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

Journal: iScience

Article Title: DAB2IP suppresses invadopodia formation through destabilizing ALK by interacting with USP10 in breast cancer

doi: 10.1016/j.isci.2023.107606

Figure Lengend Snippet:

Article Snippet: Mouse anti-ALK , Proteintech , Cat# 60321-1-Ig, RRID: AB_2881432.

Techniques: Control, Virus, Recombinant, Magnetic Beads, CCK-8 Assay, Phospho-proteomics, Ab Array, Mutagenesis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Expressing, CRISPR, Knock-Out, Software

Journal: The Journal of investigative dermatology

Article Title: Inhibition of CtBP-Regulated Proinflammatory Gene Transcription Attenuates Psoriatic Skin Inflammation.

doi: 10.1016/j.jid.2021.06.029

Figure Lengend Snippet: Figure 1. The K5.CtBP1 mice exhibited a skin inflammation phenotype with elevated Th1 and Th17 cytokine expression. (a) Representative images of CtBP1 immunohistochemistry analysis of the WT and transgenic skin (n ¼ 3). Bar ¼ 40 mm. (b‒d) H&E staining of the transgenic skin showing (b) parakeratosis and (c) subcorneal microabscesses (black arrows) and of the volar skin showing (d) epidermal down growth in the K5.CtBP1 mice. Bar ¼ 40 mm. (e) Immunofluorescence staining of the WT and transgenic skin sections for CD45, Ly-6G, and CD4 (green) with K14 counterstaining (red) and for CD31 (green) with ALK1 counterstaining (red). Bar ¼ 50 mm. (f) Relative mRNA expression of the indicated genes (mean SD) in the WT and K5.CtBP1 skin (n ¼ 4). *P < 0.05, **P < 0.01, ***P < 0.001. K14, keratin 14; Th, T helper type; WT, wild type.

Article Snippet: The primary antibodies used in this study included mouse anti-CtBP1 and anti-CtBP2 (BD Biosciences, Franklin Lakes, NJ) and anti-p300 (Santa Cruz Biotechnology, Dallas, TX); rabbit anti-mouse F4/80 and CD45 (Cell Signaling Technology, Danvers, MA), rabbit anti- CD31 (Abcam, Cambridge, United Kingdom), anti-K14 (Fitzgerald, Acton, MA), and anti-GAPDH (New England Biolabs, Ipswich, MA); rat anti-CDH1 (Sigma-Aldrich, St. Loius, MO), anti-CD4 (BF Bio- sciences, Lahore, Pakistan), and anti-Ly-6G (eBioscience, San Diego, CA); and goat anti-mouse ALK1 and chicken anti-TGFb1 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Immunohistochemistry, Transgenic Assay, Staining

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A) Schematic representation of the experimental strategy used to delete Alk1 in mice (P4-P6). (B-D) P6 retina flat mount images labeled with IB4 (blue) and GFP (white) from Alk1 f/f Mfsd2a Cre ERT2 mTmG (B), Alk1 f/f Esm1 Cre ERT2 mTmG (C) and Alk1 f/f Bmx Cre ERT2 mTmG pups (D) injected with 100 μg Tx at P4 and dissected at P6. White arrows indicate AVMs. (E-G) Quantification of AVM number. n = 6-11 mice per group. Error bars: SEM. **** P-value < 0.0001, two-tailed unpaired t-test. (H-K) Vascular labeling with latex dye (red) of retinal and brain vessels in Alk1 f/f (H and I) and Alk1 f/f Mfsd2a Cre ERT2 (J and K) P6 pups. White arrows indicate AVMs. (L) Schematic representation of the experimental strategy used to delete Alk1 in mice (P1-P6). Arrowheads indicate injection of 100 μg Tx at P1, P2 and P3 in Alk1 f/f Esm1 and Bmx Cre ERT2 mTmG pups. (M and N) IB4 (blue) and GFP (white) staining of retinal flat mount from Alk1 f/f Esm1 Cre ERT2 mTmG (M) and Alk1 f/f Bmx Cre ERT2 mTmG (N). Scale bars: 500 μm (B-D, M-N), 200 μm (H and J), 2 mm (I and K).

