Journal: Journal of oral science

Article Title: The in vitro osteogenetic characteristics of primary osteoblastic cells from a rabbit calvarium.

doi: 10.2334/josnusd.50.427

Figure Lengend Snippet: Fig. 4 Effect of rhBMP-2 on expression of osteogenesis- related proteins in the BMP signaling pathway. Rabbit primary osteoblastic cells were cultured in the absence or presence of rhBMP-2, and whole cell lysates were prepared after 1, 3, 5, 7, 10, and 14 days. The samples were normalized by the total protein concentration, and approximately 20 µg of total protein were subjected to SDS-PAGE and immunoblotted with anti- phosphorylated-Smad1 or anti-Runx2 antibodies. β- Actin served as a loading control. The blots shown are representative of at least three experiments.

Article Snippet: Western blotting was performed using goat polyclonal anti-phospho-Smad1 antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat polyclonal anti-Runx2 antibody (1:500; Santa Cruz Biotechnology), mouse monoclonal anti-β-Actin antibody (1:2000; Santa Cruz Biotechnology), and horseradish peroxidaseconjugated secondary antibody (anti-goat IgG antibody at 1:10,000 dilution, R&D Systems; or anti-mouse IgG antibody at 1:10,000 dilution, GE Healthcare, Buckinghamshire, UK).

Techniques: Expressing, Cell Culture, Protein Concentration, SDS Page, Control

Journal: PLoS ONE

Article Title: Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

doi: 10.1371/journal.pone.0072407

Figure Lengend Snippet: PC3-M cells were transiently transfected with empty vector or endoglin and the indicated siRNA as in . Two days later cells were lysed for Western blot ( A ) or luciferase promoter assay ( B ). A ) ActRIIA and BMPRII differentially regulate Smad1 protein phosphorylation. Western blot on resultant cell lysate was performed for Smad1, phospho-Smad1/5 (pSmad1/5), endoglin and GAPDH. Data are from a representative experiment (N = 4 experiments). B ) ActRIIA and BMPRII differentially regulate BRE 2 -luciferase activation. Cells were additionally co-transfected with BRE 2 -luciferase and Renilla luciferase constructs, and luciferase activity (normalized to Renilla luciferase activity) was measured. Data are the mean ± SD from a single experiment conducted in replicates of N = 2, conducted three separate times with similar results (also N = 2). *, p≤0.05 between the indicated groups. C ) BMP7- and BMP9-stimulated Smad1 phosphorylation is differentially regulated by ActRII and BMPRII. Cells were transfected as above, serum-starved, and treated with BMP7 or BMP9 as indicated. Western blot on resultant cell lysate was performed for phospho-Smad1/5 (pSmad1/5) and total Smad1. Data are from a representative experiment (N = 2 experiments).

Article Snippet: Antibodies to the following proteins were purchased: phospho-Smad1/5, phospho-Smad1/5/8, Smad1, and Myc-tag from Cell Signaling Technology (Danvers, MA), endoglin from BD Biosciences (San Jose, CA), GAPDH from Enzo Life Sciences (Farmingdale NY), FLAG-tag and Myc-tag from Sigma-Aldrich (St. Louis, MO), α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA), HRP-conjugated goat anti-mouse IgG (H+L) F(ab')2 fragment from KPL (Gaithersburg, MD), and ECL donkey anti-rabbit whole antibody from GE Healthcare Biosciences (Pittsburgh, PA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Luciferase, Promoter Assay, Activation Assay, Construct, Activity Assay

Journal: PLoS ONE

Article Title: Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

doi: 10.1371/journal.pone.0072407

Figure Lengend Snippet: PC3-M cells were transfected with endoglin, vector (Vec), or with siRNA to Smad1, (siSm1), Smad5 (siSm5), Smad8 (siSm8), BMPRII (siRII) or non-targeting (siNeg), and processed 48 hrs later as indicated. A ) Smad-targeting siRNA suppresses transcript in a Smad-specific fashion. Smad1, -5, and -8 mRNA expression was assessed via qRT-PCR, normalized to GAPDH, and expressed relative to siNeg-transfected cells (normalized to 1.0). Data represent mean ± SD from a single experiment conducted in replicates of N = 2, that was repeated 3 separate times (also in replicates of N = 2) with similar results. *, p≤0.05 compared to siNeg. B ) Effect of siRNA on phospho-Smad1/5/8, phospho-Smad1/5, and total Smad1 protein levels. Cell lysates were probed by antibody directed towards phospho-Smad1/5/8 (pSmad1/5/8) and total Smad1 protein by Western blot. The non-specific band (*) immediately under the pSmad1/5/8 band (arrow) confirms even loading. Negative control cells (Neg Ctl) were transfected with vector and serum starved overnight. Positive control cells (Pos Ctl) were transfected with endoglin, not serum starved and were treated with TGFβ for 30 min. Separate samples were similarly transfected and treated, and cell lysates were probed for phospho-Smad1/5 (pSmad1/5). Data are from one representative experiment in each case, repeated 3 separate times with similar results. C ) Endoglin-mediated BRE 2 -luciferase activity is largely mediated by Smad1. Cells were transfected with endoglin and were additionally co-transfected with BRE 2 - and Renilla luciferase construct, and luciferase assays performed. Data represent mean ± SD of a single representative experiment conducted in replicates of N = 2, repeated 3 separate times (replicates of N = 2) with similar results. *, p≤0.05 compared to Endoglin/siNeg. D ) BMPRII-mediated suppression of BRE 2 -luciferase activity is largely mediated by Smad1. Cells were transfected as in (C) with addition of indicated siRNA and luciferase activity as assessed as above. Data represent mean ± SD of a single representative experiment conducted in replicates of N = 2, repeated 2 separate times (replicates of N = 2) with similar results. *, p≤0.05 compared to Endoglin/siNeg/siBMPRII.

