Journal: Biomaterials advances

Article Title: Enhancing BMP-2-mediated osteogenesis with a synthetic heparan sulfate mimetic.

doi: 10.1016/j.bioadv.2023.213671

Figure Lengend Snippet: Fig. 2. Glycomimetic 15 binds BMP-2 and several other pro-regenerative factors with high affinity. A – SPR solution competition dosing experiments were performed with Ai - BMP-2, Aii - BMP-4, Aiii - BMP-7, Aiv BMPRII Fc chimera and Av - VEGF165 using a dose range of glycomimetic 15 (3.91 to 8000 nM). Avi – normalized response data extrapolated from sensorgrams Ai-Av. B – fluorescence polarization solution competition with BMP-2 (50 nM), 488-labelled heparin dp24 (0.5 nM) and increasing concentrations of glycomimetic 15 (red) or glycomimetic 18 (blue). Ci – DSF melt curves. Cii – first derivative of melt curve data. Ciii – BMP-2 melt temperature when incubated with multiple concentrations of glycomimetic 15, derived from the first derivative of melt curve data. Di – Normalized anti-factor Xa activity for glycomimetic 15 (black), heparin dp12 (red) and heparin dp24 (blue). Dii – anti-factor Xa activity in IU/mg, calculated using USP heparin standard curve. Bars represent mean, and error bars represent S.D. All data is representative of three independent experiments.

Article Snippet: BMP-2 (cat. # 355-BM/ CF), BMP-4 (cat. # 314-BP/CF), BMP-7 (cat. # 354-BP/CF), BMPRII Fc chimera (cat. # 811-BR), Noggin (cat. # 6057-NG/CF), VEGF165 (cat. # 293-VE/CF) polyclonal Goat anti-Human BMP-2/BMP-4 IgG (cat. # AF355), biotinylated monoclonal Mouse anti-Human BMP-2 IgG (cat. #BAM3552) were purchased from R&D systems.

Techniques: Fluorescence, Incubation, Derivative Assay, Activity Assay

Journal: Biomaterials advances

Article Title: Enhancing BMP-2-mediated osteogenesis with a synthetic heparan sulfate mimetic.

doi: 10.1016/j.bioadv.2023.213671

Figure Lengend Snippet: Fig. 5. Glycomimetic 15 influences BMPRII: BMP-2 and BMP-2: cell surface interactions. Ai – Co-immunoprecipitation and Western blot of BMP-2 (125 nM) with BMPRII Fc chimera (250 nM) ± increasing concentrations of either glycomimetic 15 or 18. Aii – Log2 fold change of densitometry values obtained from three in dependent co-immunoprecipitation experiments – bars represent mean log2 fold change ±95 % CI, normalized to BMPRII + BMP-2 alone. Bi – Flow cytometry histograms of C2C12 cells stained with BMP-2 (black histogram) ± increasing concentrations of glycomimetic 15 (red histogram) or glycomimetic 18 (blue histo gram). Bii – Cell surface binding of BMP-2 to C2C12 cells incubated with increasing concentrations of glycomimetic 15 (red) or glycomimetic 18 (blue) – data is mean geometric mean fluorescence ± S.D. normalized to BMP-2 alone. All data either constitutes (Aii, Bii) or is representative of (Ai, Bi) three independent experiments. Statistical analysis was performed via one-way ANOVA with Tukey’s post hoc testing (Aii) or two-way ANOVA with ˇSíd´ak or Bonferroni post hoc testing (Bii). Sig nificance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: BMP-2 (cat. # 355-BM/ CF), BMP-4 (cat. # 314-BP/CF), BMP-7 (cat. # 354-BP/CF), BMPRII Fc chimera (cat. # 811-BR), Noggin (cat. # 6057-NG/CF), VEGF165 (cat. # 293-VE/CF) polyclonal Goat anti-Human BMP-2/BMP-4 IgG (cat. # AF355), biotinylated monoclonal Mouse anti-Human BMP-2 IgG (cat. #BAM3552) were purchased from R&D systems.

Techniques: Immunoprecipitation, Western Blot, Flow Cytometry, Staining, Binding Assay, Incubation, Fluorescence

