ml347 alk1 2 inhibitor tocris  (Tocris)

Journal: Theranostics

Article Title: FBLN7 KO attenuates age-related cardiac fibrosis by promoting TGFBR3/ALK1/Smad1 signaling and inhibiting the profibrotic phenotypes of cardiac fibroblasts

doi: 10.7150/thno.116477

Figure Lengend Snippet: FBLN7 modulates the impaired profibrotic abilities of senescent cardiac fibroblasts through the ALK1-Smad1/5/9 pathway. A-B) Representative western blot images displaying the levels of p-Smad2, Smad2, p-Smad1/5/9, and Smad1/5/9 proteins in cardiac fibroblasts with FBLN7 silencing (siFBLN7) (A), FBLN7 overexpression (adFBLN7) (B), and corresponding control cardiac fibroblasts (siNC or adNC) following treatment with D-gal or PBS. Quantifications of band intensity are presented on the right. C-D) Representative images illustrating the nuclear translocation of p-Smad1/5/9 (red) (C) and p-Smad2 (red) (D) in siFBLN7 and siNC cardiac fibroblasts after treatment with D-gal or PBS, labeled with Vimentin (green). E) Representative western blot images showing the protein levels of p-Smad2, Smad2, p-Smad1/5/9 and Smad1/5/9 in D-gal treated cardiac fibroblasts transfected with siNC, siFBLN7, or siFBLN7 plus ML347 treatment. Quantifications are displayed on the right. F) Representative western blot images showing the protein levels of Collagen I and III, ALK1, α-SMA and P21 in D-gal treated cardiac fibroblasts transfected with siNC, siFBLN7, or siFBLN7 plus ML347 treatment. Quantifications are displayed on the right. Error bars represent the standard error of the mean (SEM). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: When indicated, CFs were pretreated with 250 nM ALK1 inhibitor (ML347, MCE, Shanghai, China) or 0.01% DMSO (Sigma-Aldrich) for 2 h prior to a 48-hour treatment with D-gal.

Techniques: Western Blot, Over Expression, Control, Translocation Assay, Labeling, Transfection

Journal: Theranostics

Article Title: FBLN7 KO attenuates age-related cardiac fibrosis by promoting TGFBR3/ALK1/Smad1 signaling and inhibiting the profibrotic phenotypes of cardiac fibroblasts

doi: 10.7150/thno.116477

Figure Lengend Snippet: TGFBR3 is essential for the differential transduction of TGF-β signals mediated by FBLN7. A) Endogenous co-immunoprecipitation (Co-IP) of FBLN7 and TGFBR3. Immunoprecipitation (IP) was performed with TGFBR3, followed by immunoblotting (IB) with FBLN7 in senescent cardiac fibroblasts (CFs). B) Representative confocal images demonstrating the co-localization of FBLN7 and TGFBR3 in senescent CFs. C) Reciprocal Co-IP of FBLN7 and TGFBR3. IP was conducted with GFP-tag and IB with Flag-TGFBR3 in 293T cells (left panel). Conversely, IP was performed with Flag-tag and IB with GFP-FBLN7 in 293T cells (right panel). D) Representative confocal images illustrating the co-localization of Myc-FBLN7 and Flag-TGFBR3 in 293T cells. E) Results of molecular docking between FBLN7 and TGFBR3. a) The optimal docking conformation of FBLN7 and TGFBR3 reveals a stable complex, with FBLN7 highlighted in blue and TGFBR3 in yellow. b) The white semi-transparent structure represents the entire molecular framework, providing a global view of the docking region. The black box in the center indicates the binding site, emphasizing critical areas on the binding interface. c) The Docking Zoom Map displays key amino acid residues at the binding interface and their interactions. F-G) Representative western blot images showing TGFBR3 and ALK1 protein levels in CFs with FBLN7 silencing (F) and overexpression (G) after treatment with D-gal or PBS. Quantifications of band intensity are presented on the right. H) Representative western blot images illustrating protein levels of FBLN7 and TGFBR3 in D-gal-treated CFs infected with either Null, adenovirus encoding FBLN7 (adFBLN7), or adFBLN7 plus Flag-TGFBR3. Quantifications are displayed on the right. I) Representative western blot images illustrating protein levels of p-Smad1/5/9, Smad1/5/9, p-Smad2 and Smad2 in D-gal-treated CFs infected with either Null, adFBLN7, or adFBLN7 plus Flag-TGFBR3. Quantifications are displayed on the bottom. J) Representative western blot images illustrating protein levels of Collagen I and III, ALK1, α-SMA, and P21 in D-gal-treated CFs infected with either Null, adFBLN7, or adFBLN7 plus Flag-TGFBR3. Quantifications are displayed around the imge. Error bars represent the standard error of the mean (SEM). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: When indicated, CFs were pretreated with 250 nM ALK1 inhibitor (ML347, MCE, Shanghai, China) or 0.01% DMSO (Sigma-Aldrich) for 2 h prior to a 48-hour treatment with D-gal.

