Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor (TGF)-β-activated Kinase 1 Mimics and Mediates TGF-β-induced Stimulation of Type II Collagen Synthesis in Chondrocytes Independent of Col2a1 Transcription and Smad3 Signaling

doi: 10.1074/jbc.m500646200

Figure Lengend Snippet: FIG. 4. TAK1a mediates the stimulation of type II collagen synthesis by TGF- and BMP2 and exhibits TAB1-independent kinase activity. A, chondrocytes were infected 24 h after plating with dominant-negative TAK1 adenoviral vectors, Ad-pC-hTAK1a-K63A, and Ad-pC-hTAK1a-K63W. Seventy-two hours after initiating infec- tion, cells were treated for 24 h with 5 ng/ml TGF-1 (T) or 100 ng/ml BMP2 (B) in the presence of [3H]proline. Samples from triplicate wells were pooled before SDS-PAGE. B, for immune complex kinase assays, chondrocytes were infected with Ad-pC-hTAK1a (TK), kinase-negative (KN) Ad-pC-hTAK1a-KWSA, and the tandem vector (TK,TB) pCEA3- hTAB1(pC-hTAK1a). Forty-eight hours after beginning infection, cells were treated with TGF-1 (40 ng/ml; T) or IL-1 (40 ng/ml; IL) for 10 min prior to lysis, immunoprecipitation with TAK-ct antibody, and TAK1a immune complex kinase assay using bacterially expressed GST- MKK6 as substrate. Lysates were equally divided prior to immunopre- cipitation to permit direct kinase assays in the presence of [-32P]ATP (upper panels) or kinase assay after pretreatment with cold ATP (lower panels). The right panels are 7-fold shorter exposures of the last two lanes. C, Western blots (IB) demonstrating activation of endogenous TAK1 and TAB1 and their overexpressed counterparts. Chondrocytes were treated for 10 or 30 min with TGF-1 (5 ng/ml) or BMP2 (100 ng/ml) and lysed with SDS sample buffer at the same time as cells exposed to 48 h of adenoviral expression of TAK1a or coexpressed TAK1a and TAB1 (tandem construct). TAK-ct and TAB-m primary antibodies were used for detection. Long dashes, unmodified TAK1 or TAB1 bands; short dashes, activated/phosphorylated bands. Right panel, shorter exposures of lanes 5 and 6.

Article Snippet: Subsequently, cells were fed with the same medium, treated with recombinant human TGF- 1 or BMP2 (R&D Systems) for 24 h and then labeled in fresh medium with tritiated proline for 24 h. Labeling medium was 0.3% ITS /DMEM, 62.5 g/ml -aminoproprionitrile, 25 g/ml ascorbate, 40 Ci/ml [5-3H]proline (Amersham Biosciences), and growth factors.

Techniques: Activity Assay, Infection, Dominant Negative Mutation, SDS Page, Immune Complex Kinase Assay, Plasmid Preparation, Lysis, Immunoprecipitation, Kinase Assay, Western Blot, Activation Assay, Expressing, Construct

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: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 1. Effect of BMP2 on DSPP expression in mouse preodontoblasts. A, quantitative RT-PCR analysis of DSPPmRNAexpressionfromMD10-F2cellstreatedwithorwithoutBMP2(100ng/ml)at12,24,48,and72h.Values were expressed as fold increase versus cells without BMP2 as 1.0-fold. * indicates significant differences between the BMP2-treatedand-untreatedcells(*,p0.05).B,qRT-PCRproductsfromoneofthreeexperimentswererunonto1.5% agarose gels and stained with ethidium bromide. M, DNA marker; Neg, negative control; Cyclo, cyclophilin serves as an internal control. Con and bmp2 lanes show MD10-F2 cells without or with BMP2 stimulation, respectively. C, DSPP protein expression in MD10-F2 cells in the presence or absence of BMP2. MD10-F2 cells treated with or without BMP2 were incubated with anti-DSP polyclonal antibody. Mouse IgG1 was used as a negative control. The samples were then incubated with the secondary antibody conjugated to Alexa Fluo 488 (Molecular Probes). Alexa Fluo 488stainingimageswereobtainedunderthesameparametersinanOlympuswidefieldmicroscopeandquantified by means of MetaMorph software. Bri and Fluo indicate bright and fluorescent fields, respectively.

