Journal: Experimental and Therapeutic Medicine

Article Title: High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation

doi: 10.3892/etm.2020.9088

Figure Lengend Snippet: TGF-β1 expression is increased in degenerated discs. (A) Representative MRI of the patients from Pfirrmann grade 2 to 5. The yellow arrow indicates the surgical segment. (B) Representative images of immunohistochemical targets TGF-β1 (magnification, x400) and (C) quantification analysis. (D) Protein expression of Col II, ALK5 and ALK1 was determined by western blotting and (E) semi-quantified. Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01. TGF-β1, transforming growth factor β1; Col II, collagen II; MRI, magnetic resonance imaging; ALK, ALK tyrosine kinase receptor.

Article Snippet: Primary antibodies against TGF-β1 (cat. no. sc-130348; 1:1,000), collagen-II (Col-II; cat. no. sc-52658; 1:1,000), ALK1 (cat. no. sc-101556; 1:1,000), GAPDH (cat. no. sc-47724; 1:3,000), and the secondary antibody m-immunoglobulin G (IgG)κ binding protein conjugated to horseradish peroxidase (cat. no. sc-516102; 1:500) were purchased from Santa Cruz Biotechnology, Inc. Primary antibody against ALK5 (cat. no. ab31013; 1:1,000) and Alexa Fluor 568 (cat. no. ab175473; 1:1,000) were from Abcam.

Techniques: Expressing, Immunohistochemical staining, Western Blot, Magnetic Resonance Imaging

Journal: Experimental and Therapeutic Medicine

Article Title: High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation

doi: 10.3892/etm.2020.9088

Figure Lengend Snippet: TGF-β1 is upregulated in TNF-α-treated NPCs. NPCs isolated from specimens in groups 2# to 5# were treated with 50 ng/ml TNF-α for 24 h. (A) TGF-β1 IF staining of NPCs from groups 2# to 5# (magnification, x400). (B) Protein expression of ALK5 and ALK1 in NPCs from groups 2# to 5# was determined by WB. (C) The quantification of IF. (D) The semi-quantification of WB. (E and F) The mRNA expression levels of smad1/2/3/5/8, Col II, TIMP-3, Col I and MMP-13 in NPCs from group 2# were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001. TGF-β1, transforming growth factor β1; TNF-α, tumor necrosis factor α; NPC, nucleus pulposus cell; IF, immunofluorescence; ALK, ALK tyrosine kinase receptor; WB, western blotting; Col, collagen; TIMP-3, tissue inhibitor of metalloproteinase-3; MMP-13, matrix metalloproteinase 13.

Article Snippet: Primary antibodies against TGF-β1 (cat. no. sc-130348; 1:1,000), collagen-II (Col-II; cat. no. sc-52658; 1:1,000), ALK1 (cat. no. sc-101556; 1:1,000), GAPDH (cat. no. sc-47724; 1:3,000), and the secondary antibody m-immunoglobulin G (IgG)κ binding protein conjugated to horseradish peroxidase (cat. no. sc-516102; 1:500) were purchased from Santa Cruz Biotechnology, Inc. Primary antibody against ALK5 (cat. no. ab31013; 1:1,000) and Alexa Fluor 568 (cat. no. ab175473; 1:1,000) were from Abcam.

Techniques: Isolation, Staining, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunofluorescence, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation

doi: 10.3892/etm.2020.9088

Figure Lengend Snippet: Inhibition of ALK5 aggravates high-dose TGF-β1-induced degeneration of NPCs. NPCs were treated with 5 nM TGF-β1 or 100 nM SB for 72 h. For the co-treated group, NPCs were treated with 5 nM TGF-β1 combined with 100 nM SB for 72 h. (A) Immunofluorescence staining of TGF-β1 (magnification, x400) and (B) quantification analysis. (C) Protein expression of ALK5 and ALK1 was determined by western blotting and (D) semi-quantified. (E and F) The mRNA expression levels of smad1/2/3/5/8, Col II, TIMP-3, Col I and MMP-13 in NPCs were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001. TGF-β1, transforming growth factor β1; NPC, nucleus pulposus cell; ALK, ALK tyrosine kinase receptor; Col, collagen; TIMP-3, tissue inhibitor of metalloproteinase-3; MMP-13, matrix metalloproteinase 13; SB, SB525334.

