Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Over-expression of BMPR-IB reduces the malignancy of glioblastoma cells by upregulation of p21 and p27Kip1

doi: 10.1186/1756-9966-31-52

Figure Lengend Snippet: Determination of BMPR-IB expression in normal human astrocytes and glioma cell lines. ( A ) Real-time-RT-PCR was used to determine the mRNA expressions of BMPR-IB and other factors involved in BMP/BMPR signaling pathway. ( B ) Western blot analyses were employed to show the protein expression of BMPR-IB, P-Smad1/5/8 and Smad1/5/8 in glioblastoma cell lines(up). Statistical analysis of results from WB analysis(down). ( C ) Alterations in the expression of BMPR-IB and P-Smad1/5/8 after 48 h of BMPR-IB overexpression, determined by WB analysis. ( D ) Immunofluorescence analysis of the activation of Smad1/5/8 after 48 h of BMPR-IB infection.

Article Snippet: After incubation with 0.1% Triton-PBS for 30 min and blocking with 1% bovine serum albumin-PBS for 2 h in room temperature, the cells were then incubated with the primary antibodies overnight in 4°C at the concentration recommended by the supplier (a rabbit anti-phospho-Smad1/5/8 antibody (Cell signal), a goat anti-BMPR-IB antibody (Santa Cruz Biotechnology) and a mouse anti-GFAP antibody (Sigma)).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Immunofluorescence, Activation Assay, Infection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Over-expression of BMPR-IB reduces the malignancy of glioblastoma cells by upregulation of p21 and p27Kip1

doi: 10.1186/1756-9966-31-52

Figure Lengend Snippet: Induction of differentiation by BMPR-IB in human glioma cell lines. ( A ) After infection and transfection with rAAV-BMPR-IB and si-BMPR-IB, the expression of GFAP of glioblastoma cells was detected by immunofluorescence (left), and the morphological alterations were examined by phase contrast microscope(right). ( B ) WB analysis showed that BMPR-IB infection induced the expression of endogenous GFAP and inhibited the expression of Nestin, whereas BMPR-IB knock-down decreased the expression of GFAP and increased the expression of Nestin.

Article Snippet: After incubation with 0.1% Triton-PBS for 30 min and blocking with 1% bovine serum albumin-PBS for 2 h in room temperature, the cells were then incubated with the primary antibodies overnight in 4°C at the concentration recommended by the supplier (a rabbit anti-phospho-Smad1/5/8 antibody (Cell signal), a goat anti-BMPR-IB antibody (Santa Cruz Biotechnology) and a mouse anti-GFAP antibody (Sigma)).

Techniques: Infection, Transfection, Expressing, Immunofluorescence, Microscopy, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Over-expression of BMPR-IB reduces the malignancy of glioblastoma cells by upregulation of p21 and p27Kip1

doi: 10.1186/1756-9966-31-52

Figure Lengend Snippet: Overexpression of BMPR-IB in human glioma cells decreased tumorigenicity in vivo. ( A ) Tumor growth in the subcutis of nude mice. ( B ) Representative H&E staining and immunohistochemistry of tumors derived from intracranial xenografts of glioma cells.a L -d L (low magnification images) L and a-d(high magnification images), HE staining of tumors derived from intracranial xenografts of glioma cells. e-h, GFAP immunohistocheistry of tumors derived from intracranial xenografts of glioma cells. i-l, CD34 immunohistocheistry of tumors derived from intracranial xenografts of glioma cells. (a, e, i, U251-AAV. b, f, j, U251-AAV-IB. c, g, k, SF763-si-control. d, h, l, SF763-si-IB). Magnification was ×20 in a-d, and ×40 in e-l. ( C ) Survival of animals intracranially injected with glioma cells that were infected or knocked down using BMPR-IB and control vectors (log rank test: p < 0.0001).

Article Snippet: After incubation with 0.1% Triton-PBS for 30 min and blocking with 1% bovine serum albumin-PBS for 2 h in room temperature, the cells were then incubated with the primary antibodies overnight in 4°C at the concentration recommended by the supplier (a rabbit anti-phospho-Smad1/5/8 antibody (Cell signal), a goat anti-BMPR-IB antibody (Santa Cruz Biotechnology) and a mouse anti-GFAP antibody (Sigma)).