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Expressing, Quantitative RT-PCR, Staining, Marker, Negative Control, Control, Incubation, Software

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 2. The DSPP promoter contains a BMP2-response element. A, schematic representation of the constructs used in the luciferase (Luc) assay. B, BMP2 stimulates DSPP promoter activity. Transient MD10-F2 transfectants in the presence or absence of BMP2 (100 ng/ml) for 12 h were used to determine transcriptional activity of those chimeric constructs. The value obtained from the control group (pGL3 basic only) was taken as 1-fold, and fold increases were calculated by dividing the individual value by the control group value and plotted as a graph showing the mean S.E. from three independent experiments in triplicate. Significant differences comparing BMP2-treated groups with BMP2-untreated groups are shown with the following prob- ability levels: *, p 0.05; **, p 0.001.

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Construct, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 3. NF-Y binding to the BMP2-response element in the mouse DSPP promoter. A, 32P-labeled double strand probe between nt 97 and 72 was incubated with MD10-F2 cell nuclear extracts in the presence or absence of 50- and 100-fold molar excesses of unlabeled competitor DNA described in Table 1. WT, wild type. B, mouse DSPP proximal promoter nucleotide sequences are shown from nt 100 to 66. Several potential transcription factor-binding sites are present in this element. C, comparison of mouse, rat, and human DNA sequences from 100 to 66 positions. Dots represent nucleotide identity. D, NF-Y antibody supershift assay. MD10-F2 cell nuclear extracts were preincubated with antibody to NF-Y subunits or serum, respectively, and then with 32P-labeled wild-type (lanes 1–9) or 32P-labeled NF-Y probes (lanes 10–18). E, NF-Y-binding site schematic in the mouse DSPP promoter. The black box shows the NF-Y- bindingbox.ThearrowsindicatespecificprimersforChIPassay.F,ChIPassay.ChIPanalysiswasperformedasdescribedunder“ExperimentalProcedures.”Lane 2 shows a negative control. Lane 3 shows the TFIIB antibody was bound to TFIIB-binding site in the GAPDH promoter region. The immunoprecipitated and purified DNA fragments as a template were amplified by GAPDH PCR primers. Lane 4 shows input DNA amplified by mouse DSPP primers. Lane 5 shows the DSPP target was effectively immunoprecipitated by the anti-NF-YB antibody.

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Binding Assay, Labeling, Incubation, Comparison, Negative Control, Immunoprecipitation, Purification, Amplification

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 4. DSPP, BMP2, and NF-Y gene expression in tooth organs during development. A–L, in situ hybridization of mouse tooth developmental stages from E15 to PN1 with BMP2 (B, D, and F) and DSPP (H, J, and L) antisense probes. Hematoxylin was shown in A, C, E, G, I, and K. od, odontoblasts; am, ameloblasts; p, dental pulp cells; c, cartilage; eo, enamel organ; dp, dental papilla; e, enamel; d, dentin. Bar scale is 200 M. M, effect of BMP2 on ALP activity in MD10-F2 cells. After the cells were treated with BMP2 (100 ng/ml) or without BMP2 (con) for 3 and 6 days, ALP activity was determined as described under “Experimental Procedures.”DataweremeansS.E.ofthreewells,significantlydifferentfromthecontrolwithoutBMP2.*,p0.001.N,RT-PCRamplificationofthethreeNF-Y subunits were carried out. Ten l of PCR products were analyzed on 1.5% agarose gels. Amplification products of mouse -actin served as an internal control. M, 100-bp DNA ladder. Arrows indicate expected PCR products of the three NF-Y subunits, NF-YA, NF-YB, and NF-YC, and -actin genes. Con, negative control; 10-F2, MD10-F2; 2 wk, 10 wk, and 13 wk indicate mouse molars at aged 2, 10, and 13 weeks after birth, respectively.