Article Snippet: Primary antibodies against TGF-β1 (cat. no. sc-130348; 1:1,000), collagen-II (Col-II; cat. no. sc-52658; 1:1,000), ALK1 (cat. no. sc-101556; 1:1,000), GAPDH (cat. no. sc-47724; 1:3,000), and the secondary antibody m-immunoglobulin G (IgG)κ binding protein conjugated to horseradish peroxidase (cat. no. sc-516102; 1:500) were purchased from Santa Cruz Biotechnology, Inc. Primary antibody against ALK5 (cat. no. ab31013; 1:1,000) and Alexa Fluor 568 (cat. no. ab175473; 1:1,000) were from Abcam.

Techniques: Inhibition, Immunofluorescence, Staining, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation

doi: 10.3892/etm.2020.9088

Figure Lengend Snippet: Inhibition of ALK1 alleviates high-dose TGF-β1-induced NPC degeneration. NPCs were treated with 5 nM TGF-β1 or 100 nM San for 72 h. For the co-treated group, NPCs were treated with 5 nM TGF-β1 combined with 100 nM San for 72 h. (A) Immunofluorescence staining of TGF-β1 (magnification, x400) and (B) quantification analysis. (C) Protein expression of ALK5 and ALK1 was determined by western blotting and (D) semi-quantified. (E and F) The mRNA expression levels of smad1/2/3/5/8, Col II, TIMP-3, Col I and MMP-13 in NPCs were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001. TGF-β1, transforming growth factor β1; NPC, nucleus pulposus cell; ALK, ALK tyrosine kinase receptor; Col, collagen; TIMP-3, tissue inhibitor of metalloproteinase-3; MMP-13, matrix metalloproteinase 13; San, San 78-130.

Article Snippet: Primary antibodies against TGF-β1 (cat. no. sc-130348; 1:1,000), collagen-II (Col-II; cat. no. sc-52658; 1:1,000), ALK1 (cat. no. sc-101556; 1:1,000), GAPDH (cat. no. sc-47724; 1:3,000), and the secondary antibody m-immunoglobulin G (IgG)κ binding protein conjugated to horseradish peroxidase (cat. no. sc-516102; 1:500) were purchased from Santa Cruz Biotechnology, Inc. Primary antibody against ALK5 (cat. no. ab31013; 1:1,000) and Alexa Fluor 568 (cat. no. ab175473; 1:1,000) were from Abcam.

Techniques: Inhibition, Immunofluorescence, Staining, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Fluid shear stress generates a unique signaling response by activating multiple TGFβ family type I receptors in osteocytes

doi: 10.1096/fj.202001998R

Figure Lengend Snippet: FSS stimulation activates multiple distinct TGFβ family type I receptors in OCY454 cells. A, qRT‐PCR analysis of TGFβ target gene Serpine1 after 60‐minute treatment with vehicle or SB‐431542 followed by 120‐minute treatment with TGFβ or FSS stimulation as indicated (n = 4 biological replicates). All values normalized to control cells. * P < .05 compared to unstimulated cells and # P < .05 compared to SB‐treated controls; † P < .05 compared to corresponding treatment group without SB‐431542, ‡ P < .05 compared to FSS‐stimulated cells. B, C, Representative western analysis of Smad phosphorylation in control cells and cells pretreated (60 minutes) with vehicle or an inhibitor of a subset of TGFβ type I receptors (SB‐431542, ALK4/5/7 inhibitor; LDN‐193189, ALK1/2/3/6 inhibitor; LDN‐214117, ALK1/2 inhibitor, as shown in B), followed by treatment (30 minutes) with TGFβ or FSS (C) (n = 3 biological replicates). D, Representative western analysis of cells pretreated (60 minutes) with ALK1Fc followed by treatment (30 minutes) with TGFβ, BMP4, or FSS (n = 2 biological replicates, non‐flow conditions were collected from cells grown in well plates)