Techniques: Transduction, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, FLAG-tag, Binding Assay, Over Expression, Infection

Journal: Theranostics

Article Title: FBLN7 KO attenuates age-related cardiac fibrosis by promoting TGFBR3/ALK1/Smad1 signaling and inhibiting the profibrotic phenotypes of cardiac fibroblasts

doi: 10.7150/thno.116477

Figure Lengend Snippet: GRO rescues the impaired profibrotic phenotypes of senescent cardiac fibroblasts and prevents age-related cardiac fibrosis and diastolic dysfunction. A) Representative Western blot images displaying protein levels of Collagen I and III, TGFBR3, ALK1, α-SMA, and P21 in senescent CFs infected with adenovirus encoding FBLN7 (adFBLN7) and treated with DMSO or various concentrations of GRO (50, 100, and 200 μM). Quantifications are presented on the right. *: adNC+D-gal+DMSO vs. adNC+PBS+DMSO, &: adFBLN7+D-gal+DMSO vs . adNC+D-gal+DMSO, $: adFBLN7+D-gal+GRO50 vs. adFBLN7+D-gal+DMSO, #: adFBLN7+D-gal+GRO100 vs. adFBLN7+D-gal+DMSO, %: adFBLN7+D-gal+GRO200 vs. adFBLN7+D-gal+DMSO. B) Schedule for FBLN7 overexpression and GRO supplementation in mice. C) Gross appearances of mice and their hearts in each group. D) Representative echocardiography images of the two groups of mice. Upper: B-mode; middle: PW Doppler mode; lower: tissue Doppler mode. Echocardiographic analysis of interventricular septal thickness in diastole (IVSd) and systole (IVSs), left ventricular posterior wall thickness in diastole (LVPWd) and systole (LVPWs), left ventricular mass, the ratio of early to late diastolic filling velocities (E/A), and the ratio of early diastolic filling velocity to left atrial pressure (E/e'), left ventricular ejection fraction (LVEF) and fractional shortening (FS%) are shown at the bottom. E) Representative images of H&E staining of hearts from aging mice (18 months old) injected with AAV-FBLN7 and administered 40 mg/kg/d of GRO or solvent control via gavage. F) Representative WGA staining images of the two groups of mice, with quantification shown on the right. G) Representative micrographs of Masson staining in heart sections from the AAV-FBLN7-18M-GRO and AAV-FBLN7-18M-CTL mice, displaying three different views: upper: perivascular; middle: global; lower: interstitial. Quantifications of fibrotic areas are presented on the right. H) FBLN7 exerts its reversing effect on the impaired profibrotic phenotypes of senescent CFs by downregulating the TGFBR3/ALK1/Smad1 pathway, which may contribute to the pathogenesis of age-related cardiac fibrosis. GRO exerts antifibrotic effects possibly through interaction with FBLN7. Error bars represent the standard error of the mean (SEM). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: When indicated, CFs were pretreated with 250 nM ALK1 inhibitor (ML347, MCE, Shanghai, China) or 0.01% DMSO (Sigma-Aldrich) for 2 h prior to a 48-hour treatment with D-gal.

Techniques: Western Blot, Infection, Over Expression, Staining, Injection, Solvent, Control

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: ( A ) Transcriptomic profile of each cell (dots) represented by a Uniform Manifold Approximation and Projection (UMAP), noted from C1 to 5 in the basal state and differential expression of BMP receptors in the five clusters (n=5), the clusters can be subdivided on two subgroups based on ALK1 expression: ALK1 high and ALK1 low . (B) TOP 10 GO terms that characterized the ALK1 high PMEC population. (C) Gene set enrichment analysis (GSEA) of differentially expressed genes (DEGs) between ALK1 high versus ALK1 low PMECs. NES: normalized enrichment score; FDR: false discovery rate.