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Gene Expression, In Situ Hybridization, Activity Assay, Amplification, Control, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 5. Effect of BMP2 on NF-Y expression in MD10-F2 cells. A, qRT-PCR analysis of NF-Y subunit mRNA expression in MD10-F2 cells. Cells were grown in the presence or absence of BMP2 at the given time points. NF-Y mRNA expression in MD10-F2 cells in the absence of BMP2 served as a 1.0-fold increase. * indicates significant differences between the BMP2-treated and -untreated cells (p 0.05). B, qRT-PCR products from one of three experiments were run on 1.5% agarose gels and stained with ethidium bromide. M, DNA marker; Neg, negative control; Cyclo, cyclophilin served as an internal control. Con and bmp2 show MD10-F2 cells without or with BMP2 induction, respectively. C, MD10-F2 cells stimulated with or without BMP2 for the indi- cated times were subjected to ChIP with anti-NF-YB antibody. Immunoprecipitated and purified DNA frag- ments were subjected to PCR with primers covering the NF-Y-binding site of the mouse DSPP promoter.

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Expressing, Quantitative RT-PCR, Staining, Marker, Negative Control, Control, Immunoprecipitation, Purification, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 6. Inhibition of BMP 2 effect of NF-Y expression in MD10-F2 cells by cycloheximide. MD10-F2 cells were treated with BMP2 (100 ng/ml) in the presence or absence of CHX (10 g/ml) at the indicated time points. Protein expression of NF-YA, NF-YB, NF-YC, DSP, and -actin was detected by Western blot analysis as described under “Experimental Procedures.”

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Inhibition, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

doi: 10.1074/jbc.m709492200

Figure Lengend Snippet: FIGURE 7. NF-Y-binding site is important in BMP2-mediated DSPP promoter activity. A, illustration of wild-type and mutant DSPP promoter-luciferase reporter gene constructs. The diagram shows the relative positions and sequences of the NF-Y-binding site with mutated sequences shown in lowercase. B, effect of BMP2 on DSPP promoter activity. MD10-F2 cells were transfected with p97wt-luc, p97mut-luc, or empty vector (pGL3-basic only). pRL-TK (Renilla luciferase) co-transfection served as an internal control. These transfectants were treated or untreated with recombinant BMP2 (100 ng/ml) for 12 h before cell harvest. The value (ratio between firefly and Renilla luciferase) was compared with the control group (pGL3-basic only). The fold lucif- erase activity was determined by dividing the individual values by the control group value. The data are expressed as the mean S.E. from at least three separate experiments performed in triplicate. Significant differences comparing BMP2-treated cells with untreated cells are shown with following probability levels: **, p 0.01. C, effect of BMP antagonist Noggin on BMP2-mediated DSPP transcription. Transient transfection p97 analysis in the presence or absence of BMP2 (100 ng/ml) for 12 h in MD10-F2 cells is shown with the effects of Noggin at different doses. The results obtained from three separate transfections show the mean S.E. Significant differences compared with groups untreated with Noggin are shown with the following probability levels: *, p 0.05; **, p 0.001.

Article Snippet: Immunohistochemistry—Mouse MD10-F2 cells were cultured on glass slides and treated with or without 100 ng/ml of recombinant BMP2 (R & D Systems Inc.) at different time points.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Luciferase, Construct, Transfection, Plasmid Preparation, Cotransfection, Control, Recombinant

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Fetal pericytes (CD146 + /CD31 − ) were infected with African strain ZIKV-MR766, Asian strains ZIKV-H/PF/2013 and PRVABC59 at MOI of 0.5 or treated with medium without virus (mock). Blue cell population is unstained control, red cell population is stained with CD146 and CD31 antibody. See for gating strategy. ( b ) At 1–4 and 8 dpi, cells were harvested for viral RNA analysis ( n =4). ( c ) Infectious virus titer was quantified in supernatants ( n =6 per group) collected from 1–4, 8 and 14 dpi. ( d ) At 3, 4 and 8 dpi, mature BMP2 protein from supernatants of mock- and ZIKV-infected pericytes was measured ( n =8). ( e ) Osteogenic gene expression in fetal pericytes was normalized to GAPDH and presented as fold changes relative to mock controls ( n =6 per group). ( f - g ) Alizarin red staining and Alizarin red concentration (mM) of ZIKV-infected fetal pericytes after 14 dpi ( n =4). Data are presented as mean ± SEM, using two-way ANOVA followed by Sidak’s multiple comparisons test ( e ) or one-way ( g ) ANOVA with Tukey’s posttest. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Data in d , e and g are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Exact P values in g compared between Mock and ZIKV H/PF/2013 ( P= 0.0046) and between Mock and PRVABC59 ( P= 0.0030). Data are representative of three independent experiments.

