Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: Msx2 is a downstream target of BMP2 signaling in the uterus during decidualization. (A) The primary cultures of mouse endometrial stromal cells (MESCs) were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points, as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of Msx2. The relative levels of gene expression were determined by setting the expression level of the GFP-treated sample at 24 h to 1.0 (n = 3). Rplp0, encoding a ribosomal protein, was used to normalize the level of RNA. *P < 0.05. (B) The nucleotide positions of the SBEs on the Msx2 promoter were analyzed by ChIP. (C) Mouse stromal cells were treated with E + P or E, P, and BMP2 (E + P + BMP2) for 90 min. ChIP, using the Smad4 antibody, was performed, as described in “Materials and Methods.” Chromatin enrichment was quantified by real-time PCR using primers flanking the potential SBE in the Msx2 promoter and also a negative control region in the ORF of Msx2. Enrichments were normalized to 1% of input DNA. The experiment was repeated twice, and representative data are shown.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression, Negative Control

Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: MSX1 and MSX2 mediate BMP2-induced HESC decidualization. The primary cultures of HESCs were established as described in “Materials and Methods.” The cells were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of MSX1 and MSX2. The relative levels of gene expression were determined by setting the expression level on day 0 of the GFP-treated sample at 1.0. RPLP0, encoding a ribosomal protein, was used to normalize the level of RNA. Data were collected from three independent clinical samples, which were subjected to the same experimental conditions. *P < 0.05; **P < 0.005.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor (TGF)-β-activated Kinase 1 Mimics and Mediates TGF-β-induced Stimulation of Type II Collagen Synthesis in Chondrocytes Independent of Col2a1 Transcription and Smad3 Signaling

doi: 10.1074/jbc.m500646200

Figure Lengend Snippet: FIG. 4. TAK1a mediates the stimulation of type II collagen synthesis by TGF- and BMP2 and exhibits TAB1-independent kinase activity. A, chondrocytes were infected 24 h after plating with dominant-negative TAK1 adenoviral vectors, Ad-pC-hTAK1a-K63A, and Ad-pC-hTAK1a-K63W. Seventy-two hours after initiating infec- tion, cells were treated for 24 h with 5 ng/ml TGF-1 (T) or 100 ng/ml BMP2 (B) in the presence of [3H]proline. Samples from triplicate wells were pooled before SDS-PAGE. B, for immune complex kinase assays, chondrocytes were infected with Ad-pC-hTAK1a (TK), kinase-negative (KN) Ad-pC-hTAK1a-KWSA, and the tandem vector (TK,TB) pCEA3- hTAB1(pC-hTAK1a). Forty-eight hours after beginning infection, cells were treated with TGF-1 (40 ng/ml; T) or IL-1 (40 ng/ml; IL) for 10 min prior to lysis, immunoprecipitation with TAK-ct antibody, and TAK1a immune complex kinase assay using bacterially expressed GST- MKK6 as substrate. Lysates were equally divided prior to immunopre- cipitation to permit direct kinase assays in the presence of [-32P]ATP (upper panels) or kinase assay after pretreatment with cold ATP (lower panels). The right panels are 7-fold shorter exposures of the last two lanes. C, Western blots (IB) demonstrating activation of endogenous TAK1 and TAB1 and their overexpressed counterparts. Chondrocytes were treated for 10 or 30 min with TGF-1 (5 ng/ml) or BMP2 (100 ng/ml) and lysed with SDS sample buffer at the same time as cells exposed to 48 h of adenoviral expression of TAK1a or coexpressed TAK1a and TAB1 (tandem construct). TAK-ct and TAB-m primary antibodies were used for detection. Long dashes, unmodified TAK1 or TAB1 bands; short dashes, activated/phosphorylated bands. Right panel, shorter exposures of lanes 5 and 6.

Article Snippet: Subsequently, cells were fed with the same medium, treated with recombinant human TGF- 1 or BMP2 (R&D Systems) for 24 h and then labeled in fresh medium with tritiated proline for 24 h. Labeling medium was 0.3% ITS /DMEM, 62.5 g/ml -aminoproprionitrile, 25 g/ml ascorbate, 40 Ci/ml [5-3H]proline (Amersham Biosciences), and growth factors.

Techniques: Activity Assay, Infection, Dominant Negative Mutation, SDS Page, Immune Complex Kinase Assay, Plasmid Preparation, Lysis, Immunoprecipitation, Kinase Assay, Western Blot, Activation Assay, Expressing, Construct

Journal: Scientific Reports

Article Title: ‘Free’ inhibin α subunit is expressed by bovine ovarian theca cells and its knockdown suppresses androgen production

doi: 10.1038/s41598-019-55829-w

Figure Lengend Snippet: Suppressive effect of BMP 2, -4, -6, and -7 (10 ng/ml) on ( A ) expression of INHA mRNA and ( B ) secretion of inhibin α subunit protein by cultured bovine theca cells. The inset ( C ) shows that co-treatment with inhibin-A (50 ng/ml) can partially reverse BMP-induced suppression of thecal INHA expression. Values are means and bars indicate SEM (n = 4 independent cultures).

Article Snippet: Recombinant human (rh) BMP2, BMP4, BMP6, BMP7, TNFα, TGFα and EGF were purchased from R&D systems (Abingdon, UK).

Techniques: Expressing, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Small molecule regulators of postnatal Nkx2.5 cardiomyoblast proliferation and differentiation

doi: 10.1111/j.1582-4934.2011.01513.x

Figure Lengend Snippet: Transforming growth factor (TGF)-β signalling regulates the proliferation and differentiation of postnatal Nkx2.5-GFP + cells. Cells were fixed and immunostained by antibodies against troponin T (TnT-(fluorescein isothiocyanate (FITC), green) and smooth muscle myosin heavy chain (smMHC-Tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC), red), and the nuclei were stained by DAPI (blue). (A) Effect of TGF-β1 and the TGF-βRI inhibitor, A83, on FACS-purified eGFP + cells from the neonatal heart. (B) TGF-β1 (100 ng/ml), but not bone morphogenetic protein (BMP)2 and activin A, induced a greater number of smMHC + cells following 7 days in culture. This effect could be abrogated by treatment with A83. (C) Effect of BMP2 (100 ng/ml) and activin (100 ng/ml) treatment on the differentiation of Nkx2.5 + cells. (D) Cell proliferation was enhanced after exposure to A83 (1 μM). Results represent the means from four independent experiments. * P < 0.05 versus DMSO in control group, and # P < 0.05 versus TGF-β + DMSO (by two-way anova with Bonferroni–Dunn's post-hoc test).

Article Snippet: Recombinant human TGF-β1, human/mouse/rat activin A and human BMP2 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Staining, Purification, Control