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Labeling, Injection, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A-P) 100 μg Tx was injected intragastrically at P4 in Alk1 f/f , Alk1 f/f Mfsd2a Cre ERT2 , Alk1 f/f Esm1 Cre ERT2 and Alk1 f/f Bmx Cre ERT2 mTmG pups, and retinas were dissected at P6. IB4 (blue), GFP (white) and ALK1 (red) staining of retinal flat mounts. GFP and ALK1 staining shows non-overlapping expression. V: vein, A: artery, Scale bar: 200 μm

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Injection, Staining, Expressing

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A) Survival curves for Alk1 f/f Mfsd2aCre ERT2 , Alk1 f/f Esm1Cre ERT2 and Alk1 f/f BmxCre ERT2 mice injected with 100 μg Tx at P4. n = 8-10 mice/group. (B) Freshly dissected small intestines from P14 mice with the indicated genotypes after 100 μg Tx injection at P4. Alk1 f/f Esm1 Cre ERT2 mTmG mice displayed intestinal hemorrhage. (C and D) GFP (white) and VE-Cad (blue) staining of mesentery and gastrointestinal (GI) tract (C) and lacteals (D) from P14 Esm1 Cre ERT2 mTmG . 100 μg Tx was injected at P4. An arrow indicates Esm1 positive capillary ECs (C). (E and F) VE-Cad (blue) and GFP (white) staining of jejunum lacteals from P14 Alk1 f/f (E) and Alk1 f/f Esm1 Cre ERT2 mTmG (F). (G-J and M-P) 100 μg Tx was injected at P4 and dissected at P12. Vascular labeling with latex dye (red) of villi, GI tracts, retinas and brains in Alk1 f/f (G, I, M and O) and Alk1 f/f Esm1 Cre ERT2 (H, J, N and P) P12 pups. (K and L) 100 μg Tx was injected at P4 and dissected at P12 (K and L). IB4 (blue) and GFP (white) staining of retinal flat mounts from Esm1 Cre ERT2 mTmG (K) and Alk1 f/f Esm1 Cre ERT2 mTmG (L) P12 mice. An arrow indicates vascular malformations (J, L and N). A: artery, V: vein, Scale bars: 1 cm (B), 400 μm (C), 1 mm (I-J and M-P), 500 μm (K-N), 200 μm (G and H), 25 μm (D-F).

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Injection, Staining, Labeling

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A-B) IB4 (Magenta) and ALK1 (white) staining of retinal flat mounts from Alk1 f/f (A) and Alk1 f/f Mfsd2a Cre ERT2 (B) pups injected with 100 μg Tx at P4 and dissected at P6. (C-F) Higher magnification of insets in A and B. GOLPH4 (green) and DAPI (blue) staining of retina flat mounts. Red arrows indicate the blood flow direction. (C’-F’) Background images from and corresponding polarity vectors (black arrows). (G) The polarity axis of each cell was defined as the angle between the direction of blood flow and the cell polarity axis, defined by a vector drawn from the center of the cell nucleus to the center of the Golgi apparatus. (H) Angular histograms showing the distribution of polarization angles of ECs in the artery, vein and capillaries from Alk1 f/f and artery, vein, capillary and AVM from Alk1 f/f Mfsd2a Cre ERT2 mouse retinas. n = 7-11 retinas. (I) PI box plots of ECs from artery, vein and capillary from Alk1 f/f and artery, vein, capillary and AVM from Alk1 f/f Mfsd2a Cre ERT2 P6 retinas. n = 7-11 retinas. (J and K) IB4 (gray) staining of retinal flat mounts from Alk1 f/f Mfsd2a Cre ERT2 pups injected with 100 μg at P4 and dissected after 24 h (P5) (J) and 36 h (P5.5) (K). (L) Angular histograms showing the distribution of polarization angles of ECs in the artery, vein and capillary from Alk1 f/f and Alk1 f/f Mfsd2a Cre ERT2 P5 retinas at 24 h after Tx injection. (M) PI box plots of ECs from artery, vein and capillary from Alk1 f/f and Alk1 f/f Mfsd2a Cre ERT2 retinas at 24 h after Tx injection. n = 5-8 retinas/group. Error bars: SEM. **P-value < 0.01, ***P-value < 0.001, ns: nonsignificant, two-tailed unpaired t-test. Scale bars: 100 μm (A-B), 20 μm (C-F) and 500 μm (J-K)

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Staining, Injection, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A-B) Representative images of wound-healing assays after 18 h showing polarity angles of HUVECs transfected with Control ( siCon ) (A) or ALK1 ( siALK1 ) (B) siRNAs under static conditions and immunolabeled with phalloidin(red), GM130 (green), and DAPI (blue). (E-F) Representative images of wound-healing assays showing polarity angles of siCon (E) or siALK1 (F) HUVECs with 18 h exposure to laminar shear stress (LSS) at 15 dynes/cm 2 . Left panels are upstream and right panels are downstream of flow. (C-D and G-H) Angular histograms showing polarization angles of siCon (C and G) or siALK1 ECs (D and H) at 18 h after scratch with (G-H) or without (C-D) LSS. Left is upstream and right is downstream of flow (G and H). (I) PI box plots of upstream (left) scratch areas from siCon or siALK1 transfected HUVECs at 18 h after with or without LSS. (C-I) n=6-8 images from 3 independent experiments. Error bars: SEM. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, two-tailed unpaired t-test. (J) Representative time lapse images of siCon or siALK1 HUVECs stably transduced with PH-AKT-mClover3 and plasma membrane targeting sequence of LCK-mRuby3. HUVEC monolayers in microfluidic chambers were exposed to 12 dynes/cm 2 LSS under the microscope. 5 min (static) and 12 min (LSS) images were selected from the movies. The surface is color-coded by the value of PH-AKT intensity. (K) Local activation of PI3K was quantified by image analysis. PH-AKT intensity was normalized with average static intensity at each time point. 0 - 5 min: static and 5 - 24.5 min: LSS, n= 61, 41 cells from 3 independent experiments, Error bar: SEM. ***P-value < 0.001, two-tailed unpaired t-test. Scale bars: 50 μm (A-B and E-F), 20 μm (J).

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Transfection, Immunolabeling, Two Tailed Test, Stable Transfection, Transduction, Sequencing, Microscopy, Activation Assay

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A-C) IB4 (Magenta) and ITGB1(A, white), ITGA5 (B, white) or ITGAV (C, white) staining of retinal flat mounts from P8 Alk1 f/f Mfsd2a Cre ERT2 pups. (D) VEGFR2 immunoprecipitation in siCon or siALK1 HUVECs and western blot analysis for ITGB1, ITGA5 and ITGAV. VEGFR2, ITGB1, ITGA5, ITGAV, ALK1 and β-actin expression from the total cell lysates are shown as loading controls. (E) Quantification of ITGB1, ITGA5 or ITGAV levels normalized to VEGFR2 from immunoprecipitation. **P<0.01, ***P-value < 0.001, two-tailed unpaired t-test. (F) Experimental strategy to assess the effects of integrin inhibitors in Alk1 deleted retinas. Arrowheads indicate the time course of Tx (100 μg) and Cilengitide (5mg/kg), ATN161 (5mg/kg) or vehicle administration. (G-I and K-M) IB4 staining of P6 retinal flat mounts from Alk1 f/f Mfsd2a Cre ERT2 (G-I) or Alk1 f/f CDH5 Cre ERT2 (K-M) injected with Cilengitide (H and L) or ATN161 (I and M) at P4 and P5. (J and N) Quantification of the AVM number. Each dot represents one retina. n = 7-16 retinas per group. Error bars: SEM. ***P-value < 0.001, One-way ANOVA with Holm-Sidak test. (O-R) IB4 (Magenta), Alk1 (white), GOLPH4 (green) and DAPI (blue) staining of retina flat mounts from Alk1 f/f (O), Alk1 f/f Mfsd2a Cre ERT2 (P), Cilengitide (Q) or ATN161 (R) injected Alk1 f/f Mfsd2a Cre ERT2 pups. A: artery, V: vein, (S) PI box plots of ECs from artery and vein from Alk1 f/f , Alk1 f/f Mfsd2a Cre ERT2 , Cilengitide or ATN161 injected Alk1 f/f Mfsd2a Cre ERT2 retinas. n=5-8 retinas/group. Error bars: SEM. *P-value < 0.05, **P-value < 0.01, ns: nonsignificant, One-way ANOVA with Holm-Sidak test. Scale bars: 500 μm (A-C, G-I and K-M), 20 μm (O-R).

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Staining, Immunoprecipitation, Western Blot, Expressing, Two Tailed Test, Injection

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A-C) IB4 (Magenta) and ITGB1(A, white), ITGA5 (B, white) or ITGAV (C, white) staining of retinal flat mounts from P8 Alk1 f/f pups. A scale bar: 500 μm (A-C)

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Staining

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A) Western blot analysis of HUVECs transfected with control and ALK1 siRNAs followed by 18 h exposure to LSS (15 dynes/cm 2 ). (B) Quantification of ITGB1, ITGA5, ITGAV, YAP or TAZ levels normalized to β-actin. *P<0.05, **P<0.01, ***P<0.001, two-tailed unpaired t-test. (C-F) YAP and TAZ (green), ALK1 (gray), IB4 (red), DAPI (blue) staining of retinal flat mounts from P8 Alk1 f/f (C-D) or Alk1 f/f Mfsd2aCre ERT2 (E-F) pups. A scale bar: 20 μm (A-F) (G) YAP or TAZ (green) and Alk1 (red) staining of siCon and siALK1 HUVECs. A scale bar: 50 μm. (H) Quantification of YAP and TAZ localization from siCon and siALK1 transfected HUVECs. ***P<0.001, n = 3 independent experiments. Multiple comparisons with Holm-Sidak test.