Article Snippet: Except where noted in the figures, cells were treated as indicated with TGFβ1 (5 ng/mL), BMP4 (50 ng/mL) (both from Peprotech); 1d11 (1.25 µg/mL, Clone 1d11.16.8, BioXCell); Noggin (100 ng/mL, SRP3227, Sigma Aldrich); SB‐431542 (10 µM), LDN‐193189 (1 µM), LDN‐214117 (1 µM), SC‐79 (10 µM) (all from Selleckchem); recombinant mouse ALK1Fc (100 ng/mL, R&D Systems); and LY294002 (50 µM, Calbiochem).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: ALK1-Fc Inhibits Multiple Mediators of Angiogenesis and Suppresses Tumor Growth

doi: 10.1158/1535-7163.mct-09-0650

Figure Lengend Snippet: Figure 1. Generation and characterization of ligand binding of ALK1-Fc compounds. A, schematic representation of (1) mouse and (2) human ALK1 constructs bearing the extracellular region (ECR) of each receptor tethered by a linker domain to the Fc region of IgG2A and IgG1, respectively. B, representative data set (black lines) for kinetic analysis of BMP10 interactions with hALK1-Fc. BMP10 was immobilized on the chip and different concentrations of hALK1-Fc were injected over immobilized ligands. Red lines, the global fit of the data to a 1:1 bimolecular interaction model with mass transport. Ligand concentrations range from 0.78 to 200 nmol/L in 2-fold dilution increments.

Article Snippet: BMP9, type I and type II receptors, ligands, VEGF (rhVEGF165), and hALK1 antibody (R&D Systems, AF770) were obtained from R&D Systems.

Techniques: Ligand Binding Assay, Construct, Injection

Journal: Molecular Cancer Therapeutics

Article Title: ALK1-Fc Inhibits Multiple Mediators of Angiogenesis and Suppresses Tumor Growth

doi: 10.1158/1535-7163.mct-09-0650

Figure Lengend Snippet: Figure 2. Characterization of mALK1-Fc on endothelial cells in vitro. BMP9 (50 ng/mL) stimulation of HMVEC cells. A, transactivation of SMAD-binding element–driven luciferase promoter in the presence and absence of hALK1-Fc (10 μg/mL) or anti-hALK1 polyclonal antibody (10 μg/mL) as determined by luciferase expression. B, Id-1 expression in BMP9 (50 ng/mL) stimulated HUVEC after 24 h in the presence or absence of human ALK1-Fc as determined by SDS-PAGE Western blot. C, HUVEC cells seeded on Matrigel substrate were assayed over 12 h for cord formation (1) in absence of any treatment, (2) in presence of 100 μg/mL hALK1-Fc, (3) in presence of 100 μg/mL mALK1-Fc, (4) in presence of 100 ng/mL endostatin, (5) in presence of 200 ng/mL ECGS, (6) in presence of 100 μg/mL hALK1-Fc + 200 ng/mL ECGS, or (7) in presence of 100 μg/mL mALK1-Fc + 200 ng/mL ECGS. D, cord quantification of single-cell width vessels was done using the IPLab software (BD Biosciences; *, P < 0.05). Scale bar, 100 μm length.

Article Snippet: BMP9, type I and type II receptors, ligands, VEGF (rhVEGF165), and hALK1 antibody (R&D Systems, AF770) were obtained from R&D Systems.

Techniques: In Vitro, Binding Assay, Luciferase, Expressing, SDS Page, Western Blot, Software

Journal: Molecular Cancer Therapeutics

Article Title: ALK1-Fc Inhibits Multiple Mediators of Angiogenesis and Suppresses Tumor Growth

doi: 10.1158/1535-7163.mct-09-0650

Figure Lengend Snippet: Figure 3. Chick CAM assay. A, quantification of vessels per disc resulting after a 14-d CAM treatment in the presence of VEGF (50 ng) and varying concentrations of mouse and human ALK1-Fc compounds in PBS. Positive (VEGF only) and negative controls (PBS only) were also assayed, along with antiangiogenic Avastin (anti-VEGF; 9 μg) treatment. (*, P < 0.05). B, VEGF- and FGF-induced vascularization was also mitigated upon treatment with hALK1-Fc as described in Materials and Methods (*, P < 0.05). C, BMP10-induced vascularization could also be reduced upon treatment with mALK1-Fc (*, P < 0.05). Concentrations of VEGF, FGF, and BMP10 used in CAMs were 50 ng per egg.