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Infection, Virus, Control, Staining, Gene Expression, Concentration Assay

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Schematic depicting the timeline for our ZIKV-vertical transmission mouse model. Time-mated immunocompetent hSTAT2 KI pregnant dams (8 to 12 weeks old) were i.p infected with PBS (mock control) or ZIKV PRVABC59 at mouse embryonic day 13.5 (E13.5) and pups were born between 19 to 21 days later. On post-delivery day (PD) 28, ZIKV-infected hSTAT2KI pups were sacrificed and brains were harvested for RNA extraction or cryo-sectioning. ( b ) Osteogenic gene expression in mock- ( n =3) or ZIKV-infected hSTAT2KI pup brains ( n =12) were normalized to GAPDH and presented as fold changes relative to mock controls. ( c ) At PD28, brain sections of pups were stained with DAPI (blue) and anti-NS2B antibody. ( d ) Brain sections were stained with Alizarin red for calcium deposit. Blue arrows indicate the locations of prominent calcium deposition. Data in b - d are representative of two independent experiments. Data in b are presented as mean ± SEM, using two-tailed unpaired Student t -test. * P < 0.05, ** P < 0.01 and **** P < 0.0001. Exact P values in b compared between PBS and ZIKV PRVABC59 ( Bmp2 P= < 0.0001; Runx2 P = 0.0027; Osx P = 0.0180; Alpl P= < 0.0001).

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Transmission Assay, Infection, Control, RNA Extraction, Gene Expression, Staining, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: (A-B) U2OS cells were infected with ZIKV at an MOI of 0.5. ( a ) Supernatants from mock- and ZIKV-infected cultures were harvested for viral plaque assays ( n =8 biologically independent samples per group), and ( b ) cells were tested for viral RNA ( n =4 biologically independent samples per group). Data in b are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. ( c ) At 1–4 dpi, cell viability was determined by flow cytometry. See for gating strategy. ( d ) At 1–3 dpi, mock- and ZIKV-infected U2OS (MR766: MR; H/PF/2013: HPF) and their supernatants were collected, and TCA precipitated for immunoblot analysis with anti-BMP2 and Ponceau staining. ( e ) Phosphorylated SMAD1/5/9, total SMAD1 and ZIKV NS3 from whole cell lysates were detected by immunoblotting with indicated antibodies. ( f - h ) Inhibition of BMP2 activity. Mock- or ZIKV-infected U2OS cells were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. ( f ) At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls ( n =3 biologically independent cells per group). ( g ) At 21 dpi, cells were stained with Alizarin red stain (calcium) and (h) Alizarin red stain concentration was quantified ( n =6). All results are representative of biological independent replicates from two independent experiments. Data are presented as mean ± SEM, using two-tailed unpaired Student t -test ( f ), two-way ANOVA followed by Sidak’s multiple comparisons test ( b ), or one-way ANOVA with Tukey’s posttest ( h ). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Exact P values in f compared between IgG and Nab group ( BMP2 P = 0.0034; RUNX2 P = 0.0267; OSX P = 0.0054; DMP1 P = 0.0113 and PDPN P = 0.0105). Significance differences of the means in h comparing between ZIKV-H/PF/2013 +IgG and ZIKV-H/PF/2013 +Nab group ( P= <0.0001)

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Infection, Flow Cytometry, Western Blot, Staining, Inhibition, Activity Assay, Control, Concentration Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a - b ) Mock- and ZIKV-infected U2OS cells were harvested at 2 and 4 dpi respectively for osteogenic gene expression. ( c ) Band intensity of pSMAD1/5/9 from U2OS whole cell lysate. ( d ) At day 1–4, IgG or nAb-treated and mock or ZIKV-infected U2OS cells ( n =6) were harvested for RNA extraction and viral load against ZIKV NS1 RNA was quantified using qRT-PCR (N.D.; Not detected) ( e ) Human primary fetal brain pericytes are infected with ZIKV PRVABC59 or PBS (mock control) that were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls ( n =4/group). Data in a , b, d and e are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons ( a ), Mann-Whitney U -test ( b ), or two-tailed unpaired Student t -test ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values in b compared between Mock and ZIKV H/PF/2013 ( BMP2 P= 0.0002; RUNX2 P = 0.0011; OSX P = 0.0002; ALPL P = 0.0002; DMP1 P = 0.0002 and PDPN P = 0.0002). Exact P values in e compared between IgG-treated and Nab-treated group ( BMP2 P= 0.0054; RUNX2 P = 0.0005; OSX P = 0.0085; DMP1 P = 0.0099 and PDPN P = 0.0005). Data are representative of two independent experiments.