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: Expressing

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: (A) UMAP illustration showing changes in the transcriptomic profile in ALK1 high and ALK1 low PMEC-clusters 4h after BMP-9 stimulation (10ng/mL). (B) Violin plots of standardized expression of the main BMP-9 receptors across ALK1 high and ALK1 low PMEC-clusters with or without BMP-9 stimulation. TOP 50 Gene Ontology/Biological Process (GO/BP) terms that characterized the BMP-9 responses in ALK1 high (C) and ALK1 high PMEC-clusters ( D) . Data are represented as mean± SEM. Significance was measured using nonparametric Mann-Whitney t-test: ****, p<0.0001 compared to ALK1 high .

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: (A) Representative images and quantifications of tube formation by human PMECs exposed to non-relevant IgG or ALK1-Fc (300ng/mL). (B) Representative images and quantifications of the surface of wound covered by human PMECs exposed to non-relevant IgG or ALK1-Fc (300ng/mL). (C) Effects of BMP-9 and siALK1 on BrdU incorporation in human PMECs. (D-F) Effects of non-relevant IgG or ALK1-Fc (300ng/mL) on tube formation, migration and proliferation of human PMECs. Data are represented as mean± SEM. Significance was measured using parametric paired t-test or 1-way ANOVA with Tukey post hoc tests: *, p<0.05; **, p<0.01; ****, p<0.0001 versus IgG, Scr sequence or 0.3% fetal calf serum (FCS). #, p<0.05; ##, p<0.01 versus Scr seq. AU: arbitrary unit. ALK1-Fc: soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment. IgG: immunoglobulin G. Nbr: number. BrdU: bromodeoxyuridine

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: BrdU Incorporation Assay, Migration, Sequencing

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: (A) Violin plots showing BMP-9 effects on mRNA levels for VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor ( PGF ), VEGFR1 ( FLT1 ), and VEGFR2 ( KDR ) in ALK1 High , with or without BMP-9 stimulation and in ALK1 Low . Representative immunoblots and densitometric analysis of ALK1 (B) , VEGFR2 and VEGFR1 expression in human PMECs upon 24h of BMP-9 stimulation (10ng/mL) (C) . (D) Representative immunoblots and densitometric analysis of p-VEGFR2, p-SRC and p-AKT in human PMECs pretreated for 24h with BMP-9 (10ng/mL) and/or stimulated for 30min with VEGF-A (20ng/mL). (E) Representative immunoblots and densitometric analysis of ALK1, VEGFR1, p-VEGFR2, VEGFR2, p-AKT and AKT in Gdf2-/- and Gdf2+/+ rat lungs. (F) Representative immunofluorescent staining of ALK1 (red), CD31 (green) and nuclei (DAPI, blue) in Gdf2-/- and Gdf2+/+ rat lungs. Scale bars=50 μm. Data are represented as mean± SEM. Significance was measured using parametric paired t-test or 1-way ANOVA with Tukey post hoc tests: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 versus basal condition or Gdf2 +/+. AKT = protein kinase B. AU = arbitrary unit. DAPI = 4’,6-diamidino-2-phenylindole. GAPDH = glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: Western Blot, Expressing, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: ALK3 and ALK6 are required for GDF2-induced SMAD1/5 phosphorylation. (A-B) Western blotting for pSMAD1/5 activation in PA1 and MCF10A cells in the presence and absence of dorsomorphin 1 μM (+) or 3 μM (++), SB431542 3 μM (+) or 5 μM (++), or ML347 500 nM (+) or 1 mM (++) as indicated with and without GDF2 (10 ng/ml) as indicated (quantification of pSMAD1/5 levels presented in Supplementary Figure S2 C ). (C) Immunoblotting of pSMAD1/5 in PA1 cells in the presence of shRNAs to ALK2, ALK3, ALK6, and BMPRII without and with GDF2 treatment (10 ng/ml) for 30 minutes. (D) QRT-PCR analyses of (C) to confirm reduced expression of ALK2, ALK3, ALK6, and BMPRII expression as indicated. (E) Kinase inactive ALK3 and ALK6 inhibit SMAD1/5 phosphorylation. Western blotting as indicated in MCF10A and PA1 cells in the presence of either mock transfected or HA-tagged kinase inactive ALK3 (ALK3 K-R) or ALK6 (ALK6 K-R) and treated with GDF2 for the time points indicated. Actin was the loading control. (F) Dorsomorphin inhibits SMAD1/5 transcriptional activation. BRE-luciferase reporter activity in indicated cells in the absence (GDF2 alone) or presence of 1 μM dorsomorphin (GDF2+DM). Fold induction of luciferase activity compared with DMSO-treated control cells is presented.

Article Snippet: ALK1/2 inhibitor ML347 (#4945) was from Tocris Bioscience.

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Quantitative RT-PCR, Expressing, Transfection, Control, Luciferase, Activity Assay