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goat anti alk1 (R&D Systems)

Name: goat anti alk1
Brand: R&D Systems
Rating: 93 (15 reviews)

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R&D Systems goat anti alk1

(A) Wholemount immunostaining of cerebral cortex slices for VE-cadherin and <t>ALK1</t> 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.

Goat Anti Alk1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/goat anti alk1/product/R&D Systems
Average 93 stars, based on 15 article reviews

goat anti alk1 - by Bioz Stars, 2026-02

93/100 stars

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1) Product Images from "Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage"

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

Journal: bioRxiv

doi: 10.1101/2024.11.25.622742

(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.

Figure Legend 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.

Techniques Used: Immunostaining, Control, Expressing

(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.

Figure Legend 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.

Techniques Used: Control

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).

Figure Legend 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).

Techniques Used: Control

(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.

Figure Legend 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.

Techniques Used: Control, Immunostaining

(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).

Figure Legend 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).

Techniques Used: Control, Immunostaining

(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.

Figure Legend 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.

Techniques Used: Immunostaining, Control

(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.

Figure Legend 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.

Techniques Used: Immunostaining, Expressing, Control

(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).

Figure Legend 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).

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

Image Search Results

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.

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).

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.

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).

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.

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.

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).

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

GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

Techniques: Expressing, Transfection, Isolation, Western Blot, MANN-WHITNEY

GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

Techniques: Transfection, MANN-WHITNEY, Western Blot, Expressing

$Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).$

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

Techniques: Expressing, Injection

Graphical representation of the role of GATA6 in coordinating cross-talk between BMP10 and oxidative stress axis in PAH. GATA6 is an activator of anti-oxidant enzymes and its deficiency in PAEC and PASMC induces oxidative stress and mitochondrial dysfunction. BMP10 induces expression of GATA6 through the ALK1, BMPRII, ENG and ERK pathway. GATA6, in turn, transcriptionally activates BMP receptors in PAEC. Endothelial GATA6 regulates PASMC function via paracrine factors. TGFβ2 secreted by GATA6 deficient PAEC induces PASMC proliferation. Administration of dimethyl fumarate (DMF) to mice with endothelial Gata6 loss restores expression of BMP receptors, resolves oxidative stress, and reverses PH.

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: Graphical representation of the role of GATA6 in coordinating cross-talk between BMP10 and oxidative stress axis in PAH. GATA6 is an activator of anti-oxidant enzymes and its deficiency in PAEC and PASMC induces oxidative stress and mitochondrial dysfunction. BMP10 induces expression of GATA6 through the ALK1, BMPRII, ENG and ERK pathway. GATA6, in turn, transcriptionally activates BMP receptors in PAEC. Endothelial GATA6 regulates PASMC function via paracrine factors. TGFβ2 secreted by GATA6 deficient PAEC induces PASMC proliferation. Administration of dimethyl fumarate (DMF) to mice with endothelial Gata6 loss restores expression of BMP receptors, resolves oxidative stress, and reverses PH.

Techniques: Expressing

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.