Techniques: Over Expression, In Vivo, Staining, Immunohistochemistry, Derivative Assay, Control, Injection, Infection

Journal: bioRxiv

Article Title: Essential role of Bone morphogenetic protein 15 in porcine ovarian and follicular development and ovulation

doi: 10.1101/724096

Figure Lengend Snippet: (A) Immunofluorescence images showed Smad1/5/8 pathway was evidently less activated in abnormal follicles in TGF ovaries, and severely inhibited in highly degraded 365-day TGS follicles, as compared to that in normal follicle of TGF and WT ovaries. (B) Immunofluorescence images demonstrated a mild decrease in Smad2/3 signaling in both normal and abnormal follicles of TG ovaries. Smad2/3 signaling was remarkably inhibited in highly degraded 365-day TGS follicles. TGFN, normal follicles in TGF ovary; TGFA, abnormal follicles in TGF ovary. Scale bar = 100μm.

Article Snippet: While -other antibodies including ALK6 (Santa cruz, sc5679), BMPR2(Santa cruz, sc5683), Smad2/3 (Santa cruz, sc8332), Smad1/5/8 (Santa cruz, sc6031R), and p-Smad2/3 (Santa cruz, sc11769) were diluted 1:50 in PBS.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Essential role of Bone morphogenetic protein 15 in porcine ovarian and follicular development and ovulation

doi: 10.1101/724096

Figure Lengend Snippet: (A) Immunofluorescence images showed Smad1/5/8 pathway was evidently less activated in abnormal follicles in TGF ovaries, and severely inhibited in highly degraded 365-day TGS follicles, as compared to that in normal follicle of TGF and WT ovaries. (B) Immunofluorescence images demonstrated a mild decrease in Smad2/3 signaling in both normal and abnormal follicles of TG ovaries. Smad2/3 signaling was remarkably inhibited in highly degraded 365-day TGS follicles. TGFN, normal follicles in TGF ovary; TGFA, abnormal follicles in TGF ovary. Scale bar = 100μm.

Article Snippet: While -other antibodies including ALK6 (Santa cruz, sc5679), BMPR2(Santa cruz, sc5683), Smad2/3 (Santa cruz, sc8332), Smad1/5/8 (Santa cruz, sc6031R), and p-Smad2/3 (Santa cruz, sc11769) were diluted 1:50 in PBS.

Techniques: Immunofluorescence

Journal: Nature medicine

Article Title: Conversion of vascular endothelial cells into multipotent stem-like cells

doi: 10.1038/nm.2252

Figure Lengend Snippet: Formation of endothelial derived multipotent stem-like cells induced by constitutively active ALK2. (a) Flow cytometry analysis showing co-expression of TIE2 and STRO-1 in endothelial cells expressing mutant ALK2. (b) Immunoblotting showing expression of the mesenchymal stem cell markers STRO-1, CD10, CD44, CD71, CD90, and CD117 in endothelial cells expressing mutant ALK2. Human bone marrow derived mesenchymal stem cells (MSC) express these markers, but human corneal fibroblasts (HCF) do not. β-actin was used as an internal control. (c) Immunoblotting showing increased expression of osteoblast (osterix), chondrocyte (SOX9), or adipocyte (PPARγ2) markers in cells treated with mutant ALK2 for 48 h followed by exposure to osteogenic, chondrogenic, or adipogenic culture medium. (d) Positive staining of osteoblast (alkaline phosphatase and alizarin red), chondrocyte (alcian blue), or adipocyte (oil red O) products in endothelial cell cultures treated with mutant ALK2, but not with vector or wild-type ALK2, for 48 h followed by growth in osteogenic, chondrogenic, or adipogenic culture medium, respectively. Scale bar, 100 μm. (e) Positive staining of osteoblast (alizarin red), chondrocyte (alcian blue), or adipocyte (oil red O) products in polylactic acid scaffolds containing endothelial cells transformed by mutant ALK2 implanted into nude mice, followed by local injection of osteogenic, chondrogenic, or adipogenic medium every 72 h for 6 weeks. Scale bar, 100 μm.

Article Snippet: Immunoblotting, immunoprecipitation, and immunofluorescence Immunoassays were performed using the following antibodies at concentrations (and using protocols) recommended by the respective manufacturers: FSP-1 (H00006275-M01; Stressgen), phospho-Smad2 (3101), Smad2 (3122), phospho-Smad5 (9516), Smad5 (9517; Cell Signaling Technology), ALK1 (sc-19547), ALK2 (sc-25449), ALK3 (sc-20736), ALK4 (sc-31297), ALK5 (sc-398), ALK6 (sc-25455), ALK7 (sc-135001), phospho-tyrosine (sc-7020), VE-cadherin (sc-6458), TIE1 (sc-342), TIE2 (sc-324, sc-9026), STRO-1 (sc-47733), CD10 (58939), CD44 (sc-71220), CD71 (sc-32272), CD90 (sc-9163), CD117 (sc-17806), osteocalcin (sc-74495, sc-23790), SOX9 (sc-20095), PPARγ2 (sc-22022; Santa Cruz Biotechnology), osterix (ab22552), adiponectin (ab22554), N-cadherin (ab76057), NG2 (ab83508), vWF (ab68545; Abcam); CD31 (IR610; Dako), His (A00174; GenScript); α-SMA (A5228), β-actin (A1978; Sigma-Aldrich).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Mutagenesis, Western Blot, Control, Staining, Plasmid Preparation, Transformation Assay, Injection

Journal: Nature medicine

Article Title: Conversion of vascular endothelial cells into multipotent stem-like cells

doi: 10.1038/nm.2252

Figure Lengend Snippet: Endothelial cells transformed by treatment with TGF-β2 or BMP4 express mesenchymal stem cell markers and exhibit multipotency. (a) Flow cytometry showing co-expression of TIE2 and STRO-1 in endothelial cells treated with TGF-β2 or BMP4 for 48 h. (b) Immunoblotting confirming increased protein expression of mesenchymal stem cell markers STRO-1, CD10, CD44, CD71, CD90, and CD117 in cells treated with TGF-β2 or BMP4. β-actin was used as an internal control. (c) Immunoblotting showing increased expression of osteoblast (osterix), chondrocyte (SOX9), or adipocyte (PPARγ2) markers in cells treated with TGF-β2 or BMP4 for 48 h followed by exposure to osteogenic, chondrogenic, or adipogenic culture medium, respectively. (d) Positive staining of osteoblast (alkaline phosphatase and alizarin red), chondrocyte (alcian blue), or adipocyte (oil red O) products in endothelial cell cultures treated with TGF-β2 or BMP4 for 48 h followed by growth in osteogenic, chondrogenic, or adipogenic culture medium. Scale bar, 100 μm. (e) Positive staining of osteoblast (alizarin red), chondrocyte (alcian blue), or adipocyte (oil red O) products in polylactic acid scaffolds containing endothelial cells transformed by TGF-β2 or BMP4 subcutaneously implanted into nude mice, followed by local injection of osteogenic, chondrogenic, or adipogenic medium every 72 h for 6 weeks. Scale bar, 100 μm.

Article Snippet: Immunoblotting, immunoprecipitation, and immunofluorescence Immunoassays were performed using the following antibodies at concentrations (and using protocols) recommended by the respective manufacturers: FSP-1 (H00006275-M01; Stressgen), phospho-Smad2 (3101), Smad2 (3122), phospho-Smad5 (9516), Smad5 (9517; Cell Signaling Technology), ALK1 (sc-19547), ALK2 (sc-25449), ALK3 (sc-20736), ALK4 (sc-31297), ALK5 (sc-398), ALK6 (sc-25455), ALK7 (sc-135001), phospho-tyrosine (sc-7020), VE-cadherin (sc-6458), TIE1 (sc-342), TIE2 (sc-324, sc-9026), STRO-1 (sc-47733), CD10 (58939), CD44 (sc-71220), CD71 (sc-32272), CD90 (sc-9163), CD117 (sc-17806), osteocalcin (sc-74495, sc-23790), SOX9 (sc-20095), PPARγ2 (sc-22022; Santa Cruz Biotechnology), osterix (ab22552), adiponectin (ab22554), N-cadherin (ab76057), NG2 (ab83508), vWF (ab68545; Abcam); CD31 (IR610; Dako), His (A00174; GenScript); α-SMA (A5228), β-actin (A1978; Sigma-Aldrich).

Techniques: Transformation Assay, Flow Cytometry, Expressing, Western Blot, Control, Staining, Injection