Article Snippet: BMP9, type I and type II receptors, ligands, VEGF (rhVEGF165), and hALK1 antibody (R&D Systems, AF770) were obtained from R&D Systems.

Techniques: Chick Chorioallantoic Membrane Assay

Journal: Nature communications

Article Title: Clonal hematopoiesis with JAK2V617F promotes pulmonary hypertension with ALK1 upregulation in lung neutrophils.

doi: 10.1038/s41467-021-26435-0

Figure Lengend Snippet: Fig. 7 JAK2V617F transcriptionally upregulates ACVRL1 by STAT3-binding. a Western blot analysis of STAT3 in JAK2V617F/+ knock-in HCT116 cells. p-STAT3 and t-STAT3 indicate phosphorylated and total STAT3, respectively. p-STAT3 to t-STAT3 ratios are shown in the bar graph (n = 3, *P = 0.0296). The average value of JAK2+/+ HCT116 cells was set to 1. b mRNA expression in ACVRL1 in JAK2V617F/+ cells. The data were normalized to 18 s rRNA levels. The average value of JAK2+/+ cells was set to 1 (n = 3, *P = 0.0001). c Western blot analysis of the ALK1-SMAD pathway. The graphs show the densitometric analysis for ALK1, p-Smad1/5/8 and t-Smad1 (n = 3 in each, *P = 0.0070, 0.0004, respectively). p-Smad1/5/8 and t-Smad1 indicate phosphorylated Smad1/5/8 and total Smad1, respectively. GAPDH was used as the loading control. d Sequence alignments of putative STAT3 binding sites of Acvrl1 in human (hg19) and mouse (m10). Numbers are given according to the genomic sequence from transcriptional start site (TSS). The sequences of the STAT3 binding motifs are highlighted in red. Sequence logos for the motifs analyzed by TRANSFAC and JASPAR databases are displayed. e ChIP- quantitative PCR analysis for STAT3 binding to the putative ACVRL1 promoter. Chromatin was extracted from JAK2+/+ and JAK2V617F/+ HCT116 cells, and then precipitated with an anti-STAT3 antibody or IgG (negative control). The genomic DNA fragments of ACVRL1 promoter were evaluated for enrichment by quantitative PCR using the specific primers to the Acvrl1 promoter given from TSS. Data are expressed as the respective DNA inputs (n = 3 independent experiments, *P = 0.0015, 0.0026, respectively). f Dual luciferase reporter assays for the ACVRL1 gene promoter. The pGL3-basic vector containing the putative ACVRL1 promoter region (TSS −875 bp) and pNL1.1.TK [Nluc/TK] as a control vector were co-transfected in JAK2V617F/+ HCT116 cells. Twenty- four h after transfection, cell lysates were collected, and relative luciferase activity was determined by the ratio of firefly luciferase to Nano luciferase activity (n = 3 independent experiments, *P = 0.0051). g, h Inhibition of JAK1/2 or STAT3 reduced the elevated ACVRL1 promoter activity in JAK2V617F/+ cells. Twenty-four h after transfection, the JAK2V617F/+ HCT116 cells were incubated with a specific JAK1/2 inhibitor, ruxolitinib or a specific STAT3 inhibitor, stattic, at the indicated concentration for a further 24 h, and then luciferase activity was measured (n = 4 independent experiments, g, *P = 0.0059; h, n = 4 independent experiments, *P = 0.0164 [left], 0.0027 [right]). All data are presented as mean ± SEM. *P < 0.05 versus JAK2+/+ cells or vehicle by the unpaired Student’s t test (two-sided) or the one-way ANOVA with Tukey post-hoc analysis. Source data are provided as a Source Data file.

