Journal: PLoS ONE

Article Title: Gremlin Enhances the Determined Path to Cardiomyogenesis

doi: 10.1371/journal.pone.0002407

Figure Lengend Snippet: (A) Phase contrast micrograph of CL6 cells with exposure to DMSO alone (a), Grem1 (125 ng/ml) and DMSO (b) for 14 days. The medium, including Grem1 and DMSO, was changed every day. CL6 cells exhibited apparent spontaneous beating between days 9–11. Beating CL6 cell colonies are outlined by white lines. (B) Percentage of beating area in differentiated CL6 cells. CL6 cell treated with Grem1 (125 ng/ml) and DMSO exhibited the strongest contraction. (C) RT-PCR analysis of the genes encoding cardiac-specific transcriptional factors ( Csx/Nkx2.5 , Gata4 , Mef2c , Hand2 ), circulating hormone ( ANP , BNP ), cardiac-specific proteins ( MyLC-2a , MyLC-2v , β-MyHC ), cytokines ( Bmp2 , Bmp4 , Fgf8 , Grem1 , Wnt1 , Wnt3a , Wnt5a , Wnt7a , Wnt11 ), SM-MyHC , and Gapdh (From top to bottom). Mouse total heart RNA for the Csx/Nkx2.5 , Gata4 , Mef2c , Hand2 , ANP , BNP , MyLC-2a , MyLC-2v , β-MyHC , Bmp2 , Bmp4 , Grem1 , Wnt11 , SM-MyHC , and Gapdh genes, mouse embryonic stem cell RNA for the Fgf8 gene, and mouse total skeletal muscle RNA for the Wnt1 , Wnt3a , Wnt5a , and Wnt7a genes were used for positive controls. H 2 O (without RNA) served as a negative control. (D) Immunocytochemistry of CL6 cells 14 days after exposure to Grem1 (125 ng/ml) and DMSO with MF20 and cTnT (a), and α-actinin (b). Cell nuclei are stained with DAPI. Clear striations are evident. (E) Immunocytochemistry of CL6 cells 14 days after exposure to Grem1 and DMSO with cardiac troponin T (cTnT) and sarcomeric myosin (MF20). CL6 cells treated with Grem1 (125 ng/ml) and DMSO (a), and DMSO alone (b) stained positive for cTnT and MF20. Untreated CL6 cells, i.e. not exposed to Grem1(125 ng/ml) or DMSO, stained negative for cTnT and MF20. Cell nuclei were stained with DAPI. (F) Percentage of MF20- and cTnT-double positive area.

Article Snippet: Recombinant human bone morphogenetic protein-2 (BMP2) was purchased from R&D systems.

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control, Immunocytochemistry, Staining

Journal: PLoS ONE

Article Title: Gremlin Enhances the Determined Path to Cardiomyogenesis

doi: 10.1371/journal.pone.0002407

Figure Lengend Snippet: (A) RT-PCR analysis of the gene encoding BrachyuryT , Tbx6 , cardiac-specific transcriptional factor ( Csx/Nkx2. 5), cardiac-specific protein ( β-MyHC ), and Gapdh (From top to bottom) of CL6 cells treated with DMSO alone, or DMSO and BMP2 (100 ng/ml) for the first 3 days (days 1–3). The medium, including BMP2 and DMSO, was changed every day. (B) Percentage of MF20-positive area. Immunocytochemistry was carried out on CL6 cells 14 days after cells had been exposed to DMSO and BMP2 (100 ng/ml) for the first 3 days (days 1–3). The asterisk indicates a significant statistical difference ( P <0.05). (C) Quantitative real-time RT-PCR analysis of the gene encoding Bmp2 (left), and Bmp4 (right) in CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3). (D) BMP signaling activity of CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3) were determined by luciferase activity analysis using Id1 promoter-Lux (a firefly luciferase reporter plasmid driven by the Id1 binding sites), pRL-CMV as co-transfected control, and Dual luciferase reporter assay system. Relative luciferase unit of the CL6 cells untreated with inducers at day 3 is regarded as 0.1 (data not shown). (E) Quantitative real-time RT-PCR analysis of the gene encoding Wnt3a in CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3). (F) Wnt/β-catenin signaling activity of CL6 cells 48 h after exposure to DMSO, or DMSO and BMP2 (100 ng/ml) was determined by luciferase activity analysis using TOPflash (a firefly luciferase reporter plasmid driven by two sets of three copies of the TCF binding site and herpes simple virus thymidine kinase minimal promoter), pRL-CMV as co-transfected control, and Dual luciferase reporter assay system. Relative luciferase unit of the CL6 cells untreated with inducers is regarded as 0.1. The asterisk indicates a significant statistical difference ( P <0.05). (G) Timeframe of Wnt/β-catenin signaling activity in CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3). Relative luciferase unit of the CL6 cells untreated with inducers at day 3 is regarded as 0.1 (data not shown).