Article Snippet: Antibodies to the following proteins were purchased: phospho-Smad1/5, phospho-Smad1/5/8, Smad1, and Myc-tag from Cell Signaling Technology (Danvers, MA), endoglin from BD Biosciences (San Jose, CA), GAPDH from Enzo Life Sciences (Farmingdale NY), FLAG-tag and Myc-tag from Sigma-Aldrich (St. Louis, MO), α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA), HRP-conjugated goat anti-mouse IgG (H+L) F(ab')2 fragment from KPL (Gaithersburg, MD), and ECL donkey anti-rabbit whole antibody from GE Healthcare Biosciences (Pittsburgh, PA).

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Positive Control, Luciferase, Activity Assay, Construct

Journal: PLoS ONE

Article Title: Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

doi: 10.1371/journal.pone.0072407

Figure Lengend Snippet: A) Schematic depiction of ActRIIA and BMPRII constructs. Signal peptide (hatched), transmembrane domain (light gray), kinase domain (black), and BMPRII tail domain (dark gray) are indicated with the amino acid position that begins each portion. Also indicated are five sequential Myc tags or single FLAG tag at the C-terminus of ΔKD ActRIIA and BMPRII constructs, respectively (checkered). The small white stripe in KI BMPRII’s kinase domain represents the site of kinase-inactivating mutation. Segment lengths are to scale. B ) ActRIIA promotes Smad1 signaling dependent on the kinase domain. PC3-M cells were transfected with BRE 2 -luciferase and Renilla luciferase, endoglin, wild type (WT) or kinase domain deletion (ΔKD) ActRIIA constructs, and siRNA to ActRIIA or non-targeting as indicated. Luciferase assay performed as in . Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 compared to Eng/siNeg. C ) BMPRII suppresses Smad1 signaling independent of kinase function but dependent on the tail domain. PC3-M cells were transfected as above except that BMPRII constructs and siRNA were used. WT = wild type; KI = kinase inactive; Δtail = tail domain deleted. Luciferase assay performed as in . Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 compared to Eng/siBMPRII.

Article Snippet: Antibodies to the following proteins were purchased: phospho-Smad1/5, phospho-Smad1/5/8, Smad1, and Myc-tag from Cell Signaling Technology (Danvers, MA), endoglin from BD Biosciences (San Jose, CA), GAPDH from Enzo Life Sciences (Farmingdale NY), FLAG-tag and Myc-tag from Sigma-Aldrich (St. Louis, MO), α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA), HRP-conjugated goat anti-mouse IgG (H+L) F(ab')2 fragment from KPL (Gaithersburg, MD), and ECL donkey anti-rabbit whole antibody from GE Healthcare Biosciences (Pittsburgh, PA).

Techniques: Construct, FLAG-tag, Mutagenesis, Transfection, Luciferase

Journal: PLoS ONE

Article Title: Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

doi: 10.1371/journal.pone.0072407

Figure Lengend Snippet: Based upon our current and prior findings, we propose the model depicted in this schema. A ligand-stimulated endoglin-ActRIIA-ALK2 signaling axis promotes Smad1 signaling to decrease the invasiveness of PCa cells. BMPRII is simultaneously required through additional, noncanonical regulatory elements (depicted as a dashed arrow). See text for expanded discussion. BMPRII plays a bimodal role in Smad1 signaling, promoting it via the kinase domain while inhibiting it in a tail-domain-dependent manner, potentially through a tail-domain-interacting protein or by direct interaction with ActRIIA. Endoglin physically interacts with both ActRIIA and BMPRII, and BMPRII interacts with ActRIIA in the absence of endoglin (bidirectional arrows). Previous work from our group has demonstrated that TGFβ signals through Smad3 to promote PCa invasion, that the balance between Smad3 and Smad1 regulates motility and invasion, and that endoglin acts as a gatekeeper in this regard.

Article Snippet: Antibodies to the following proteins were purchased: phospho-Smad1/5, phospho-Smad1/5/8, Smad1, and Myc-tag from Cell Signaling Technology (Danvers, MA), endoglin from BD Biosciences (San Jose, CA), GAPDH from Enzo Life Sciences (Farmingdale NY), FLAG-tag and Myc-tag from Sigma-Aldrich (St. Louis, MO), α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA), HRP-conjugated goat anti-mouse IgG (H+L) F(ab')2 fragment from KPL (Gaithersburg, MD), and ECL donkey anti-rabbit whole antibody from GE Healthcare Biosciences (Pittsburgh, PA).

Techniques: