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ZenBio bmp9 antibody
Bmp9 Antibody, supplied by ZenBio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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bmp9 antibody - by Bioz Stars, 2026-03

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( A ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in primary human foreskin fibroblasts (HFF-1) were determined by RT-qPCR. ( B ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated with IFNβ (5 ng/ml), BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), followed by either immunoblot analysis 1 h post stimulation or RNA extraction from cell lysates and RT-qPCR 6 h post stimulation. GAPDH transcript levels were used for normalization. ( C ) Immunoblot analysis of HFF-1 upon stimulation to determine phosphorylation levels of respective signaling components. ( D ) Transcript levels of the BMP-responsive genes Id1 and Id3 upon stimulation with the indicated ligands. ( E ) Transcript levels of the ISGs Isg15 , Irf9 , Ifi6 , and Stat2 upon stimulation with the indicated ligands. Data information: ( A , B ) The Experiment was performed two independent times, one representative is shown. ( D , E) The experiment was performed three independent times, one representative is shown. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in primary human foreskin fibroblasts (HFF-1) were determined by RT-qPCR. ( B ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated with IFNβ (5 ng/ml), BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), followed by either immunoblot analysis 1 h post stimulation or RNA extraction from cell lysates and RT-qPCR 6 h post stimulation. GAPDH transcript levels were used for normalization. ( C ) Immunoblot analysis of HFF-1 upon stimulation to determine phosphorylation levels of respective signaling components. ( D ) Transcript levels of the BMP-responsive genes Id1 and Id3 upon stimulation with the indicated ligands. ( E ) Transcript levels of the ISGs Isg15 , Irf9 , Ifi6 , and Stat2 upon stimulation with the indicated ligands. Data information: ( A , B ) The Experiment was performed two independent times, one representative is shown. ( D , E) The experiment was performed three independent times, one representative is shown. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Western Blot, RNA Extraction, Phospho-proteomics

( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.

Techniques: Western Blot, Control, Quantitative RT-PCR, Transfection, Expressing, Luciferase, Incubation

( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or co-stimulated with IFNβ (5 ng/ml), followed by HCMV infection (MOI 0.5) for 16 h. Cells were fixed, nuclei were stained and cells were labeled for HCMV IE1 + cells as a readout for infection. ( B ) HCMV IE1 + cells normalized to total cell numbers and the untreated control (white column) in BMP/Activin stimulated samples (left panel) or with IFNβ co-stimulated samples (right panel). ( C ) HCMV IE1 + cells normalized to total cell numbers in cells pre-stimulated with either low (0.25 nM) or high (3 nM) concentrations of BMP9 (green symbols) or IFNβ co-stimulated with low and high concentrations of BMP9 (beige symbols). ( D ) HFF-1 were infected by centrifugal enhancement with HCMV WT (MOI 0.5) and supernatants of infected cells were collected in 6 h increments. 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were either stimulated with supernatants from HCMV-infected cells, or supernatants from HCMV-infected cells incubated for 15 min at RT with an α-BMP9 antibody, for 16 h, followed by a dual-luciferase assay readout. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. Data information: ( B ) Experiment was performed three independent times, one representative is shown. ( C , D ) Data are combined from two independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or co-stimulated with IFNβ (5 ng/ml), followed by HCMV infection (MOI 0.5) for 16 h. Cells were fixed, nuclei were stained and cells were labeled for HCMV IE1 + cells as a readout for infection. ( B ) HCMV IE1 + cells normalized to total cell numbers and the untreated control (white column) in BMP/Activin stimulated samples (left panel) or with IFNβ co-stimulated samples (right panel). ( C ) HCMV IE1 + cells normalized to total cell numbers in cells pre-stimulated with either low (0.25 nM) or high (3 nM) concentrations of BMP9 (green symbols) or IFNβ co-stimulated with low and high concentrations of BMP9 (beige symbols). ( D ) HFF-1 were infected by centrifugal enhancement with HCMV WT (MOI 0.5) and supernatants of infected cells were collected in 6 h increments. 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were either stimulated with supernatants from HCMV-infected cells, or supernatants from HCMV-infected cells incubated for 15 min at RT with an α-BMP9 antibody, for 16 h, followed by a dual-luciferase assay readout. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. Data information: ( B ) Experiment was performed three independent times, one representative is shown. ( C , D ) Data are combined from two independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SD. .

Techniques: Infection, Staining, Labeling, Control, Transfection, Expressing, Luciferase, Incubation, Two Tailed Test

( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR. ( B ) Transcript levels of BMP receptors Acvrl1 (ALK1, type I receptor) and Bmpr2 (type II receptor), which are the main receptors for BMP9, and the BMP-responsive gene Id1 . ( C ) Transcript levels of the ISGs Isg15 , Irf7 , Ifi6 , Irf9 , Stat2 , and Irf1 . ( D ) Transcript levels of the negative regulators Usp18 and Smurf1 . ( E ) HFF-1 were incubated with either Ruxolitinib or DMH1 for 1 h, then stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for transcript levels of Id3 , Stat2 , and Irf9 . Data information: ( B – D ) Data are combined from three independent experiments with the exception of Ifi6 transcript levels where three independent experiments were performed and data was combined from two independent experiments. ( E ) The experiment was performed two independent times, one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR. ( B ) Transcript levels of BMP receptors Acvrl1 (ALK1, type I receptor) and Bmpr2 (type II receptor), which are the main receptors for BMP9, and the BMP-responsive gene Id1 . ( C ) Transcript levels of the ISGs Isg15 , Irf7 , Ifi6 , Irf9 , Stat2 , and Irf1 . ( D ) Transcript levels of the negative regulators Usp18 and Smurf1 . ( E ) HFF-1 were incubated with either Ruxolitinib or DMH1 for 1 h, then stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for transcript levels of Id3 , Stat2 , and Irf9 . Data information: ( B – D ) Data are combined from three independent experiments with the exception of Ifi6 transcript levels where three independent experiments were performed and data was combined from two independent experiments. ( E ) The experiment was performed two independent times, one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Techniques: RNA Extraction, Quantitative RT-PCR, Incubation, Two Tailed Test

( A ) After 2 h of serum starvation, HFF-1 were stimulated for either one or 8 h with IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or were co-stimulated with IFNβ and BMP9, followed by cell lysis and immunoblot analysis with antibodies for phospho-STAT1, STAT1, phospho-SMAD1/5/9, and β-Actin. Phospho-STAT1 band intensities were first normalized to corresponding total STAT1 levels, then to IFNβ stimulation only, and are shown below. ( B ) After 2 h of serum starvation, HFF-1 were stimulated for 1 h with either IFNα2 (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2 or IFNγ and BMP9, followed by cell lysis, immunoblot analysis and quantification as in ( A ). ( C ) After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNα2 (5 ng/ml), IFNβ (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2, IFNβ, IFNγ, and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Irf9 , Stat2 , Irf1 , and Id3 transcripts. Data information: ( A , B ) Experiment was performed three independent times, one representative immunoblot is shown. Quantified data for the STAT1 phosphorylation levels are combined from three independent experiments. ( C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) After 2 h of serum starvation, HFF-1 were stimulated for either one or 8 h with IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or were co-stimulated with IFNβ and BMP9, followed by cell lysis and immunoblot analysis with antibodies for phospho-STAT1, STAT1, phospho-SMAD1/5/9, and β-Actin. Phospho-STAT1 band intensities were first normalized to corresponding total STAT1 levels, then to IFNβ stimulation only, and are shown below. ( B ) After 2 h of serum starvation, HFF-1 were stimulated for 1 h with either IFNα2 (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2 or IFNγ and BMP9, followed by cell lysis, immunoblot analysis and quantification as in ( A ). ( C ) After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNα2 (5 ng/ml), IFNβ (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2, IFNβ, IFNγ, and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Irf9 , Stat2 , Irf1 , and Id3 transcripts. Data information: ( A , B ) Experiment was performed three independent times, one representative immunoblot is shown. Quantified data for the STAT1 phosphorylation levels are combined from three independent experiments. ( C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are shown as mean ± SD. .

Techniques: Lysis, Western Blot, RNA Extraction, Quantitative RT-PCR, Phospho-proteomics, Two Tailed Test

( A ) 293T were co-transfected with expression plasmids for empty vector (EV) or V5-tagged US18, US20, or M27 (a known inhibitor of IFNAR signaling), together with either a BRE-Luciferase or MX1-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were stimulated for 16 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, co-stimulated with IFNβ and BMP9, or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( B ) Results from the Dual-luciferase assay with the BRE-Luciferase (top panel) and MX1-Luciferase (bottom panel) reporter. ( C ) Cell lysates from ( B ) were analyzed by immunoblot for the expression of M27, US18 and US20 with a V5-specific antibody, β-Actin served as loading control. ( D ) 293T were co-transfected with expression plasmids for the BRE-Luciferase and Renilla reporters as in ( A ), together with V5-tagged US18, US20 or co-transfected with US18 and US20 in combination. 24 h post transfection 293 T were stimulated for 16 h with BMP9 (3 nM), or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( E ) 293T were co-transfected with expression plasmids for either Cherry-STING and cGAS-GFP (left panel), RIG-I N (middle panel), or IRF3-5D (a constitutively activate IRF3 mutant; right panel), together with the murine IFNβ-luciferase reporter (IFNβ-Luc) and the Renilla reporter as normalization control. Cells were additionally transfected with expression plasmids for EV, V5-tagged M35 (a known inhibitor of PRR-mediated signaling pathways, (Chan et al, ), US18 or US20. Twenty hours post-transfection, cells were lysed and a dual-luciferase assay was performed. Immunoblot analysis of cell lysates from the respective experiments for M35, US18, and US20 detected with a V5-specific antibody, and β-Actin as a loading control, are shown below. Data information: ( B – E ) Data are combined from three independent experiments, for the immunoblots one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) 293T were co-transfected with expression plasmids for empty vector (EV) or V5-tagged US18, US20, or M27 (a known inhibitor of IFNAR signaling), together with either a BRE-Luciferase or MX1-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were stimulated for 16 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, co-stimulated with IFNβ and BMP9, or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( B ) Results from the Dual-luciferase assay with the BRE-Luciferase (top panel) and MX1-Luciferase (bottom panel) reporter. ( C ) Cell lysates from ( B ) were analyzed by immunoblot for the expression of M27, US18 and US20 with a V5-specific antibody, β-Actin served as loading control. ( D ) 293T were co-transfected with expression plasmids for the BRE-Luciferase and Renilla reporters as in ( A ), together with V5-tagged US18, US20 or co-transfected with US18 and US20 in combination. 24 h post transfection 293 T were stimulated for 16 h with BMP9 (3 nM), or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( E ) 293T were co-transfected with expression plasmids for either Cherry-STING and cGAS-GFP (left panel), RIG-I N (middle panel), or IRF3-5D (a constitutively activate IRF3 mutant; right panel), together with the murine IFNβ-luciferase reporter (IFNβ-Luc) and the Renilla reporter as normalization control. Cells were additionally transfected with expression plasmids for EV, V5-tagged M35 (a known inhibitor of PRR-mediated signaling pathways, (Chan et al, ), US18 or US20. Twenty hours post-transfection, cells were lysed and a dual-luciferase assay was performed. Immunoblot analysis of cell lysates from the respective experiments for M35, US18, and US20 detected with a V5-specific antibody, and β-Actin as a loading control, are shown below. Data information: ( B – E ) Data are combined from three independent experiments, for the immunoblots one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. .

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Lysis, Western Blot, Mutagenesis, Protein-Protein interactions, Two Tailed Test

( A ) HFF-1 with doxycycline-inducible expression of US18-V5, US20-HA, or US18-V5 and US20-HA with corresponding control cell lines (EV1, EV2, EV1 + 2) were generated. Protein expression was induced with 1 µg/ml doxycycline for 20 h prior stimulation and verified by immunoblot with V5- and HA-specific antibodies, and β-Actin as a loading control. ( B ) HFF-1 US18-V5, HFF-1 US20-HA, or HFF-1 US18-V5 US20-HA were left untreated or protein expression was induced with 1 µg/ml doxycycline to the medium for 20 h. Cell lysates were either left untreated or treated with PNGase F for 3 h at 37 °C, followed by immunoblot analysis with V5-, HA-, and Calnexin-specific antibodies. ( C , D ) Indicated HFF-1 lines were treated with 1 µg/ml doxycycline to induce protein expression for 18 h, followed by 2 h of serum starvation. ( C ) Cells were stimulated for 1 h with BMP9 (3 nM), followed by cell lysis and immunoblot analysis with phospho-SMAD1/5/9, SMAD1, V5, HA, and Calnexin antibodies. ( D ) Cells were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Id3 and Stat2 transcripts. Data information: ( A – C ) Experiment was performed three independent times, one representative is shown. ( D ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) HFF-1 with doxycycline-inducible expression of US18-V5, US20-HA, or US18-V5 and US20-HA with corresponding control cell lines (EV1, EV2, EV1 + 2) were generated. Protein expression was induced with 1 µg/ml doxycycline for 20 h prior stimulation and verified by immunoblot with V5- and HA-specific antibodies, and β-Actin as a loading control. ( B ) HFF-1 US18-V5, HFF-1 US20-HA, or HFF-1 US18-V5 US20-HA were left untreated or protein expression was induced with 1 µg/ml doxycycline to the medium for 20 h. Cell lysates were either left untreated or treated with PNGase F for 3 h at 37 °C, followed by immunoblot analysis with V5-, HA-, and Calnexin-specific antibodies. ( C , D ) Indicated HFF-1 lines were treated with 1 µg/ml doxycycline to induce protein expression for 18 h, followed by 2 h of serum starvation. ( C ) Cells were stimulated for 1 h with BMP9 (3 nM), followed by cell lysis and immunoblot analysis with phospho-SMAD1/5/9, SMAD1, V5, HA, and Calnexin antibodies. ( D ) Cells were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Id3 and Stat2 transcripts. Data information: ( A – C ) Experiment was performed three independent times, one representative is shown. ( D ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01. Data are shown as mean ± SD. .

Techniques: Expressing, Control, Generated, Western Blot, Lysis, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

( A ) Schematic representation of the workflow. Recombinant HCMV US18stop, HCMV US20stop, and HCMV US18/20stop were constructed by introducing a 16 base pair (bp) stop cassette within the respective coding region. HFF-1 were infected by centrifugal enhancement with HCMV WT, HCMV US18stop, HCMV US20stop, or HCMV US18/20stop (MOI 4 for the 3 h time point, MOI 0.5 for the 48 h time point). Three or 48 h post infection, cells were stimulated with BMP9 (3 nM) for (1) 2 h, followed by cell lysis and immunoblot analysis, or (2) 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B , C ) Cell lysates from ( A ) were subjected to immunoblot analysis with p-SMAD1/5/9, SMAD1, HCMV UL35, and GAPDH-specific antibodies (top panel), and transcript levels of Id3 and Stat2 are shown below. Data information: ( B , C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. Recombinant HCMV US18stop, HCMV US20stop, and HCMV US18/20stop were constructed by introducing a 16 base pair (bp) stop cassette within the respective coding region. HFF-1 were infected by centrifugal enhancement with HCMV WT, HCMV US18stop, HCMV US20stop, or HCMV US18/20stop (MOI 4 for the 3 h time point, MOI 0.5 for the 48 h time point). Three or 48 h post infection, cells were stimulated with BMP9 (3 nM) for (1) 2 h, followed by cell lysis and immunoblot analysis, or (2) 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B , C ) Cell lysates from ( A ) were subjected to immunoblot analysis with p-SMAD1/5/9, SMAD1, HCMV UL35, and GAPDH-specific antibodies (top panel), and transcript levels of Id3 and Stat2 are shown below. Data information: ( B , C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Techniques: Recombinant, Construct, Infection, Lysis, Western Blot, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

( A ) HFF-1 were infected by centrifugal enhancement with HCMV WT or HCMV US18/20stop (MOI 4). Three hours post infection, cells were stimulated with IFNβ (1 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9 for 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B – D ) Transcript levels of Id3 ( B ), the ISGs Irf9 and Stat2 ( C ), and HCMV transcripts HCMV IE1 and HCMV UL44 ( D ). Data information: Three independent experiments with similar results were performed and data shown is combined from two of the three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) HFF-1 were infected by centrifugal enhancement with HCMV WT or HCMV US18/20stop (MOI 4). Three hours post infection, cells were stimulated with IFNβ (1 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9 for 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B – D ) Transcript levels of Id3 ( B ), the ISGs Irf9 and Stat2 ( C ), and HCMV transcripts HCMV IE1 and HCMV UL44 ( D ). Data information: Three independent experiments with similar results were performed and data shown is combined from two of the three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Techniques: Infection, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Fig. 8 Summary of the results. After the 10 weeks tilorone treatment, the HFD-induced increases in complex II-linked oxidative phosphorylation and com- plex IV activity reduced and the HFD-induced decreases in BMP9, pSmad1/5/8, and PPARγ levels were normal- ized. PET/MRI showed increased tissue uptake of 18FDG. As a consequence, lipid content of the liver decreased, the glycogen content increased, and blood glucose level, body mass, and liver and adipose tissue weights were normal- ized. HFD, high-fat diet; BMP, bone morphogenetic protein; PPARγ, peroxi- some proliferator-activated receptor gamma; 18FDG, 18F-fluoro-2-deoxyglucose

Journal: GeroScience

Article Title: Tilorone attenuates high-fat diet-induced hepatic steatosis by enhancing BMP9-Smad1/5/8 signaling.

doi: 10.1007/s11357-025-01685-8

Figure Lengend Snippet: Fig. 8 Summary of the results. After the 10 weeks tilorone treatment, the HFD-induced increases in complex II-linked oxidative phosphorylation and com- plex IV activity reduced and the HFD-induced decreases in BMP9, pSmad1/5/8, and PPARγ levels were normal- ized. PET/MRI showed increased tissue uptake of 18FDG. As a consequence, lipid content of the liver decreased, the glycogen content increased, and blood glucose level, body mass, and liver and adipose tissue weights were normal- ized. HFD, high-fat diet; BMP, bone morphogenetic protein; PPARγ, peroxi- some proliferator-activated receptor gamma; 18FDG, 18F-fluoro-2-deoxyglucose

Article Snippet: Then, the membranes were blocked in Tris buffered saline containing 5% skimmed milk and 0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature and were incubated at 4 °C overnight with the following rabbit polyclonal primary antibodies: phospho-Smad1/5/8 (Smad1 [Ser463/465]/Smad5 [Ser463/465]/Smad9 [Ser465/467]; #AB-3848-J; Sigma-Aldrich); Smad1/5/8 (#56,656; Novus Biologicals; Centennial, CO, USA); PPARγ (#2435; Cell Signaling Technology; Danvers; MA; USA); PGC-1α (#2178; Cell Signaling Technology); TOM20 (#42,406; Cell Signaling Technology); BMP6 (#55,421–1-AP; Proteintech; Rosemont; IL, USA); BMP9 (#17,769–1-AP; Proteintech); and GAPDH (#2188; Cell Signaling Technology).

Techniques: Phospho-proteomics, Activity Assay

Figure 1 Effect of LGR4 on bone formation in BMP9-KO mice. (A) The micro-CT images showing BMP9’s effect on bone formation. (B) Quantitative results of micro-CT assay showing BMP9’s effect on bone formation. (C) The effect of BMP9 on LGR4 in BMSCs from 4-week-old WT and BMP9-KO mice cultured in an osteogenic medium for 7 days shown by Western blotting. (D) The effect of BMP9 on bone formation shown by H&E staining. (E) The effect of BMP9 on LGR4 in femurs from 4-week-old WT and BMP9-KO mice shown by immunohistochemistry staining. (F, G) The effect of LGR4 on the ALP activity of BMSCs shown by histochemical staining. (H) The effect of LGR4 on the mineralization of BMSCs shown by histochemical staining. BV/TV, the ratio of bone volume to total volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. *P < 0.05, **P < 0.01; n Z 6.

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 1 Effect of LGR4 on bone formation in BMP9-KO mice. (A) The micro-CT images showing BMP9’s effect on bone formation. (B) Quantitative results of micro-CT assay showing BMP9’s effect on bone formation. (C) The effect of BMP9 on LGR4 in BMSCs from 4-week-old WT and BMP9-KO mice cultured in an osteogenic medium for 7 days shown by Western blotting. (D) The effect of BMP9 on bone formation shown by H&E staining. (E) The effect of BMP9 on LGR4 in femurs from 4-week-old WT and BMP9-KO mice shown by immunohistochemistry staining. (F, G) The effect of LGR4 on the ALP activity of BMSCs shown by histochemical staining. (H) The effect of LGR4 on the mineralization of BMSCs shown by histochemical staining. BV/TV, the ratio of bone volume to total volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. *P < 0.05, **P < 0.01; n Z 6.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Micro-CT, Cell Culture, Western Blot, Staining, Immunohistochemistry, Activity Assay

Figure 2 Effect of BMP9 on LGR4 in multiple progenitor cells. (A) The level of LGR4 in the progenitor cells shown by Western blotting. (B) The effect of osteogenic BMP9 on LGR4 shown by Western blotting. (C) The effect of BMP9 on LGR4 over time shown by Western blotting. (D) The images of C3H10T1/2 cells showing the transfection rates of BMP9 at 24 h (scale bar, 100 mm; original magniﬁcation, 100). (E) Relative quantiﬁcation of BMP9 adenovirus infection rate in C3H10T1/2 cells. (F) Western blotting shows BMP9 recombinant adenovirus affects the protein level of BMP9 at 24 h. (G) RT-qPCR assay showing BMP9 affects LGR4 expression. (H) Western blotting shows BMP9 affects the protein level of LGR4 at 24 h and 48 h. *P < 0.05, **P < 0.01; n Z 3.

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 2 Effect of BMP9 on LGR4 in multiple progenitor cells. (A) The level of LGR4 in the progenitor cells shown by Western blotting. (B) The effect of osteogenic BMP9 on LGR4 shown by Western blotting. (C) The effect of BMP9 on LGR4 over time shown by Western blotting. (D) The images of C3H10T1/2 cells showing the transfection rates of BMP9 at 24 h (scale bar, 100 mm; original magniﬁcation, 100). (E) Relative quantiﬁcation of BMP9 adenovirus infection rate in C3H10T1/2 cells. (F) Western blotting shows BMP9 recombinant adenovirus affects the protein level of BMP9 at 24 h. (G) RT-qPCR assay showing BMP9 affects LGR4 expression. (H) Western blotting shows BMP9 affects the protein level of LGR4 at 24 h and 48 h. *P < 0.05, **P < 0.01; n Z 3.

Techniques: Western Blot, Transfection, Infection, Recombinant, Quantitative RT-PCR, Expressing

Figure 3 Effect of LGR4 on BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) The effects of BMP9 and/or LGR4 on RUNX2 mRNA expression shown by RT-qPCR. (B) The effects of BMP9 and/or LGR4 on RUNX2 protein level shown by Western blotting. (C) The effects of BMP9 and/or LGR4 on ALP activity shown by alkaline phosphatase assay. (D) The effects of BMP9 and/or LGR4 on OPN mRNA expression shown by RT-qPCR. (E) The effects of BMP9 and/or LGR4 on OPN level shown by Western blotting. (F) The effects of BMP9 and/or LGR4 on mineralization shown by alizarin red S staining. (G) The effects of BMP9 and/or LGR4 knockdown on RUNX2

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 3 Effect of LGR4 on BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) The effects of BMP9 and/or LGR4 on RUNX2 mRNA expression shown by RT-qPCR. (B) The effects of BMP9 and/or LGR4 on RUNX2 protein level shown by Western blotting. (C) The effects of BMP9 and/or LGR4 on ALP activity shown by alkaline phosphatase assay. (D) The effects of BMP9 and/or LGR4 on OPN mRNA expression shown by RT-qPCR. (E) The effects of BMP9 and/or LGR4 on OPN level shown by Western blotting. (F) The effects of BMP9 and/or LGR4 on mineralization shown by alizarin red S staining. (G) The effects of BMP9 and/or LGR4 knockdown on RUNX2

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, ALP Assay, Staining, Knockdown

Figure 4 Effect of mTOR on BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) The effect of BMP9 on Raptor shown by RT- qPCR. (B) The effects of BMP9 and/or Raptor knockdown on RUNX2 mRNA expression shown by RT-qPCR. (C) The effects of BMP9 and/or Raptor knockdown on RUNX2 protein level shown by Western blotting. (D) The effects of BMP9 and/or Raptor knockdown on OPN mRNA level shown by RT-qPCR. (E) The effects of BMP9 and/or Raptor knockdown on OPN protein level shown by Western blotting. (F) The effects of BMP9 and/or Raptor knockdown on mineralization shown by alizarin red S staining. (G) The effect of BMP9 on Rictor mRNA level shown by RT-qPCR. (H) The effects of BMP9 and/or Rictor on RUNX2 mRNA level shown by RT-qPCR. (I) The effects of BMP9 and/or Rictor knockdown on RUNX2 protein level shown by Western blotting. (J) The effects of BMP9 and/or Rictor knockdown on OPN mRNA level shown by RT-qPCR. (K) The effects of BMP9 and/or Rictor knockdown on OPN protein level shown by Western blotting. (L) The effects of BMP9 and/or Rictor knockdown on mineralization shown by alizarin red S staining. *P < 0.05, **P < 0.01; n Z 3.