Article Snippet: Recombinant human bone morphogenetic protein-2 (BMP2) was purchased from R&D systems.

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry, Quantitative RT-PCR, Activity Assay, Luciferase, Plasmid Preparation, Binding Assay, Transfection, Reporter Assay, Virus

Journal: Animals : an Open Access Journal from MDPI

Article Title: Association Between BMP2 Functional Polymorphisms and Sheep Tail Type

doi: 10.3390/ani10040739

Figure Lengend Snippet: BMP2 gene pool sequencing and genotyping results. (A) Site of g.48401619 T > A. (B) Site of g.48401272 C > A. (C) Site of g.48401136 C > T. Yellow region, blue region, and green region represent different genotypes. Numbers in brackets indicate number of individuals of the three genotypes. Chromatograms from DNA pool sequencing are shown in the top row, while the mass spectrometry results are presented in the bottom row.

Article Snippet: On the following day, cells were cultured in new fresh medium containing the BMP2-overexpression lentiviral vector (Genechem, Shanghai, China) for 12 h. Preadipocytes were then cultured in new differentiation medium containing 10% FBS, 1 μM dexamethasone (Macklin, Shanghai, China), 0.5 mM isobutylmethylxanthine (Macklin), and 10 mg/mL insulin (Macklin) for 2 days, followed by 10 mg/mL insulin alone for 2 days.

Techniques: Sequencing, Mass Spectrometry

Journal: Animals : an Open Access Journal from MDPI

Article Title: Association Between BMP2 Functional Polymorphisms and Sheep Tail Type

doi: 10.3390/ani10040739

Figure Lengend Snippet: Diversity parameters for the sheep BMP2 gene.

Article Snippet: On the following day, cells were cultured in new fresh medium containing the BMP2-overexpression lentiviral vector (Genechem, Shanghai, China) for 12 h. Preadipocytes were then cultured in new differentiation medium containing 10% FBS, 1 μM dexamethasone (Macklin, Shanghai, China), 0.5 mM isobutylmethylxanthine (Macklin), and 10 mg/mL insulin (Macklin) for 2 days, followed by 10 mg/mL insulin alone for 2 days.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: Association Between BMP2 Functional Polymorphisms and Sheep Tail Type

doi: 10.3390/ani10040739

Figure Lengend Snippet: Linkage disequilibrium analysis of the BMP2 gene.

Article Snippet: On the following day, cells were cultured in new fresh medium containing the BMP2-overexpression lentiviral vector (Genechem, Shanghai, China) for 12 h. Preadipocytes were then cultured in new differentiation medium containing 10% FBS, 1 μM dexamethasone (Macklin, Shanghai, China), 0.5 mM isobutylmethylxanthine (Macklin), and 10 mg/mL insulin (Macklin) for 2 days, followed by 10 mg/mL insulin alone for 2 days.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: Association Between BMP2 Functional Polymorphisms and Sheep Tail Type

doi: 10.3390/ani10040739

Figure Lengend Snippet: mRNA expression of the PPARγ and LPL genes. ( A ) mRNA expression of the BMP2 gene. ( B ) mRNA expression of the PPARγ gene. ( C ) mRNA expression of the LPL gene. * p < 0.05, ** p < 0.01, NS: no significant difference. ACTB was used as a reference gene.

Article Snippet: On the following day, cells were cultured in new fresh medium containing the BMP2-overexpression lentiviral vector (Genechem, Shanghai, China) for 12 h. Preadipocytes were then cultured in new differentiation medium containing 10% FBS, 1 μM dexamethasone (Macklin, Shanghai, China), 0.5 mM isobutylmethylxanthine (Macklin), and 10 mg/mL insulin (Macklin) for 2 days, followed by 10 mg/mL insulin alone for 2 days.

Techniques: Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Association Between BMP2 Functional Polymorphisms and Sheep Tail Type

doi: 10.3390/ani10040739

Figure Lengend Snippet: Oil Red O staining when overexpressing the BMP2 gene in sheep preadipocytes after 7 days of induced differentiation. (A) Control group. (B) BMP2 overexpressed group.