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

Figure 1. ALK1 is a target of miR-31-5p in human colonic epithelial cells. (A) Correlation of expression in the colonic mucosa of CD patients (N ¼ 10) between miR-31-5p (reads per million miRNAs mapped, RPMMM) and predicted targets of miR-31-5p (E2F2, ALK1, PRKAB1, and DCBLD2; DESeq normalized). (B) Association between the expression of miR-31-5p and the expression of ALK1 or E2F2 in isolated colonic epithelial cells (N ¼ 27). Gene expression was quantiﬁed by qPCR and samples were split into 3 equally sized groups (N ¼ 9 per group) according to the relative miR-31-5p expression levels. (C) Representative blot of ALK1 expression in the colonic tissue of NIBD and CD patients (left). Correlation between ALK1 protein expression and miR-31-5p in the colonic mucosa (right, N ¼ 18). (D) ALK1 expression by immunohistochemistry in the colonic mucosa of NIBD controls and CD patients. The values shown at the bottom are the matched miR-31-5p expression level normalized to NIBD. (E) 3’UTR reporter assay for ALK1 in the presence or absence of 30 nmol/L miRNA mimics for hsa-miR- 31-5p (m31), hsa-miR-122a-5p (m122), or hsa-miR-215-5p (m215) or negative control mimics (NC). N ¼ 6 per group. (F) Schematic representation of the miR-31-5p binding sites in the reporter plasmid. (G) Site-directed mutagenesis assay with 10 nmol/L of m31 or NC mimics (N ¼ 6 per group). All correlation values were calculated by the Spearman correlation coefﬁcient. Each gene expression was normalized to GAPDH (ALK1, E2F2) or RNU48 (miR-31-5p). *P < .05. P values were determined by the Kruskal–Wallis test, followed by the Dunn multiple comparison test. Mut, mutation; NC, negative control mimics.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 1. ALK1 is a target of miR-31-5p in human colonic epithelial cells. (A) Correlation of expression in the colonic mucosa of CD patients (N ¼ 10) between miR-31-5p (reads per million miRNAs mapped, RPMMM) and predicted targets of miR-31-5p (E2F2, ALK1, PRKAB1, and DCBLD2; DESeq normalized). (B) Association between the expression of miR-31-5p and the expression of ALK1 or E2F2 in isolated colonic epithelial cells (N ¼ 27). Gene expression was quantiﬁed by qPCR and samples were split into 3 equally sized groups (N ¼ 9 per group) according to the relative miR-31-5p expression levels. (C) Representative blot of ALK1 expression in the colonic tissue of NIBD and CD patients (left). Correlation between ALK1 protein expression and miR-31-5p in the colonic mucosa (right, N ¼ 18). (D) ALK1 expression by immunohistochemistry in the colonic mucosa of NIBD controls and CD patients. The values shown at the bottom are the matched miR-31-5p expression level normalized to NIBD. (E) 3’UTR reporter assay for ALK1 in the presence or absence of 30 nmol/L miRNA mimics for hsa-miR- 31-5p (m31), hsa-miR-122a-5p (m122), or hsa-miR-215-5p (m215) or negative control mimics (NC). N ¼ 6 per group. (F) Schematic representation of the miR-31-5p binding sites in the reporter plasmid. (G) Site-directed mutagenesis assay with 10 nmol/L of m31 or NC mimics (N ¼ 6 per group). All correlation values were calculated by the Spearman correlation coefﬁcient. Each gene expression was normalized to GAPDH (ALK1, E2F2) or RNU48 (miR-31-5p). *P < .05. P values were determined by the Kruskal–Wallis test, followed by the Dunn multiple comparison test. Mut, mutation; NC, negative control mimics.

Article Snippet: Western blot analyses were performed on whole-cell extracts.50 Goat anti-human ALK1 antibody (AF370-SP) and goat IgG horseradish-peroxidase–conjugated antibody (HAF109) were purchased from R&D Systems.

Techniques: Expressing, Isolation, Gene Expression, Immunohistochemistry, Reporter Assay, Negative Control, Binding Assay, Plasmid Preparation, Mutagenesis, Comparison