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: Figure 1. Endoglin requires ActRIIA and BMPRII to suppress invasion. A) The effect of type II receptors on endoglin mediated suppression of invasion (EMSI). PC3-M (left) or DU-145 (right) cells were transiently transfected with empty vector (Vec) or with endoglin along with siRNA, as indicated. The following siRNAs were used: siNeg – non-targeting negative control, si2A - targets ActRIIA, si2B - targets ActRIIB, siBMP - targets BMPRII, siTGF - targets TGFbRII. After 48 hours, cell invasion was measured. Data represent the mean 6 SEM of 3 independent experiments, each in replicates of 3. *, p#0.05 compared to Vec/siNeg. Micrographs are representative images of cells that have invaded through Matrigel, were stained for bgal, and imaged (magnification 100X). B) ActRIIA and BMPRII siRNA is specific. PC3-M cells were transiently transfected with endoglin along with the indicated siRNAs as in (A). After 48 hours mRNA expression was assessed via qRT-PCR, normalized to GAPDH, and expressed relative to siNeg- transfected cells (normalized to 1.0). Data represent the mean 6 SD of a single experiment, performed in replicates of N = 2; similar results were seen in an independent experiment (N = 2 replicates). *, p#0.05 compared to siNeg. C) ActRIIA and BMPRII siRNA suppresses target protein. PC3-M cells were transfected with ActRIIA-myc (upper panels) or BMPRII-flag (lower panels), as well as with the indicated siRNAs, followed by Western blot for indicated proteins. Data are from a representative experiment (N = 2 separate experiments). D) Blocking ActRIIA or BMPRII ligand binding inhibits EMSI. PC3-M cells were transiently transfected with empty vector or endoglin as above. After 2 days, cells were pretreated for 5 hrs ligand traps comprised of ActRIIA or BMPRII extracellular domain fused to immunoglobulin Fc region (Fc-A2 or Fc-B2 respectively). Treatment continued during the subsequent conduct of cell invasion assays. Data represent mean 6 SEM of 3 independent experiments, each in replicates of N = 3. *, p#0.05 compared to Vec/-. doi:10.1371/journal.pone.0072407.g001

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Transfection, Plasmid Preparation, Negative Control, Staining, Expressing, Quantitative RT-PCR, Western Blot, Blocking Assay, Ligand Binding 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: Figure 2. ActRIIA promotes Smad1 signaling while BMPRII is inhibitory. PC3-M cells were transiently transfected with empty vector or endoglin and the indicated siRNA as in Figure 1. 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 BRE2- luciferase activation. Cells were additionally co-transfected with BRE2-luciferase and Renilla luciferase constructs, and luciferase activity (normalized to Renilla luciferase activity) was measured. Data are the mean 6 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). doi:10.1371/journal.pone.0072407.g002

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Transfection, Plasmid Preparation, Western Blot, Luciferase, Promoter Assay, Phospho-proteomics, 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: Figure 3. Smad1 is the main downstream target of endoglin. 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 6 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 TGFb 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 BRE2-luciferase activity is largely mediated by Smad1. Cells were transfected with endoglin and were additionally co- transfected with BRE2- and Renilla luciferase construct, and luciferase assays performed. Data represent mean 6 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 BRE2-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 6 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. doi:10.1371/journal.pone.0072407.g003

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

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: Figure 4. ActRIIA promotion of Smad1 signaling is kinase dependent, while BMPRII inhibition occurs via the tail domain. 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 DKD 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 BRE2-luciferase and Renilla luciferase, endoglin, wild type (WT) or kinase domain deletion (DKD) ActRIIA constructs, and siRNA to ActRIIA or non-targeting as indicated. Luciferase assay performed as in Figure 2B. Data represent mean 6 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; Dtail = tail domain deleted. Luciferase assay performed as in Figure 2B. Data represent mean 6 SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p#0.05 compared to Eng/siBMPRII. doi:10.1371/journal.pone.0072407.g004

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Inhibition, 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: Figure 6. BMPRII suppresses ActRIIA-mediated Smad1 activity. PC3-M cells were transfected with BRE2-luciferase, Renilla luciferase, and indicated plasmid DNA with or without indicated siRNA. siRNA lanes marked with a hyphen were transfected with non-targeting siRNA. Two days later cells were lysed and luciferase activity was assessed as in Figure 2B. A) Increased BRE2-luciferase activity upon silencing BMPRII is mediated by ActRIIA. Neg = non-targeting siRNA. 2A = siActRIIA. 2B = siActRIIB. Data represent mean 6 SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p#0.05 for indicated comparison. B) BMPRII-mediated suppression of BRE2-luciferase activity is dependent on ActRIIA expression. BMPRII construct abbreviations as in Figure 4. Data represent mean 6 SD of a single representative experiment (N = 2 replicates), repeated 3 times (N = 2 replicates) with similar results. *, p#0.05 for indicated comparison. C) BMPRII suppresses signaling from ActRIIA. Data represent mean 6 SD from one representative experiment (N = 2 replicates) of three repeated separately (N = 2). *, p,0.05 for indicated comparison. doi:10.1371/journal.pone.0072407.g006