Article Snippet: On the following day, cells were cultured in new fresh medium containing the BMP2-overexpression lentiviral vector (Genechem, Shanghai, China) for 12 h. Preadipocytes were then cultured in new differentiation medium containing 10% FBS, 1 μM dexamethasone (Macklin, Shanghai, China), 0.5 mM isobutylmethylxanthine (Macklin), and 10 mg/mL insulin (Macklin) for 2 days, followed by 10 mg/mL insulin alone for 2 days.

Techniques: Staining

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: (A) The C2C12 cells were treated with Adv-BMP2 (150 pfu/cell) or Adv-β-Gal (150 pfu/cell) for one, three, and five days and SATB2 expression was examined by western blot and then underwent densitometric analysis. (B, C, D) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell) or various concentrations of Adv-BMP2 for three days. The SATB2 relative mRNA levels (B) and protein levels (C) were assessed and densitometric analysis of C (D) was graphed. (E) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), or 10 ng/mL to 60 ng/mL of TNF-α for 72 h, then followed by MTT assay. (F) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), or 60 ng/mL of TNF-α for five days. ALP activity were measured. (G) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), or 10 ng/mL to 60 ng/mL of TNF-α for five days and ALP staining was performed. (H _ J) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), or 10 ng/mL to 60 ng/mL of TNF-α for 72 h. The SATB2 gene expressions were assessed by real-time PCR (H) and western blot (I) and densitometric analysis (J). The data are presented as mean ± S.D. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001), these western blot images were uncropped.

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Expressing, Western Blot, MTT Assay, Activity Assay, Staining, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: (A) SATB2 over-expression lentivirus and control lentivirus were transfected into C2C12 cells to construct cell lines that over-express SATB2 constantly, two cell lines were chosen and uncropped western blot images were shown. (B _ E) Control cell lines and SATB2 over-expression cell lines from (A) were treated with or without 250 ng/ml BMP2 and TNF-α as indicated for 3 days, followed by MTT assay (B) and ALP staining (C). The ALP staining density in figure B were analyzed with image J software (D) and the SATB2 expression levels were detected using western blotting and uncropped western blot images were shown (E). (D) C2C12 cells were transfected with pCDNA3.1+ vector or pCDNA3.1+-SATB2 vector and then treated with TNF-α or BMP2 (250ng/ml) as indicated for 5 days, the OCN mRNA levels were measured with real time RT-PCR. The data are presented as mean ± S.D. ( n=3, * p < 0.05; ** p < 0.01 ; *** p < 0.001, T10 means 10 ng/ml TNF-α, BT10 means 250 ng/ml BMP2 and 10 ng/ml TNF-α).

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Over Expression, Transfection, Construct, Western Blot, MTT Assay, Staining, Software, Expressing, Plasmid Preparation, Quantitative RT-PCR

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: (A, C) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), 10 ng/mL or 60 ng/mL of TNF-α for 2 or 12 h. Cell lysates were used to analyze p-smad1/5/8 activation. The GAPDH were used as loading controls. (B, D) Densitometric analysis of A and C respectively. (E) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), 10 ng/mL or 30 ng/mL of TNF-α, 0.1μM of LDN193189 for 72 h. The SATB2 and OSX expression levels were detected by western blotting. (F, G) Densitometric analysis of E. The data are presented as mean ± S.D. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.01; ## p < 0.001 compared with the related same treatment without LDN193189 administration). Uncropped western blot images corresponding to Figure were shown in (A, B and C respectively).

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: (A) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), or 60 ng/mL of TNF-α for 2 or 12 h. Cell lysates were used to analyze p-ERK, p-p38, and p-JNK activation. The total-ERK, total-p38, and total-JNK were used as loading controls, respectively. The relative densitometric analysis of p-p38, p-ERK and p-JNK were shown. These western blot images were uncropped. (B, C) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), 30 ng/mL of TNF-α or 10 μM to 50 μM of U0126 for 72 h. The SATB2 expression level was detected by real-time PCR (B) and western blotting (C). (D) Densitometric analysis of C. The data are presented as mean ± S.D. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001). Uncropped western blot images corresponding to Figure was shown in .