Figure 2. Decreased ALK1 expression is associated with reduced NOTCH activity and NOTCH target gene expression in the colonic epithelial cells of CD patients. (A) Representative blot (left) and the difference of JAG1 and NOTCH intra- cellular domain (NICD) protein expression between NIBD and CD patients (right). (B) BMP9 concentration in the serum of NIBD controls (N ¼ 17) and CD patients (N ¼ 23). (C) NOTCH target gene expression in colonic epithelial cells from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). (D) NOTCH target gene expression in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. N ¼ 6 per group. Each gene expression was normalized to (C) GAPDH or (D) RPLP0. *P < .05, **P < .01, and ***P < .001. P values were determined by the (A–C) Mann–Whitney test or the (D) Friedman test followed by the Dunn multiple comparison test.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 2. Decreased ALK1 expression is associated with reduced NOTCH activity and NOTCH target gene expression in the colonic epithelial cells of CD patients. (A) Representative blot (left) and the difference of JAG1 and NOTCH intra- cellular domain (NICD) protein expression between NIBD and CD patients (right). (B) BMP9 concentration in the serum of NIBD controls (N ¼ 17) and CD patients (N ¼ 23). (C) NOTCH target gene expression in colonic epithelial cells from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). (D) NOTCH target gene expression in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. N ¼ 6 per group. Each gene expression was normalized to (C) GAPDH or (D) RPLP0. *P < .05, **P < .01, and ***P < .001. P values were determined by the (A–C) Mann–Whitney test or the (D) Friedman test followed by the Dunn multiple comparison test.

Techniques: Expressing, Activity Assay, Targeted Gene Expression, Concentration Assay, Derivative Assay, Cell Culture, Gene Expression, MANN-WHITNEY, Comparison

Figure 3. Expression of miR-31-5p and ALK1 in primary- cultured colonic epithelial monolayers derived from NIBD controls and CD patients. N ¼ 6 per group. Each gene expression was normalized to RNU48 (miR-31-5p) or GAPDH (ALK1). Statistical signiﬁcance was determined by the Mann–Whitney test.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 3. Expression of miR-31-5p and ALK1 in primary- cultured colonic epithelial monolayers derived from NIBD controls and CD patients. N ¼ 6 per group. Each gene expression was normalized to RNU48 (miR-31-5p) or GAPDH (ALK1). Statistical signiﬁcance was determined by the Mann–Whitney test.

Techniques: Expressing, Cell Culture, Derivative Assay, Gene Expression, MANN-WHITNEY

Figure 4. BMP9–ALK1 signaling restricts the stemness of human colonic IECs. (A) EdU assay in NIBD patient-derived colonic epithelial cell monolayers (N ¼ 4–8 per group). Expanded cells were cultured in EM in the presence or absence of BMP9 and ALK1–Fc chimera protein. Red, EdU; blue, Hoechst 33342. (B) Proliferation- and stemness-related gene expression in NIBD patient-derived colonic epithelial cell monolayers (N ¼ 6 per group). (C) Proliferation- and stemness-related gene expression in colonic epithelial cells isolated from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). (D) Representative immunohistochemical images of OLFM4 expression in the colonic crypts of NIBD controls (left) and CD patients (right). The percentage of OLFM4 staining area in colonic crypts was compared between CD patients and NIBD controls (N ¼ 4 per group). Each gene expression was normalized to (B) RPLP0 or (C) GAPDH. *P < .05, **P < .01, and ***P < .001. P values were determined by the (A) Kruskal–Wallis test or the (B) Friedman test followed by the Dunn multiple comparisons test, or the (C and D) Mann–Whitney test.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 4. BMP9–ALK1 signaling restricts the stemness of human colonic IECs. (A) EdU assay in NIBD patient-derived colonic epithelial cell monolayers (N ¼ 4–8 per group). Expanded cells were cultured in EM in the presence or absence of BMP9 and ALK1–Fc chimera protein. Red, EdU; blue, Hoechst 33342. (B) Proliferation- and stemness-related gene expression in NIBD patient-derived colonic epithelial cell monolayers (N ¼ 6 per group). (C) Proliferation- and stemness-related gene expression in colonic epithelial cells isolated from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). (D) Representative immunohistochemical images of OLFM4 expression in the colonic crypts of NIBD controls (left) and CD patients (right). The percentage of OLFM4 staining area in colonic crypts was compared between CD patients and NIBD controls (N ¼ 4 per group). Each gene expression was normalized to (B) RPLP0 or (C) GAPDH. *P < .05, **P < .01, and ***P < .001. P values were determined by the (A) Kruskal–Wallis test or the (B) Friedman test followed by the Dunn multiple comparisons test, or the (C and D) Mann–Whitney test.