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Comparison, Expressing, 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: Figure 7. Endoglin physically interacts with ActRIIA and BMPRII. After transient transfection, the surface proteins of PC3-M cells were crosslinked, cells lysed, immunoprecipitation performed, crosslinking reversed, and Western blot performed. A) ActRIIA coprecipitates with endoglin. Cells were transfected with Myc-ActRIIA and FLAG-endoglin, FLAG (endoglin) immunoprecipitated, and Western blots probed for ActRIIA (with anti- Myc) and endoglin. Controls for immunoprecipitation included agarose beads alone (no Ig) and nonspecific isotype control IgG (IgG isotype). Input lysate, lysate post-immunoprecipitation (i.e. supernatant), and immunoprecipitation (IP) samples were loaded as indicated. Data are from a representative experiment (N = 2 experiments). B) The kinase domain of ActRIIA is dispensable for interaction with endoglin. Cells transfected with Myc-WT or -DKD-ActRIIA and FLAG-endoglin as indicated, FLAG or Myc was immunoprecipitated as indicated, and Western blots probed as indicated. Data are from a representative experiment (N = 4 experiments). (C) BMPRII precipitates with endoglin. Cells were transfected with FLAG-BMPRII and untagged endoglin, FLAG immunoprecipitated, and Western blots probed as indicated. Data are from a representative experiment (N = 2 experiments). (D) The kinase activity and tail domain of BMPRII are dispensable for interaction with endoglin. Cells transfected with FLAG-WT, -KI, or -Dtail-BMPRII and untagged endoglin as indicated, FLAG-BMPRII immunoprecipitated, and Western blots probed as indicated. In some instances surface proteins were crosslinked (+), which was reversed after immunoprecipitation, while in other instances proteins were not crosslinked (-) Data are from a representative experiment (N = 3 experiments). doi:10.1371/journal.pone.0072407.g007

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, 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: Figure 8. ActRIIA and BMPRII physically interact. Coimmunoprecipitation experiments were performed as in Figure 7. In all experiments, cells were transfected, cell surface proteins crosslinked, immunoprecipitation (IP) performed, crosslinks reversed, and Western blot (IB) performed as indicated. In some studies, cells were transfected with FLAG-endoglin as a positive control. Input lysate, post IP lysate, and IP samples are loaded as indicated. (A and B) ActRIIA precipitates with BMPRII. (C and D) ActRIIA kinase domain is dispensable for interaction with BMPRII. (E and F) BMPRII kinase activity and tail domain are dispensable for interaction with BMPRII. All data are from a representative experiment, repeated at least N = 5 separate times. doi:10.1371/journal.pone.0072407.g008

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Transfection, Immunoprecipitation, Western Blot, Positive Control, 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: Figure 9. Endoglin-Mediated Suppression of Invasion is Dependent on ActRIIA Kinase Domain and Independent of BMPRII Kinase Activity or Tail Domain. Cells were transfected with endoglin, ActRIIA (A) or BMPRII constructs (B), and ActRIIA or BMPRII directed siRNA, as indicated, and cell invasion assays conducted as in Figure 1. Data represent the mean 6 SEM of N = 2 independent experiments (A) or N = 3 independent experiments (B), each in replicates of 3. * denotes p#0.05 compared to cells transfected with empty vector and non-targeting siRNA. doi:10.1371/journal.pone.0072407.g009

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques: Activity Assay, Transfection, Construct, Plasmid Preparation

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: Figure 10. Proposed model for the regulation of endoglin- mediated suppression of invasion by ActRIIA and BMPRII. 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-interact- ing 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 TGFb 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. doi:10.1371/journal.pone.0072407.g010

Article Snippet: Reagents Neutralizing antibody to ActRIIA, Fc-ActRIIA and Fc-BMPRII ligand traps, and recombinant human TGFb were purchased from R&D Systems (Minneapolis MN).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Epigenetic repression of bone morphogenetic protein receptor II expression in scleroderma

doi: 10.1111/jcmm.12105

Figure Lengend Snippet: Effects of BMP2 and reduced BMPRII expression on MVECs survival. ( A ) BMP2 shows the anti-apoptotic effects on starvation-induced apoptosis in normal-MVECs, but not in SSc-MVECs. Normal and SSc-MVECs cells were cultured in serum and growth factor free (SGF) medium with and without BMP2 (200 ng/ml) for 24 hrs. The cell viability was measured by CellTiter Proliferation Assay. Viability of untreated control cells was set at 100%. Data are expressed as mean ± SD from triplicate experiments, * P < 0.01 versus control; ** P < 0.05 versus SGF without BMP2 treatment. ( B – D ) Knockdown of BMPRII in normal-MVECs by siRNA led to sensitize the cells to apoptotic stimuli. ( B and C ) reduced BMPRII mRNA and protein expression by siRNA. Normal-MVECs were transfected with IR-iRNA or BMPRII-siRNA. After 24 hrs, BMPRII mRNA expression levels were measured by real-time PCR (see B ; the data are normalized for GAPDH, data are presented as mean ± SD, n = 5, * P < 0.01 versus control and IR-siRNA control), and BMPRII protein expression levels were assessed by Western blot analysis ( C ). ( D ) Effect of reduced BMPRII expression on MVECs survival. Normal endothelial cells were transfected with IR-siRNA or BMPRII-siRNA and cultured in EBM-2 medium without FCS and growth factors for 24 hrs (SGF). Cell viability was determined using the CellTiter 96 Cell Proliferation Assay. Data showed that reduction in BMPRII expression in Normal-MVECs significantly increased cell apoptosis induced by SGF. P < 0.01 versus control and IR-siRNA control.

Article Snippet: Protein detection was carried out with primary antibodies using goat anti-human BMPRII antibody (1:200; R&D systems, Minneapolis, MN, USA) or monoclonal antibodies against β-actin (Abcam Inc., Cambridge, MA, USA) or β-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Cell Culture, Proliferation Assay, Control, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Western Blot