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Infection, Gene Expression, RNA Extraction, Quantitative RT-PCR, Control, MANN-WHITNEY, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Primary fetal pericytes transduced with lentivirus encoding individual ZIKV protein. Lenti-transduced cells were harvested for analysis of osteogenic gene expression normalized to GAPDH and expressed as fold changes relative to vector control ( n =8). ( b ) Schematic of ZIKV NS3 protease WT and H51A/S135A mutant (ZIKV NS3 Mut ). ( c ) Osteogenic gene expressions in WT and mutant (Mut) NS3-transduced pericytes ( n =4). Data in a and c are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are presented as mean ± SEM, using two-tailed unpaired Student t -test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values in c compared between WT and Mut NS3 group ( BMP2 P= 0.0012 ; RUNX2 P = 0.0013; OSX P = 0.0002; DMP1 P= 0.0030; PDPN P= <0.0001). Data are representative of three independent experiments.

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Transduction, Gene Expression, Plasmid Preparation, Control, Mutagenesis, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Sequence alignment comparison between BMP2 gene in human (homo sapiens; amino acid residues 1–396) and hamster (Cricetulus; amino acid residues 1–399). ( b ) Purity of BL21 strain-derived recombinant ZIKV NS3 protease, CHO-FD11 cells-derived full-length BMP2 and BMP4 were determined by coomassie blue stain. MW: molecular weight. ( c ) Sequence alignment comparison between human BMP2 (amino acid residues 1–396) and BMP4 (amino acid residues 1–408). ( d ) Sequence alignment of NS3 protease domain (amino acid residues 1–177) was compared across two African ZIKV (MR766, IbH30656) and two Asian ZIKV (PRVABC59 and H/PF/2103). The red-colored letters indicate protease catalytic triad. ( e ) In vitro cleavage assay of carboxyl terminal HA-tagged purified human BMP4 and purified ZIKV NS3 protease were performed at 37 °C for 3 h, followed by immunoblot analysis. Data in b and e are representative of three independent repeats.

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Sequencing, Comparison, Derivative Assay, Recombinant, Staining, Molecular Weight, In Vitro, Cleavage Assay, Purification, Western Blot

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) At 3 days after transfection with Flag-tagged WT or mutant NS3, the supernatants and lysates of U2OS cells were subjected to immunoblotting analysis or Ponceau staining. ( b ) U2OS stably-expressing WT or mutant NS3 were cultured and stained with Alizarin red. ( c ) Schematic of predicted BMP2 cleavage products. ( d ) In vitro cleavage assay of carboxyl terminal HA-tagged purified human BMP2 and purified ZIKV NS3 protease were performed at 37 °C for 3 h, followed by immunoblot analysis. ( e ) Cleaved BMP2 products from in vitro reaction were added to U2OS cells for 0 or 15 min and cells were harvested for immunoblot analysis. Data are representative of two independent experiments. ( f ) Schematic model of ZIKV NS3 protease-induced BMP2 maturation and osteogenic gene expression for fetal brain calcification. ZIKV NS3 protease efficiently cleaves pro-BMP2 to generate secreted mature BMP2 that induces its receptor-mediated osteogenic gene expression and calcification.

Article Snippet: Human BMP2 ELISA assay (R&D systems, #DY355) were performed according to manufacturer’s instructions.

