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: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Microarray, Software, Microscopy

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: British Journal of Pharmacology

Article Title: The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1‐bone morphogenic protein 2 longevity axis

doi: 10.1111/bph.14477

Figure Lengend Snippet: eNOS and NO‐donors stimulate bone growth. eNOS mRNA expression in MC3T3 (A) and 2T3 (B) cells treated with RSV (1–100 μM) or vehicle for 24 h ( n = 5) determined by real time PCR. Nitrite (NO 2 − ) levels (C) or total eNOS protein (D) from 2T3 or MC3T3 cells treated with RSV (1–100 μM) or vehicle ( n = 5) for 48 h, determined by colorimetric assay. ALP levels from primary osteoblasts treated with NO‐donor (NOC22, 0.1–10 μM) or vehicle for 4 days ( n = 5) normalized to total cell protein (E). Calvariae from newborn mice cultured with NO‐donor (NOC22, 0.1–1 μM) or vehicle control for 4 days ( n = 5) processed for histology and H&E staining (F). mRNA expression of osteoblast marker genes Runx2, osteocalcin (OCN) and BMP2 determined in 2T3 cells treated with NOC22 (1.0 μM) or vehicle for 24 h ( n = 5) by real time PCR (G). BMP2 protein levels assessed in conditioned media from NO‐donor‐treated osteoblasts (NOC22 or SNP, 3–200 μM, 48 h, n = 5), by ELISA (with rhBMP2 as standard) (H). The μCT analysis of dissected tibia from homozygous eNOS knockout ( −/− ) or wild‐type ( +/+ ) control mice (4 month, n = 10) (I) and trabecular bone volume (J) and BMD (K) analysis (* P < 0.05 vs. vehicle; * P < 0.01 vs. wild‐type control animals).

Article Snippet: Recombinant human BMP2 (hBMP2; R&D Systems Inc.) was used as a standard.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Colorimetric Assay, Cell Culture, Control, Staining, Marker, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: British Journal of Pharmacology

Article Title: The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1‐bone morphogenic protein 2 longevity axis

doi: 10.1111/bph.14477

Figure Lengend Snippet: The eNOS‐SIRT1 axis is necessary for the pro‐osteogenic effects of RSV. Different effects on ALP levels (A) BMP2 gene expression (qPCR, B) and promoter activity (C) in primary osteoblasts from eNOS knockout ( −/− ) or wild‐type ( +/+ ) control mice ( n = 5) treated with RSV (5 μM) or vehicle (24 h). RSV‐induced ALP levels (5 μM) in the presence of the BMP inhibitor noggin (500 ng·mL −1 , 48 h, n = 5), normalized to total protein (D). SIRT1 gene expression in 2T3 osteoblasts following RSV (1–100 μM) treatment, quantified by real time PCR (E), along with eNOS (F) and BMP2 (G) mRNA levels transfected with SIRT1 siRNA or scramble control ( n = 5) with and without RSV treatment (5 μM). RSV‐induced ALP levels (5 μM) in the presence of SIRT1 (or scrambled control) siRNA (H) ( n = 5) (* P < 0.05 vs. vehicle treated WT/scrambled ctrl; # P < 0.001 vs. RSV‐treated WT/scrambled ctrl).

Article Snippet: Recombinant human BMP2 (hBMP2; R&D Systems Inc.) was used as a standard.

Techniques: Gene Expression, Activity Assay, Knock-Out, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: British Journal of Pharmacology

Article Title: The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1‐bone morphogenic protein 2 longevity axis

doi: 10.1111/bph.14477

Figure Lengend Snippet: Ageing decreases bone volume and eNOS‐BMP2 expression. μCT analysis of tibia of young (3 month) or aged (12 month) mice ( n = 10) (A) determining BV/TV (B) and BMD (C). Gene expression changes (real time PCR) of eNOS (D), BMP2 (E) in ageing long bones ( n = 10). Proposed mechanism of RSV action within osteoblasts (F) (* P < 0.05).

Article Snippet: Recombinant human BMP2 (hBMP2; R&D Systems Inc.) was used as a standard.

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

Journal: Oncotarget

Article Title: Overexpression of colorectal cancer oncogene CHRDL2 predicts a poor prognosis.

doi: 10.18632/oncotarget.14039

Figure Lengend Snippet: Figure 6: CHRDL2 binds to BMP2 and inhibits the phosphorylation of Smad1/5. A. Coimmunoprecipitation (co-IP) of CHRDL2 and BMPs. HCT116 cell culture media were performed using anti-CHRDL2 and control (mouse IgG) antibodies. Immunoprecipitates and culture media were subjected to western blot analysis using anti-BMP2, anti-BMP4, and anti-BMP6 antibodies. B. Culture media were performed using anti-BMP2, anti-BMP4, anti-BMP6 and control (rabbit IgG) antibodies, and western blot analysis using anti-CHRDL2 antibody. C. Co-IP of recombinant CHRDL2 protein and the recombinant BMP2 protein. a: BMP-2 IP with anti-CHRDL2; b: CHRDL2 IP with anti-CHRDL2; c: mixture of BMP2 and CHRDL2; d: mixture of BMP2 and CHRDL2 IP with anti-CHRDL2. D. CHRDL2 inhibited the Smad1/5 phosphorylation of HCT116 cell induced by BMP2. The cytoplasm and nucleus protein extracts from HCT116 cells treated with BMP2, CHRDL2 or both for 1h were probed for p-Smad1/5, Smad1. E. Immunofluorescence array for p-Smad1/5 and Smad4 in HCT116 cell. Merged images of HCT116 cells treated with different concentrations of BMP2, CHRDL2 or both for 1 h, stained with DAPI and immunofluorescence stained with p-Smad1/5 and Smad4 respectively. Corresponding anti-rabbit secondary antibodies were conjugated with Alexa Fluor 488 and 555.

Article Snippet: Purified recombinant protein of Homo sapiens CHRDL2 (TP320245) and BMP2 (TP750012) were purchased from ORIGENE (USA).

Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, Cell Culture, Control, Western Blot, Recombinant, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Overexpression of colorectal cancer oncogene CHRDL2 predicts a poor prognosis.

doi: 10.18632/oncotarget.14039

Figure Lengend Snippet: Figure 7: CHRDL2 blocks the BMP2 and attenuates the effect of promoting proliferation and inhibiting apoptosis induced by BMP2 in HCT116 cells. A. Cell-cycle analysis by flow cytometry in HCT116 cells treated with 100 ng/ml BMP2 or 100 ng/ml CHRDL2. The histograms show the ratio of different cell phase populations (G0/G1, S and G2/M cells) in treated HCT116 cells. B. Apoptosis analysis by flow cytometry in HCT116 cells treated with 100 ng/ml BMP2 or 100 ng/ml CHRDL2. The histograms show the apoptosis rate in treated HCT116 cells. C. Western blot analysis was used to measure the P21, Cyclin D1, Cleaved caspase 9 and Cleaved caspase 3 in the lysis of HCT116 cells treated with 100 ng/ml BMP2 or 100 ng/ml CHRDL2.

Article Snippet: Purified recombinant protein of Homo sapiens CHRDL2 (TP320245) and BMP2 (TP750012) were purchased from ORIGENE (USA).

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot, Lysis