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: (A) The C2C12 cells were cotransfected with the pGL4.32[luc2P/NF-κB-RE/Hygro] vector and Rluc control reporter vector. 24 hours after transfection, the cells were treated with 40 ng/mL of TNF-α or PBS for 4h, 12h, 24h. Cell lysates were used to assess the luciferase activity. (B) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), or 60 ng/mL of TNF-α for 2 h. The cytoplasmic and nuclear proteins were isolated to assess the p65 level, uncropped western blot images and densitometric analysis of p65 were shown. (C) C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), 60 ng/mL of TNF-α or 8 μM of BAY11-7082 for 2 h and assayed to detect p65 protein (green) nuclear localization via a confocal microscopic method. DAPI (blue) was used for nuclear dyeing. (Bar=25μm) (D, E, F) The C2C12 cells were treated with Adv-β-Gal (150 pfu/cell), Adv-BMP2 (150 pfu/cell), 30 ng/mL of TNF-α, BAY11-7082 for 72 h. The SATB2 RNA expression levels (D) and protein levels (E, F) were examined. (F) The densitometric analysis for (E). The data are presented as mean ± S.D. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; N.S means no significant difference). Uncropped western blot images corresponding to Figure was shown in .

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Plasmid Preparation, Transfection, Luciferase, Activity Assay, Isolation, Western Blot, RNA Expression

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: (A) Time course of TNF-α action. C2C12 cells were treated with BMP2 and with or without TNF-a (10ng/ml) for indicated times, SATB2 mRNA was measured by real time RT-PCR. (B) SATB2 mRNA stability was not changed by TNF-α. C2C12 cells were treated with BMP2 (150ng/ml) for one day and then treated with actinomycin D (0.5ug/ml) 2 hours before TNF-a (10ng/ml) were added, followed by detecting SATB2 mRNA relative levels at indicated time. (C) New protein synthesis is not required for TNF-α inhibition of SATB2 expression. Cycloheximide (CHX) (1 ug/ml, 5 ug/ml) was added 2 h prior to TNF-α (10 ng/ml, 20 ng/ml). SATB2 mRNA was measured 24 h after the addition of TNF-α. (D) C2C12 cells were treated with Adv-BMP2 and 10ng/ml TNF-α for indicated times and CHIP assay were performed with NF-kB/P65 antibody, followed by PCR with indicated primers. The gels have been run under the same experimental conditions. Uncropped images corresponding to Figure were shown in . (E) NF-κB associate sites on SATB2 promoter and the schema of luciferase reporter gene plasmids construction. (F, G, H, I) Luciferase assay. Luciferase assay with plasmid pGL3-SATB2-5 (F) or pGL3-SATB2-6 (G) and treated with TNF-α. Luciferase assay with plasmid pGL3-SATB2-5 (H) or pGL3-SATB2-6 (I) and combined with NF-κB/p65 over-expression. The data are presented as mean ± S.D. ( n =3; * p < 0.05. ** p < 0.001, *** p < 0.0001).

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Quantitative RT-PCR, Inhibition, Expressing, Luciferase, Plasmid Preparation, Over Expression

Journal: Oncotarget

Article Title: TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways

doi: 10.18632/oncotarget.23373

Figure Lengend Snippet: BMP2 stimulates SATB2 expression and this up-regulation could be significantly inhibited by TNF-α, TNF-α inhibits BMP2 induced smad1/5/8 signaling, activates pERK1/2 and p38 signaling, in addition, TNF-α stimulates NF-κB signaling which results in translocation of p65 into nuclear and binds to SATB2 promoter to suppress SATB2 expression. Furthermore, the expression of osteoblast differentiation marker genes and osteoblastogenesis were inhibited.

Article Snippet: After transfection, cells were incubated in a CO2 incubator at 37°C for 12 hours and then 150 ng/ml of human recombinant BMP2 (R&D) were added in each well.