Techniques: Transfection, Mutagenesis, Western Blot, Staining, Stable Transfection, Expressing, Cell Culture, In Vitro, Cleavage Assay, Purification, Gene Expression

Journal: Pharmaceuticals

Article Title: FOXO3-Activated circFGFBP1 Inhibits Extracellular Matrix Degradation and Nucleus Pulposus Cell Death via miR-9-5p/BMP2 Axis in Intervertebral Disc Degeneration In Vivo and In Vitro

doi: 10.3390/ph16030473

Figure Lengend Snippet: BMP2 is identified as a target of miR-9-5p. ( A ) BMP2 expression under IL-1β stimulation using qPCR. ( B ) The potential site of miR-9-5p located in BMP2 by starBase. ( C ) The relationship between miR-9-5p and BMP2 via dual luciferase reporter gene assay. ( D ) BMP2 expression by qPCR and Western blot. Data were estimated as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: The downregulation plasmid of BMP2 based on the short hairpin RNA (shRNA) sequence (sh-BMP2) and its negative control (sh-NC) were purchased from RiboBio (Guangzhou, China).

Techniques: Expressing, Luciferase, Reporter Gene Assay, Western Blot

Journal: Pharmaceuticals

Article Title: FOXO3-Activated circFGFBP1 Inhibits Extracellular Matrix Degradation and Nucleus Pulposus Cell Death via miR-9-5p/BMP2 Axis in Intervertebral Disc Degeneration In Vivo and In Vitro

doi: 10.3390/ph16030473

Figure Lengend Snippet: Knockdown of miR-9-5p suppresses apoptosis and ECM degradation by targeting BMP2 in NP cells. ( A ) MiR-9-5p expression by qPCR. ( B ) BMP2 expression by qPCR and Western blot. ( C ) Cell viability by CCK-8 assay. ( D ) Apoptosis in NP cells by flow cytometry. ( E , F ) Expressions of ECM proteins by Western blot ( E ) and immunofluorescence ( F ). Data were estimated as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The downregulation plasmid of BMP2 based on the short hairpin RNA (shRNA) sequence (sh-BMP2) and its negative control (sh-NC) were purchased from RiboBio (Guangzhou, China).

Techniques: Knockdown, Expressing, Western Blot, CCK-8 Assay, Flow Cytometry, Immunofluorescence

Journal: Pharmaceuticals

Article Title: FOXO3-Activated circFGFBP1 Inhibits Extracellular Matrix Degradation and Nucleus Pulposus Cell Death via miR-9-5p/BMP2 Axis in Intervertebral Disc Degeneration In Vivo and In Vitro

doi: 10.3390/ph16030473

Figure Lengend Snippet: Overexpression of circFGFBP1 alleviates the progression of IVDD in vivo. ( A ) CircFGFBP1 expression in NP tissues by qPCR. ( B ) Histopathological changes by H&E staining. ( C ) Apoptosis in NP tissues by TUNEL staining. ( D ) Expressions of ECM proteins in NP tissues by Western blot. ( E ) Expressions of BMP2 and MMP-13 in in NP tissues by immunohistochemistry. ( F ) Expression of miR-9-5p by qPCR. Data were estimated as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The downregulation plasmid of BMP2 based on the short hairpin RNA (shRNA) sequence (sh-BMP2) and its negative control (sh-NC) were purchased from RiboBio (Guangzhou, China).

Techniques: Over Expression, In Vivo, Expressing, Staining, TUNEL Assay, Western Blot, Immunohistochemistry

Journal: Pharmaceuticals

Article Title: FOXO3-Activated circFGFBP1 Inhibits Extracellular Matrix Degradation and Nucleus Pulposus Cell Death via miR-9-5p/BMP2 Axis in Intervertebral Disc Degeneration In Vivo and In Vitro

doi: 10.3390/ph16030473

Figure Lengend Snippet: The primer sequences used in this study for qPCR.

Article Snippet: The downregulation plasmid of BMP2 based on the short hairpin RNA (shRNA) sequence (sh-BMP2) and its negative control (sh-NC) were purchased from RiboBio (Guangzhou, China).

Techniques:

Journal: Pharmaceuticals

Article Title: FOXO3-Activated circFGFBP1 Inhibits Extracellular Matrix Degradation and Nucleus Pulposus Cell Death via miR-9-5p/BMP2 Axis in Intervertebral Disc Degeneration In Vivo and In Vitro

doi: 10.3390/ph16030473

Figure Lengend Snippet: The primary antibodies used in this study for Western blot.

Article Snippet: The downregulation plasmid of BMP2 based on the short hairpin RNA (shRNA) sequence (sh-BMP2) and its negative control (sh-NC) were purchased from RiboBio (Guangzhou, China).

Techniques: Western Blot