Millipore, Billerica, MA, USA), BMP4 (Santa Cruz Biotechnology, Inc., Dallas, Texas USA), and GAPDH (Abcam).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Recombinant

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