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 4 Effect of mTOR on BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) The effect of BMP9 on Raptor shown by RT- qPCR. (B) The effects of BMP9 and/or Raptor knockdown on RUNX2 mRNA expression shown by RT-qPCR. (C) The effects of BMP9 and/or Raptor knockdown on RUNX2 protein level shown by Western blotting. (D) The effects of BMP9 and/or Raptor knockdown on OPN mRNA level shown by RT-qPCR. (E) The effects of BMP9 and/or Raptor knockdown on OPN protein level shown by Western blotting. (F) The effects of BMP9 and/or Raptor knockdown on mineralization shown by alizarin red S staining. (G) The effect of BMP9 on Rictor mRNA level shown by RT-qPCR. (H) The effects of BMP9 and/or Rictor on RUNX2 mRNA level shown by RT-qPCR. (I) The effects of BMP9 and/or Rictor knockdown on RUNX2 protein level shown by Western blotting. (J) The effects of BMP9 and/or Rictor knockdown on OPN mRNA level shown by RT-qPCR. (K) The effects of BMP9 and/or Rictor knockdown on OPN protein level shown by Western blotting. (L) The effects of BMP9 and/or Rictor knockdown on mineralization shown by alizarin red S staining. *P < 0.05, **P < 0.01; n Z 3.

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Staining

Figure 5 Effect of LGR4 and/or Raptor knockdown on osteoblastic and adipogenic markers induced by BMP9 in C3H10T1/2 cells. (A) The effects of LGR4 and/or Raptor knockdown on BMP9-induced RUNX2 mRNA level shown by RT-qPCR. (B) The effects of LGR4 and/or Raptor knockdown on BMP9-induced RUNX2 shown by Western blotting. (C) The effects of LGR4 and/or Raptor knockdown on BMP9-induced ALP activity shown by histochemical staining. (D) The effects of LGR4 and/or Raptor knockdown on BMP9-induced OPN mRNA level shown by RT-qPCR. (E) The effects of LGR4 and/or Raptor knockdown on BMP9-induced OPN level shown by Western blotting. (F) The effects of LGR4 and/or Raptor knockdown on BMP9-induced matrix mineralization shown by histo- chemical staining. (G) The effects of LGR4 and/or Raptor knockdown on BMP9-induced PPARg mRNA expression shown by RT-qPCR. (H) The effects of LGR4 and/or Raptor knockdown on BMP9-induced PPARg protein level shown by Western blotting. (I) The effects of LGR4 and/or Raptor knockdown on BMP9-induced lipid droplets formation shown by oil red O staining. *P < 0.05, **P < 0.01; n Z 3.

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 5 Effect of LGR4 and/or Raptor knockdown on osteoblastic and adipogenic markers induced by BMP9 in C3H10T1/2 cells. (A) The effects of LGR4 and/or Raptor knockdown on BMP9-induced RUNX2 mRNA level shown by RT-qPCR. (B) The effects of LGR4 and/or Raptor knockdown on BMP9-induced RUNX2 shown by Western blotting. (C) The effects of LGR4 and/or Raptor knockdown on BMP9-induced ALP activity shown by histochemical staining. (D) The effects of LGR4 and/or Raptor knockdown on BMP9-induced OPN mRNA level shown by RT-qPCR. (E) The effects of LGR4 and/or Raptor knockdown on BMP9-induced OPN level shown by Western blotting. (F) The effects of LGR4 and/or Raptor knockdown on BMP9-induced matrix mineralization shown by histo- chemical staining. (G) The effects of LGR4 and/or Raptor knockdown on BMP9-induced PPARg mRNA expression shown by RT-qPCR. (H) The effects of LGR4 and/or Raptor knockdown on BMP9-induced PPARg protein level shown by Western blotting. (I) The effects of LGR4 and/or Raptor knockdown on BMP9-induced lipid droplets formation shown by oil red O staining. *P < 0.05, **P < 0.01; n Z 3.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Expressing

Figure 6 Effect of LGR4 and Raptor on the BMP9-induced skull defect repair. (A) The effect of LGR4 and Raptor knockdown on the bone formation induced by BMP9 (the area circled in red is the area of the defect site) shown by reconstruction of micro-CT analysis. (BeD) The effects of LGR4 and Raptor knockdown on bone formation induced by BMP9 indicated by quantitative results of the micro-CT assay of BV/TV, Tb.N, and BS/TV. BV/TV, bone volume per tissue volume; Tb.N, trabecular number; BS/TV, bone volume per bone surface area. *P < 0.05, **P < 0.01 vs. AdGFP control group; #P < 0.05, ##P < 0.01 vs. AdBMP9 group; DP < 0.05, DDP < 0.01 vs. AdBMP9 þ AdsiRaptor group; n Z 6.