Techniques: Expressing, Translocation Assay, Marker

Journal: British Journal of Cancer

Article Title: The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC

doi: 10.1038/bjc.2014.560

Figure Lengend Snippet: Activation of BMP signalling differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. ( A ) CRC cell lines HCT116, LS174T, HT-29, SW480, RKO and DLD-1 and the embryonic kidney cell line HEK-293 (used as non-cancer cell line) were transfected with WRE-luc and MRE-luc. The next day, the cells were transfected with either BMPR2 (to activate BMP signalling) or the empty control vector pcDNA4/TO. After 24 h, the cells were lysed and luciferase activity was measured. The control (pcDNA) condition was set to 1. ( B ) An overview of APC/ β -catenin, KRAS/BRAF, p53 and SMAD4 mutation status in the cell lines. ( C ) HCT116, HCT116 SMAD4−/−, HCT116 R248 (p53 mutated) and HCT116 p53−/− cells were transfected with WRE-luc and MRE-luc. The next day, the cells were transfected with either BMPR2 (to activate BMP signalling) or the empty control vector pcDNA4/TO. After 24 h, the cells were lysed and luciferase activity was measured. ( D ) DLD-1 (harbouring a p53 mutation) and DLD-1 SIL/+ (one WT copy p53) were transfected with WRE-luc/MRE-luc. The next day, the cells were treated with either 100 ng ml −1 BMP2 or the control vehicle (both diluted in medium with 0,5% FCS). After 24 h, the cells were lysed and luciferase activity was measured. ( E ) SMAD4 was stably knocked down in LS174T cell using lentiviral shRNA transduction. The empty shRNA SHC002 was used to create a control cell line. The cells were transfected with WRE-luc/MRE-luc. The next day the cells were treated with either 100 ng ml −1 BMP2 or the control vehicle (both diluted in medium with 1% FCS). After 24 h, the cells were lysed and luciferase activity was measured. (All the experiments were performed three times. Mean±s.e.m. is shown, * P <0.05, ** P <0.01, *** P <0.001)

Article Snippet: Stock solutions of recombinant human BMP2 ligands (R&D systems, Minneapolis, MN, USA) were prepared in phosphate-buffered saline (PBS) and subsequently dissolved in culture medium (100 ng ml −1 ) containing 0.5% FCS.

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Stable Transfection, shRNA, Transduction

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: Treatment Groups for Cotransfection/Coculture Experiment

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Cotransfection

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: Quantitative RT-Polymerase Chain Reaction Gene Expression Primer/Probe Information

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Gene Expression

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: Adenoviral transfection without proliferative alterations or signs of toxicity. Adenoviral vectors carrying the gene for BMP2, VEGF, or LacZ were used to transfect bone marrow–derived rat MSCs. Combinations of the genes were introduced to the cells at a total MOI of 100 PFU/cell. (A) Relative quantities of adenovirus Hexon mRNA expression were then analyzed (n = 3/group) and normalized by concurrent analysis of ribosomal 18S RNA. Similar levels of adenovirus-specific Hexon mRNA expression suggest that the viral load and efficacy of transfection were similar in all groups. Values represent mean ± SD of replicates (n = 3). (B) Alterations in growth was assessed at various time-points using the 3-(4,5-diamethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and compared to nontransfected controls. The growth curves were similar in all groups, and at the final day of the assay, no significant difference in optical density (proportional to cell number) was found between the groups. Labels in the graph represent the introduced genes and the corresponding viral MOI. Values represent mean ± SD of replicates (n = 5/group). BMP2, bone morphogenetic protein 2; VEGF, vascular endothelial growth factor; MOI, multiplicity of infection; PFU, plaque forming units; SD, standard deviation; MSCs, mesenchymal stem cells.

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Transfection, Derivative Assay, Expressing, MTS Assay, Infection, Standard Deviation

