Journal: Advanced Science

Article Title: Automated, High‐Throughput Phenotypic Screening and Analysis Platform to Study Pre‐ and Post‐Implantation Morphogenesis in Stem Cell‐Derived Embryo‐Like Structures

doi: 10.1002/advs.202304987

Figure Lengend Snippet: Library of signaling pathway modulators.

Article Snippet: ML347 , ALK1/2 inhibitor , Tocris 4945 , 1.5 µ m.

Techniques: Concentration Assay

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: Frontiers in Pharmacology

Article Title: Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway

doi: 10.3389/fphar.2017.00111

Figure Lengend Snippet: ALK1 receptor gene silencing by siRNA abrogates Smad1/5/8 phosphorylation, VEGF upregulation and angiogenesis induced by Dkk-3. (A) RTPCR analysis of ALK1 mRNA level in both control and ALK1 silenced cells. ** p < 0.001 vs. control-siRNA treated cells. (B) Western blot analysis of phospho-Smad1,5,8 expression in both control and ALK1 silenced cells exposed to hrDkk-3 (10 ng/ml, 60 min). HUVECs incubated with TGF-β1 were used as positive controls. Values are means ± SEM. ** p < 0.001 vs. baseline conditions, ## p < 0.001 vs. hrDkk-3. Immunocytochemical (C) and western blot analysis (D) of VEGF expression in both control and ALK1 silenced cells exposed to hrDkk-3 (10 ng/ml, 12 h). Values are means ± SEM. ** p < 0.001 vs. baseline conditions, # p < 0.05 vs. hrDkk-3, ## p < 0.001 vs. hrDkk-3. (E) Endothelial cell tube formation of both control and ALK1 silenced cells treated with hrDkk-3 (10 ng/ml, 18 h). Values are means ± SEM. * p < 0.05 vs. baseline conditions, # p < 0.05 vs. hrDkk-3.

Article Snippet: To better assess the role of ALK1 in Dkk-3 mediated VEGF upregulation, the VEGF intracellular expression was assessed by western blot in HUVECs following incubation with hrDkk-3 (10 ng/ml, 12 h) either in the presence or in the absence of the selective ALK1 antagonist ML347 (0.5 or 1 μM, Tocris Bioscience).

Techniques: Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Incubation

Journal: Frontiers in Pharmacology

Article Title: Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway

doi: 10.3389/fphar.2017.00111

Figure Lengend Snippet: ALK1 receptor pharmacological blockade abrogates VEGF upregulation induced by Dkk-3 in HUVECs. (A) Western blot analysis of VEGF expression in HUVECs exposed to hrDkk-3 (10 ng/ml, 12 h) either in the presence or in the absence of the selective inhibitor ML347 (0.5 M or 3 μM). (B) Corresponding densitometric analysis. Values are means ± SEM. ** p < 0.001 vs. baseline conditions, ## p < 0.001 vs. hrDkk-3.

Article Snippet: To better assess the role of ALK1 in Dkk-3 mediated VEGF upregulation, the VEGF intracellular expression was assessed by western blot in HUVECs following incubation with hrDkk-3 (10 ng/ml, 12 h) either in the presence or in the absence of the selective ALK1 antagonist ML347 (0.5 or 1 μM, Tocris Bioscience).

Techniques: Western Blot, Expressing

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