Article Snippet: HCT116 cells were collected and incubated with an anti-ALK1 antibody (14745-1-AP, Proteintech) followed by R-PE-conjugated donkey anti-rabbit secondary antibody (711-116-152, Jackson ImmunoResearch).

Techniques: Binding Assay, Western Blot, Knock-In, Expressing, Control, Sequencing, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Inhibition, Incubation, Concentration Assay

Journal: Nature communications

Article Title: Clonal hematopoiesis with JAK2V617F promotes pulmonary hypertension with ALK1 upregulation in lung neutrophils.

doi: 10.1038/s41467-021-26435-0

Figure Lengend Snippet: Fig. 8 Inhibition of ALK1/2 improves chronic hypoxia-induced pulmonary hypertension in JAK2V617F mice. a Schematic protocol. Vehicle (DMSO) or an ALK1/2 inhibitor, K02288 was administered via an intraperitoneal injection of 12 mg/kg body weight during 2-week chronic hypoxia-exposure, as indicated. b Peripheral blood cell counts in DMSO- or K02288-treated WT mice and JAK2V617F mice after exposure to chronic hypoxia for 2 weeks (n = 7, 7, 5, 6, *P = 0.0381 for WBC, n = 7, 8, 5, 7, *P = 0.0074 [left], 0.0037 [right] for Hb, n = 7, 8, 5, 7, *P = 0.0401 [left], 0.0120 [right] for PLT). c RVSP and RV hypertrophy determined by RV/LV + S in DMSO- or K02288-treated WT mice and JAK2V617F mice (n = 6, 8, 8, 7, *P = 0.0238 for RVSP, n = 8, 8, 8, 7, *P = 0.0112, †P = 0.0240 for RV/LV + S). d Representative images of EM-stained sections and sections immunostained with anti-αSMA antibody from DMSO- or K02288-treated WT mice and JAK2V617F mice. Scale bar, 25 µm. e Quantitative analysis of medial wall thickness in EM-stained sections (left, n = 6 in each group, *P < 0.0001, †P < 0.0001) and the percentage of muscularized distal pulmonary vessels in αSMA-immunostained sections (right, n = 6 in each group, *P < 0.0001, †P < 0.0001). f Representative immunofluorescence images of lung sections stained with anti-Ly6G (green) antibody and DAPI (blue). Scale bars, 50 μm. g Quantitative analysis of the numbers of Ly6G+ cells in the perivascular regions (n = 3 in each group, *P = 0.0001 [left], 0.0103 [right], †P = 0.0074). h Elastase activity in the lung extracts from DMSO- or K02288-treated WT mice and JAK2V617F mice. The average value for DMSO- treated WT mice was set to 1 (n = 3 in each group, *P = 0.0017, †P = 0.0075). All data are presented as mean ± SEM. *P < 0.05 versus the corresponding WT mice and †P < 0.05 versus DMSO-treated JAK2V617F mice by the one-way ANOVA with Tukey post-hoc analysis. WBC white blood cell count, Hb hemoglobin concentration, PLT platelet count. Source data are provided as a Source Data file.

Article Snippet: HCT116 cells were collected and incubated with an anti-ALK1 antibody (14745-1-AP, Proteintech) followed by R-PE-conjugated donkey anti-rabbit secondary antibody (711-116-152, Jackson ImmunoResearch).