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Western Blot, Transfection, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A) YAP and TAZ staining of siCon , ALK1 , SMAD4 or ENG siRNAs transfected HUVECs treated with DMSO or Verteporfin (VP, 5 μM) for 6 h. Nuclear YAP/TAZ localization in siALK1 , siSMAD4 or siENG ECs is blocked by VP treatment. A scale bar: 50 μm. (B) Experimental strategy to assess the effects of YAP/TAZ inhibition in EC specific Alk1 deleted vasculature. Arrowheads indicate the time course of Tx (100 μg) and VP (50mg/kg) or vehicle administration. (C) IB4 staining of P6 retinal flat mounts from VP injected Alk1 f/f , Alk1 f/f CDH5 Cre ERT2 or Alk1 f/f Mfsd2a Cre ERT2 mice. (D) Stereomicroscopy images of vehicle or VP injected Alk1 f/f Mfsd2a Cre ERT2 retinas. (E) Quantification of the AVM number/retina. Each dot represents one retina. n = 6-8 retinas per group. Error bars: SEM. ***P-value < 0.001, two-tailed unpaired t-test. (F) Angular histograms showing polarization angles of artery and vein from Alk1 f/f Mfsd2a Cre ERT2 with VP. (G) PI box plots of Alk1 f/f Mfsd2a Cre ERT2 with vehicle or VP. n=5-6 retinas, Error bars: SEM, ***P-value < 0.001, ns: nonsignificant, two-tailed unpaired t-test. Scale bars: 50 μm (A), 500 μm (C), 300 μm (D)

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Staining, Transfection, Inhibition, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A) YAP and TAZ staining for control, ALK1 siRNAs transfected HUVECs treated with PBS, Cilengitide (Cil, 5 μM) or ATN161 (ATN, 5 μM) for 12 h. Nuclear YAP/TAZ localization in siALK1 ECs is blocked by Cilengitide and ATN161 treatment. (B) Quantification of YAP and TAZ localization from siCon and siALK1 transfected HUVECs. ***P<0.001, n = 3 independent experiments. Error bars: SEM. ***P-value < 0.001, ns: nonsignificant, Multiple comparisons with Holm-Sidak test. (C) YAP and TAZ (green), ALK1 (white), IB4 (red) and DAPI (blue) staining of retinal flat mounts from Cilengitide or ATN161 injected Alk1 f/f Mfsd2a Cre ERT2 P6 mice. (D) A model for ALK1-integrin-YAP/TAZ signaling in maintenance of vascular quiescence. In quiescence, ALK1 signaling represses PI3K activation downstream of integrin-VEGFR2 signaling, through inhibition of YAP/TAZ expression and localization. ALK1 deletion results in increased integrin-VEGFR2 signaling, and consequently in excessive YAP/TAZ expression and localization to the nucleus, thereby inducing vascular defects. Blocking integrin-ECM interaction with integrin inhibitors or YAP/TAZ localization with YAP/TAZ inhibitor rescues vascular malformations in Alk1 deficient mice. Scale bars: 50 μm (A), 20 μm (C)

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Staining, Transfection, Injection, Activation Assay, Inhibition, Expressing, Blocking Assay

Journal: bioRxiv

Article Title: Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2021.05.06.442985

Figure Lengend Snippet: (A) YAP and TAZ staining for control, ALK1 siRNAs transfected HUVECs treated with PBS, Wortmannin (100 nM) for 12 h. A scale bar: 50 μm

Article Snippet: For immunostaining: IB4 ([IsolectinB4] #121412, 10 μg/mL; Life Technologies), GFP Polyclonal Antibody, Alexa Fluor 488 (#A-21311, 1:1000; Invitrogen), GOLPH4 (#ab28049, 1:400; abcam), anti-YAP (#14074, 1:300; Cell Signaling), anti-TAZ (#HPA007415, 1:300 Sigma), mouse anti-ALK1 (#AF770, 1:300; R&D) human anti-ALK1(#AF370, 1:300; R&D), VE-Cadherin (#555289, 1:200; BD) GM-130 (#610822, 1:500; BD), DAPI (#D1306, 1:1000; Life Technologies), anti-integrin β1 Alexa Fluor 647 (#303047,1:500; BioLegend), anti-integrin α5 and αv (From Martin A Schwartz) For western blotting: anti-ALK1 (7R-49334, 1:1000; Fitzgerald), anti-integrin β1 (#34971, Cell Signaling), anti-integrin α5 and αv, anti-VEGFR2 (#9698, Cell Signaling), β-actin (#A1978 1:3000; Sigma), anti-YAP (#14074, 1:1000; Cell Signaling), anti-TAZ (#HPA007415, 1:2000 Sigma).

Techniques: Staining, Transfection