Journal: The Journal of Experimental Medicine

Article Title: Bone morphogenetic protein 7 in dormancy and metastasis of prostate cancer stem-like cells in bone

doi: 10.1084/jem.20110840

Figure Lengend Snippet: The expression of BMPR2 receptor is inversely correlated with recurrence and bone metastasis of prostate cancer patients. (A) The relation between recurrence-free survival and the expression (positive or negative) of the indicated BMP receptors was analyzed using an existing cohort data of prostate cancer patients ( n = 21; ). *, P = 0.0485 (Log-rank test). (B, top) Representative images of immunohistochemical staining for BMPR2 on prostate cancer patient samples with or without bone metastasis. Bar, 50 µm. (bottom) The correlation between BMPR2 expression and bone metastasis (met) status was evaluated by Fisher’s exact test.

Article Snippet: The cells were lysed and analyzed by immunoblotting using antibodies specific for the following proteins: total and phospho-p38, p21, p27 (Cell Signaling Technology), NDRG1 (gift from T. Commes, Université de Montpellier 2, Montpellier, France), BMP7, BMPR2 (R&D Systems), and β-tubulin (Millipore).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: The Journal of Experimental Medicine

Article Title: Bone morphogenetic protein 7 in dormancy and metastasis of prostate cancer stem-like cells in bone

doi: 10.1084/jem.20110840

Figure Lengend Snippet: BMPR2 mediates BMP7-induced reversible senescence. (A) The expression of BMPR2 and β-tubulin in PC3 mm that had either scrambled shRNA (scramble) or shRNA for BMPR2 (shBMPR2) was examined by Western blot (top) and qRT-PCR (bottom; n = 3). (B) Kaplan-Meier analysis for bone metastasis–free survival of mice after intracardiac injection of PC3 mm cells that carried either scrambled shRNA (scramble; n = 9) or shRNA for BMPR2 (shBMPR2; n = 10) followed by treatment with vehicle or BMP7. Scramble versus Scramble + BMP7: ***, P < 0.0001; shBMPR2 versus shBMPR2 + BMP7: NS; Scramble + BMP7 versus shBMPR2 + BMP7: ***, P = 0.0002 by Log-rank test. (C) PC3 mm/scramble cells and PC3 mm/shBMPR2 cells were treated with or without BMP7, and the expression of p-p38, p38, p21, NDRG1, and β-tubulin was examined by Western blot. (D) The PC3 mm cells stably expressing Tet-shBMPR2 were cultured with (+) or without (−) induction of shBMPR2 in the presence or absence of BMP7, followed by assaying SA–β-gal. −/−, no induction; +/+, continuous induction; +/−, 48-h induction followed by 48-h withdrawal of tetracycline ( n = 3). ***, P < 0.001. The expression of BMPR2 and β-tubulin was examined by Western blot (inset). Experiments in A, C, and D were performed three times, the experiment in B was performed twice independently, and representative data are shown. Results are shown as mean ± SEM.

Article Snippet: The cells were lysed and analyzed by immunoblotting using antibodies specific for the following proteins: total and phospho-p38, p21, p27 (Cell Signaling Technology), NDRG1 (gift from T. Commes, Université de Montpellier 2, Montpellier, France), BMP7, BMPR2 (R&D Systems), and β-tubulin (Millipore).

Techniques: Expressing, shRNA, Western Blot, Quantitative RT-PCR, Injection, Stable Transfection, Cell Culture

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: Human fetal spinal cord BMPRII immunostaining at gestational ages of 15–16 weeks. Cryostat section (12 μm) of the human fetal spinal cord estimated to be at gestational ages of 15–16 weeks showing intense BMPRII + immunoreactivity (green) along the ventral surface, as well as the dorsal (sensory) and ventral (motor) horns. Insets A–G shown at higher magnification, scale bar: 50 μm in D; nuclei counterstain DAPI (blue). Staining was observed along the dorsal surface and the sensory and motor neurons in the dorsal and ventral horns. The dotted red line demarcates regions of higher intensity BMPRII-immunoreactivity in dorsal horns. Magenta marks the outer margin of higher-intensity BMPRII-immunoreactivity in the ventral funiculus, while orange demarcates the extent of the structure. (A) BMPRII + immunoreactivity in the dorsal horn (B) BMPRII + immunoreactivity in the ventral horn (C) BMPRII + immunoreactivity at the ventral funiculus. (D–F) Vascular tube-like structures showed strong immunoreactivity to BMPRII staining, while (G) shows an example of staining of the meninges surrounding the spinal cord. The main anatomical structures are identified by labeling. 5× magnification, scale bar: 200 μm. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Immunostaining, Staining, Labeling