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 6 Effect of LGR4 and Raptor on the BMP9-induced skull defect repair. (A) The effect of LGR4 and Raptor knockdown on the bone formation induced by BMP9 (the area circled in red is the area of the defect site) shown by reconstruction of micro-CT analysis. (BeD) The effects of LGR4 and Raptor knockdown on bone formation induced by BMP9 indicated by quantitative results of the micro-CT assay of BV/TV, Tb.N, and BS/TV. BV/TV, bone volume per tissue volume; Tb.N, trabecular number; BS/TV, bone volume per bone surface area. *P < 0.05, **P < 0.01 vs. AdGFP control group; #P < 0.05, ##P < 0.01 vs. AdBMP9 group; DP < 0.05, DDP < 0.01 vs. AdBMP9 þ AdsiRaptor group; n Z 6.

Techniques: Knockdown, Micro-CT, Control

Figure 7 Effect of mTORC1/Stat3 signal and LGR4 on BMP9-induced osteogenesis in C3H10T1/2 cells. (A) The effects of BMP9 and/or Raptor knockdown on LGR4 mRNA expression shown by RT-qPCR. (B) The effects of BMP9 and/or Raptor knockdown on LGR4 protein level shown by Western blotting. (C) The effects of BMP9 and/or Raptor knockdown on total and phosphorylated Stat3 levels shown by Western blotting. (D) The effects of AG490 and/or BMP9 on LGR4 mRNA shown by RT-qPCR. (E) The effects of AG490 and/or BMP9 on LGR4 shown by Western blotting. (F) The effects of AG490 and/or LGR4 on BMP9-induced mineralization shown by histochemical staining. (G) The effects of AG490 and/or LGR4 on BMP9-induced RUNX2 mRNA expression shown by RT-qPCR. (H) The effects of AG490 and/or LGR4 on BMP9-induced RUNX2 shown by Western blotting. (I) The effects of AG490 and/or LGR4 on BMP9- induced ALP activity shown by histochemical staining. (J) The effects of AG490 and/or LGR4 on BMP9-induced OPN mRNA expression shown by RT-qPCR. (K) The effects of AG490 and/or LGR4 on BMP9-induced OPN shown by Western blotting. AG490: Jak2/Stat3 inhibitor. *P < 0.05, **P < 0.01; n Z 3.

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 7 Effect of mTORC1/Stat3 signal and LGR4 on BMP9-induced osteogenesis in C3H10T1/2 cells. (A) The effects of BMP9 and/or Raptor knockdown on LGR4 mRNA expression shown by RT-qPCR. (B) The effects of BMP9 and/or Raptor knockdown on LGR4 protein level shown by Western blotting. (C) The effects of BMP9 and/or Raptor knockdown on total and phosphorylated Stat3 levels shown by Western blotting. (D) The effects of AG490 and/or BMP9 on LGR4 mRNA shown by RT-qPCR. (E) The effects of AG490 and/or BMP9 on LGR4 shown by Western blotting. (F) The effects of AG490 and/or LGR4 on BMP9-induced mineralization shown by histochemical staining. (G) The effects of AG490 and/or LGR4 on BMP9-induced RUNX2 mRNA expression shown by RT-qPCR. (H) The effects of AG490 and/or LGR4 on BMP9-induced RUNX2 shown by Western blotting. (I) The effects of AG490 and/or LGR4 on BMP9- induced ALP activity shown by histochemical staining. (J) The effects of AG490 and/or LGR4 on BMP9-induced OPN mRNA expression shown by RT-qPCR. (K) The effects of AG490 and/or LGR4 on BMP9-induced OPN shown by Western blotting. AG490: Jak2/Stat3 inhibitor. *P < 0.05, **P < 0.01; n Z 3.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Staining, Activity Assay

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