Techniques: Inhibition, Injection, Staining, Activity Assay, Cell Counting, Concentration Assay

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Wholemount immunostaining of cerebral cortex slices for VE-cadherin and ALK1 from P31 control and Alk1 iΔAEC mice (25µg tamoxifen on P2). (B) Wholemount immunostaining for CD31 and ALK1 in cerebral cortex slices of P10 control and Alk1 iΔAEC mice expressing Efnb2 H2B-GFP (50µg tamoxifen on P2). A and V label arteries and veins, respectively. Dashed outlines in ALK1 images indicate artery position.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Immunostaining, Control, Expressing

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Photograph of P33 Alk1 iΔAEC mouse with blood on nose (arrowhead). (B) Photograph of blood present (arrowhead) in cage housing P81 Alk1 iΔAEC mice. (C) Gross pathological images demonstrating multi-focal hemorrhages (arrowheads) present in Alk1 iΔAEC mice brains but absent in control at P7. All mice administered 25µg tamoxifen on P2.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: Kaplan-Meier analysis showed that time to moribundity in Alk1 iΔAEC mice administered 100µg tamoxifen on P2 and P3 (red triangles) was significantly faster (median 23 days) than control (black circles) and Alk1 iΔAEC mice administered 25µg tamoxifen on P2 (N = 45-80 mice per condition).

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) MICROFIL casting of nasal cavity and cranial exterior of control (P16) and Alk1 iΔAEC (P19) mice revealed enlarged and tortuous vessels in Alk1 iΔAEC mouse (100µg tamoxifen on P2 & P3). Black and white arrowheads indicate the facial artery/vein pair and a tangle of blood vessels in the distal nasal vestibule, respectively. Mouse schematic created in BioRender.com. (B) Wholemount immunostaining for CD31 and Rosa26 Ai75 Cre reporter signal in nasal mucosa of P21 control ( Bmx-Cre ERT2 ; Alk1 fx/+ ) and Alk1 iΔAEC mice (25µg tamoxifen on P2). Blue dashed line indicates the most dorsal crease of the nasal cavity. Caudal (C), rostral (R), dorsal (D), and ventral (V) directions are indicated.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control, Immunostaining

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) MICROFIL casting of the brain demonstrating tortuous vessels in control and Alk1 iΔAEC mice at P24 (100µg tamoxifen on P2 & P3). (B) Wholemount immunostaining for CD31 in cerebral cortex slices of P31 control and Alk1 iΔAEC mice (75µg tamoxifen on P2).

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control, Immunostaining

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Wholemount immunostaining for CD31 and α-smooth muscle actin (αSMA) in nasal mucosa of P103 control and Alk1 iΔAEC mice (100µg tamoxifen per g body weight on P13 & P14). (B) Photomicrographs of wholemount immunostaining for CD31 and αSMA in cerebral cortex slices of P31 control and Alk1 iΔAEC mice (75µg tamoxifen on P2). Red arrows indicate gaps in smooth muscle coverage. Blue arrow indicates regions of misaligned smooth muscle cells. Brain schematic created in BioRender.com.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Immunostaining, Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Wholemount immunostaining for CD31 and expression of Efnb2 H2B-GFP arterial endothelial reporter in nasal mucosa of P13 control and Alk1 iΔAEC mice (100µg tamoxifen per g body weight on P13 & P14). Red boxed indicates location of inset images. Black dashed line indicates position of artery. (B) Wholemount immunostaining for CD31 and expression of Rosa26 Ai75 Cre recombinase reporter and Efnb2 H2B-GFP arterial endothelial reporter in nasal mucosa of P13 control and Alk1 iΔAEC mice (25µg tamoxifen on P2). Red boxed indicates location of inset images. (C) Wholemount immunostaining for CD31 and expression of Efnb2 H2B-GFP arterial endothelial reporter in cerebral cortex slices of P13 control and Alk1 iΔAEC mice (25µg tamoxifen on P2). Mouse schematic created in BioRender.com. A and V label arteries and veins, respectively.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Immunostaining, Expressing, Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Schematic demonstrating location of pial vessel imaging during in vivo arterial tone assay. (B) Representative images of pial vasculature in live P13 control and Alk1 iΔAEC mice (100µg tamoxifen on P2 & P3) visualized through an open cranial window. aCSF, artificial cerebrospinal fluid. (C) Quantification of changes in acetylcholine-induced vasodilation between control and Alk1 iΔAEC mice (unpaired Student’s t-test). (D) Quantification of changes in arterial tone between control and Alk1 iΔAEC mice (Mann-Whitney U test).

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Imaging, In Vivo, Control, MANN-WHITNEY