Techniques: EdU Assay, Derivative Assay, Cell Culture, Gene Expression, Isolation, Immunohistochemical staining, Expressing, Staining, MANN-WHITNEY

Figure 5. BMP9–ALK1 signaling is associated with epithelial cell differentiation toward colonocytes. (A) Lineage-speciﬁc gene expression in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. Each gene expression was normalized to RPLP0 (N ¼ 6 per group). (B) CA1 protein expression in NIBD patient-derived colonic epithelial monolayers (N ¼ 4 per group). (C) Colonocyte marker expression in colonic epithelial cells isolated from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). Each gene expression was normalized to GAPDH. (D) CA1 expression by immunohistochemistry in the colonic mucosa of NIBD controls (left) and CD patients (right). (E) CA1 protein expression in the colonic mucosa of NIBD controls and CD patients (N ¼ 5 per group). *P < .05, **P < .01, ***P < .001, and ****P < .0001. P values were determined by the (A and B) Friedman test followed by the Dunn multiple comparison test, or the (C and E) Mann–Whitney test.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 5. BMP9–ALK1 signaling is associated with epithelial cell differentiation toward colonocytes. (A) Lineage-speciﬁc gene expression in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. Each gene expression was normalized to RPLP0 (N ¼ 6 per group). (B) CA1 protein expression in NIBD patient-derived colonic epithelial monolayers (N ¼ 4 per group). (C) Colonocyte marker expression in colonic epithelial cells isolated from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). Each gene expression was normalized to GAPDH. (D) CA1 expression by immunohistochemistry in the colonic mucosa of NIBD controls (left) and CD patients (right). (E) CA1 protein expression in the colonic mucosa of NIBD controls and CD patients (N ¼ 5 per group). *P < .05, **P < .01, ***P < .001, and ****P < .0001. P values were determined by the (A and B) Friedman test followed by the Dunn multiple comparison test, or the (C and E) Mann–Whitney test.

Techniques: Cell Differentiation, Gene Expression, Derivative Assay, Cell Culture, Expressing, Marker, Isolation, Immunohistochemistry, Comparison, MANN-WHITNEY

Figure 7. BMP9–ALK1 signaling enhances human colonic IEC barrier integrity. (A) Gene expression of junctional proteins in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. Each gene expression was normalized to RPLP0 (N ¼ 6 per group). (B) Gene expression of tight junction proteins in colonic epithelial cells isolated from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). Each gene expression was normalized to GAPDH. (C) Epithelial permeability assay in NIBD patient-derived colonic epithelial cells cultured on a collagen scaffold. TEER was measured over time (N ¼ 3 per group). (D) Cells were stimulated with BMP9 in EM in the presence or absence of ALK1–Fc chimera protein or cultured in DM on day 4. The changes in TEER between days 4 and 6 are shown as DTEER% (N ¼ 4–8 per group). *P < .05, **P < .01, and ***P < .001. P values were determined by the (A and D) Kruskal–Wallis test followed by the Dunn multiple comparison test, and the (B) Mann–Whitney test.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 7. BMP9–ALK1 signaling enhances human colonic IEC barrier integrity. (A) Gene expression of junctional proteins in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. Each gene expression was normalized to RPLP0 (N ¼ 6 per group). (B) Gene expression of tight junction proteins in colonic epithelial cells isolated from CD patients (N ¼ 15) and NIBD controls (N ¼ 12). Each gene expression was normalized to GAPDH. (C) Epithelial permeability assay in NIBD patient-derived colonic epithelial cells cultured on a collagen scaffold. TEER was measured over time (N ¼ 3 per group). (D) Cells were stimulated with BMP9 in EM in the presence or absence of ALK1–Fc chimera protein or cultured in DM on day 4. The changes in TEER between days 4 and 6 are shown as DTEER% (N ¼ 4–8 per group). *P < .05, **P < .01, and ***P < .001. P values were determined by the (A and D) Kruskal–Wallis test followed by the Dunn multiple comparison test, and the (B) Mann–Whitney test.