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: Cotransfection of AdV-VEGF decreases BMP2 expression. Adenoviral vectors carrying the gene for BMP2, VEGF, or LacZ were used to transfect bone marrow–derived rat MSCs. Combinations of the genes were introduced to the cells at a total MOI of 100 PFU/cell. (A) When BMP2 was coexpressed with LacZ in various ratios, while keeping the total viral load constant, the mRNA expression varied according to the AdV-BMP2 MOI. When the same ratios were applied to the AdV-BMP2 and AdV-VEGF cotransfection, however, the BMP2 expression was drastically reduced, demonstrating an exponential distribution that seemed correlated to the AdV- BMP2:VEGF ratio rather than the AdV-BMP2 MOI alone. One group was plated as a coculture composed of cells, separately transfected with AdV-BMP2 and AdV-VEGF. The total number of cells as well as the total viral load and average AdV-BMP2 vector per cell was equal to the groups dually transfected with BMP2 (MOI 50) + VEGF (MOI 50) or BMP2 (MOI 50) + LacZ (MOI 50). The levels of BMP2 were approximately three times higher in cultures composed of separately transfected cells compared to cells dually transfected with AdV-BMP2 and AdV-VEGF. Separately transfected but cocultured cells expressed, however, less BMP2 compared to cells dually transfected with AdV-BMP2 and AdV-LacZ. (B) When the levels of BMP2 and VEGF were measured in the cell medium through real time PCR, the general patterns corresponded to the ELISA results and demonstrated that the phenomenon occurs at the mRNA level. (C, D) The expression of VEGF correlated to the AdV-VEGF MOI, relatively independent of any AdV-BMP2 or AdV-LacZ cotransfection, when evaluated with ELISA and rtPCR. Relative quantities of RNA were obtained by normalization against ribosomal 18S RNA as well as viral-specific Hexon RNA to weigh in any variations in viral transfection between groups. Labels in the graph represent the introduced genes and the corresponding viral MOI. Values represent mean ± SD of replicates (n = 3). ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Cotransfection, Expressing, Derivative Assay, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: Adenoviral-mediated BMP2 overexpression and MSC differentiation can be suppressed by exogenous VEGF. (A) Bone marrow–derived rat MSCs transfected with Adv-BMP2 at an MOI of 100 PFU/cell were exposed to the medium supplemented with 10 or 50 ng/mL recombinant VEGF protein. After 48 h, cultures were analyzed with ELISA and showed a dose-dependant decrease in BMP2 production dependent on the VEGF concentration. BMP2 production decreased fivefold when VEGF was supplemented to a concentration of 50 ng/mL, indicating that exogenous VEGF is a potent inhibitor of BMP2 expression in MSCs. (B) MSCs were cultured in the differentiation medium and assessed for expression of early bone differentiation marker RUNX-2. At 12 h RUNX-2 expression was markedly reduced in cells exposed to recombinant VEGF. (C) While positive staining for calcium deposits was found in cell cultures grown for 21 days in the differentiation medium without rhVEGF (left), little signs of bone formation were present in cultures grown under the same conditions but with VEGF-supplement (right).

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Over Expression, Derivative Assay, Transfection, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Cell Culture, Marker, Staining

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: BMP2 inhibition occurs at the transcriptional level. To determine if the inhibition of BMP2 by VEGF occurred as a result of changes in transcription or translation, these processes were inhibited using RNA-polymerase II inhibitor actinomycin-D or the protein synthesis inhibitor cycloheximide. (A) Rat MSCs were transfected with AdV-BMP2 at an MOI of 50 PFU/cell and exposed to the medium containing 5 ng/mL actinomycin-D, with or without 10 ng/mL VEGF. A time course of BMP2 transcript expression demonstrated a similar rate of BMP2 transcript degradation (slope −0.4872 ± 0.03695 vs. −0.5059 ± 0.1170), implying that VEGF reduces BMP2 expression by a method other than transcriptional degradation. (B) MSCs were transfected with the combination of either AdV-BMP2 and AdV-VEGF or AdV-BMP2 and AdV-LacZ at a 1:1 ratio and then exposed to the medium containing 10 ng/mL cycloheximide. Six hours after cycloheximide supplementation analysis with rtPCR showed no increase in BMP2 expression, indicating that protein translation is not necessary for inhibition of BMP2 expression by VEGF. Values represent mean ± SD of replicates (n = 3).

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Inhibition, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Tissue Engineering. Part A

Article Title: Vascular Endothelial Growth Factor Inhibits Bone Morphogenetic Protein 2 Expression in Rat Mesenchymal Stem Cells

doi: 10.1089/ten.tea.2009.0426

Figure Lengend Snippet: VEGF-mediated BMP2 inhibition impairs bone formation in vivo. Bone marrow–derived MSCs from green fluorescent transgenic rats were seeded on collagen scaffolds and transfected with adenoviral vectors carrying the gene for BMP2, VEGF, or LacZ. Combinations of the genes were introduced to the cells at a 1:1 ratio with a total MOI of 100 PFU/cell. Constructs were then implanted in the groin of syngeneic green fluorescent protein–negative rats, and the superficial epigastric artery and vein were incorporated into the implant. While constructs transfected with AdV-BMP2 and AdV-VEGF formed little or no bone (A), implants transfected with AdV-BMP2 and AdV-LacZ were completely or in part replaced by bone (arrow) (B) as shown in the microcomputed tomography images. Decalcified sections of the AdV-BMP2:LacZ implants showed formation of cancellous bone surrounding the epigastric pedicle (arrow) (C). Donor cells expressing green fluorescent protein could be found within the osseous tissue (D). Color images available online at www.liebertonline.com/ten.

Article Snippet: Bone morphogenetic protein 2 ( BMP2 ) , Mm01340178_m1.

Techniques: Inhibition, In Vivo, Derivative Assay, Transgenic Assay, Transfection, Construct, Tomography, Expressing