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: BMPRII + hNPCs (red) can also be identified in the human fetal brain cortex (A–C) and are identified by a lack of vascular marker expression (D–H). Scattered BMPRII + putative hNPCs (white arrows) are present in the parenchyma of a second-trimester human fetal brain cortex (B), nuclei counterstain DAPI (blue; A), shown merged in (C). By virtue of lower expression, these cells are clearly distinct from BMPRII + cells in vascular tubes (yellow arrow). 63× magnification, scale bar: 20 μm. Quadruple-labeled immunofluorescence of an intact second-trimester spinal cord, utilizing antibodies against BMPRII (G) combined with two established vascular lineage markers (CD34; green; D) and ecto-ADPase CD39 (magenta; E), showing a colocalized, triple-labeled vascular segment (yellow arrow in H). BMPRII (red) is highly expressed by vascular cells (yellow arrow), while scattered BMPRII + single cells (putative hNPCs) negative for vascular markers are evident in the tissue parenchyma (e.g., white arrow in (H); merge of all images). Nuclear counterstain DAPI (blue; F) labels nuclei. 63× magnification, scale bar: 20 μm. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole; hNPCs: human neural precursor cells.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Marker, Expressing, Labeling, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: Effects of tissue culture condition on organotypic reaggregate neurosphere phenotype. (A) A comparison of sphere size showed that uniform stratification significantly favored populations of medium size spheres (200–500 μm diameter, e.g., J, ) compared with small (< 200 μm diameter, e.g. F, ) and large spheres (> 500 μm diameter). *** P < 0.001. (B) The effects of high-density reaggregation compared with low-density aggregation in combination with the presence or absence of LIF were assayed for their effects on neurosphere stratification. Both high-density and LIF-containing culture conditions were significantly different from low-density spheres or those grown in the absence of LIF. (C–F) Immunocharacterization of organotypic reaggregate neurospheres. Labeling with βIII-tubulin (C, green) shows an outer layer of immunoreactivity typical of organotypic neurospheres, while being negative for GFAP (D, red). The inside of organotypic neurospheres shows heterogenous βIII-tubulin and GFAP labeling. Co-staining with BMPRII (E) confirmed that βIII-tubulin + cells are double positive; merge shown in (F), and white boxed areas in C–F are zoomed below each sphere to show the fine detail of expressing cells. Reaggregate neurospheres were also characterized by positive immunoreactivity for LIF (G) and LIFR (H) but with no obvious indication of stratification; whereas endogenous BMP (I) and BMPRII (J) (all green) were both localized to the cell surface layer. All nuclei were counterstained with DAPI (magenta or blue, as indicated). Scale bar in F and J is 25 μm, and in F (inset below), 12.5 μm. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; LIF: leukemia inhibitory factor; LIFR: LIF receptor; ORN: organotypic reaggregate neurosphere.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Comparison, Labeling, Staining, Expressing

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: Characterization of fluorescence-assisted cell sorting-sorted BMPRII + hNPCs. (A–E) Sorted cells were plated, fixed, and subjected to immunofluorescence staining with antibodies to O4 (blue), GFAP (red), MAP2ab (green), and the nuclei counterstained with DAPI (magenta). Image (A) shows sorted cells at low magnification. Images in C through E are shown with polarized bright field and merged at a higher magnification in B (from the area of the white box in A). (F) A representative single-label FACS histogram plot of fluorescence intensity versus cell number for collected BMPRII + cells (green trace) and control (2° antibody only; red trace). “Collected” shows the fluorescence intensity gate used to collect the BMPRII + cells. (G) Sorted cells were plated and the proportion of each phenotype was quantified. A significant increase was found for the number of MAP2ab + cells from approximately 24% in control to 83% in collected. (H) Cells were characterized using antibodies against each indicated marker where ‘+’ indicates > 90% of the cells with neuronal morphology were positive and ‘–’ indicates < 10% of these cells were positive. Data are expressed as the mean ± SEM, and scale bars are as indicated. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; hNPCs: human neural precursor cells; MAP: microtubule-associated protein; NeuN: neuron-specific nuclear protein; PSA-NCAM: polysialylated-neural cell adhesion molecule.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Fluorescence, FACS, Immunofluorescence, Staining, Control, Marker

Journal: JCI Insight

Article Title: Stem cell–associated osteogenic deficiency causes craniofacial deformities with progeroid accumulation of prelamin A

doi: 10.1172/jci.insight.196932

Figure Lengend Snippet: ( A ) Limiting dilution analysis of stem cell–mediated bone formation with renal capsule transplantation. Representative images of whole-mount von Kossa staining detecting ectopic bone formation in the mouse recipients transplanted by the indicated number of suture cells into the renal capsule. Arrowheads indicate the ectopic bones. ( B ) Representative images showing the analysis of Axin2-expressing cells using the Axin2 mGFP allele in the indicated 1-month-old (1M) suture. ( C ) Representative images examining the BMPR1A + and GLI1 + cell population within the indicated 1-month-old (1M) suture. ( D ) Graphs indicate the quantitation of the average percentage of BMPR1A + and GLI1 + cells in 3 independent experiments ( P < 0.005 or 0.05, n = 3, mean ± SEM, 2-tailed Student’s t test). SAG, sagittal; COR, coronal; AF, anterior frontal. Scale bars: 1 mm ( A ) and 50 μm ( B and C ).

Article Snippet: Mouse monoclonal antibodies against Bmpr1a (NBP2-37421, 1:75, Novus Biologicals), fascin1 (SC-21743, 1:100, Santa Cruz), and Alexa Fluor 568 phalloidin (A12380, 1:400, Invitrogen); rabbit polyclonal antibodies Osx (ab22552, 1:800, Abcam), OCN (23418-1-AP, 1:50, Proteintech), Gli1 (NBP1-78259, 1:100, Novus Biologicals), Myl2 (3671, 1:100, Cell Signaling Technology), and GM130 (2296, 1:200, Cell Signaling Technology); rabbit monoclonal antibodies lamin A/C (MA5-35284, 1:200, Invitrogen) and SUN2(EPR6557, 1:100, Abcam); anti-rabbit secondary antibody (BA-1000, 1:200, Vector laboratories); and anti-mouse IgG secondary antibodies (PK-2200, 1:250, Vector laboratories), were used in the immunostaining studies.

Techniques: Transplantation Assay, Staining, Expressing, Quantitation Assay