Techniques: Gene Expression, Derivative Assay, Cell Culture, Isolation, Permeability, Comparison, MANN-WHITNEY

Figure 8. Decreased colonic ALK1 is associated with a poor clinical outcome in CD patients. (A) ALK1 expression was quantiﬁed in colonic biopsy samples obtained from NIBD controls (N ¼ 10) and CD patients (N ¼ 28) by qPCR. (B) ALK1 expression in the colonic mucosa of CD patients at the time of surgery and after surgery (N ¼ 5 per group). (C) Percentages of CD patients who were diagnosed as CD before and after age 40 years in low-ALK1 (N ¼ 15) and hi-ALK1 (N ¼ 13) CD subsets. (D) Kaplan–Meier survival analysis to evaluate the impact of colonic ALK1 expression on endoscopic relapse in patients with CD (N ¼ 12 for low-ALK1 and 9 for hi-ALK1 CD subgroups). ALK1 expression was normalized to GAPDH. ***P < .001. P values were determined by the (A) Mann–Whitney test, (B) Wilcoxon test, and (D) log-rank test.

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

doi: 10.1016/j.jcmgh.2020.06.005

Figure Lengend Snippet: Figure 8. Decreased colonic ALK1 is associated with a poor clinical outcome in CD patients. (A) ALK1 expression was quantiﬁed in colonic biopsy samples obtained from NIBD controls (N ¼ 10) and CD patients (N ¼ 28) by qPCR. (B) ALK1 expression in the colonic mucosa of CD patients at the time of surgery and after surgery (N ¼ 5 per group). (C) Percentages of CD patients who were diagnosed as CD before and after age 40 years in low-ALK1 (N ¼ 15) and hi-ALK1 (N ¼ 13) CD subsets. (D) Kaplan–Meier survival analysis to evaluate the impact of colonic ALK1 expression on endoscopic relapse in patients with CD (N ¼ 12 for low-ALK1 and 9 for hi-ALK1 CD subgroups). ALK1 expression was normalized to GAPDH. ***P < .001. P values were determined by the (A) Mann–Whitney test, (B) Wilcoxon test, and (D) log-rank test.

Techniques: Expressing, MANN-WHITNEY

Effects of BMP-9 on the expression of BMP receptors. (A) Expression of BMPR-IA, BMPR-IB, BMPR-II and ALK1 receptors in cells treated with BMP-9 (100 ng/ml) using ELISA. **P<0.01 and *P<0.05, vs. other receptors. (B) Immunofluorescence of (a) ALKI receptor, (b) counterstaining with DAPI and (c) the two stains merged in cells without BMP-9 treatment. Immunofluorescence of (d) ALKI receptor (e) counterstaining with DAPI and (f) the two stains merged in cells treated with BMP-9 (100 ng/ml). Positive expression of ALK1 receptor is indicated by white arrows. BMP-9, bone morphogenetic protein-9; BMPR, BMP receptor; ALK1, anaplastic lymphoma kinase 1; OD, optical density.

Journal: Molecular Medicine Reports

Article Title: Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

doi: 10.3892/mmr.2016.5803

Figure Lengend Snippet: Effects of BMP-9 on the expression of BMP receptors. (A) Expression of BMPR-IA, BMPR-IB, BMPR-II and ALK1 receptors in cells treated with BMP-9 (100 ng/ml) using ELISA. **P<0.01 and *P<0.05, vs. other receptors. (B) Immunofluorescence of (a) ALKI receptor, (b) counterstaining with DAPI and (c) the two stains merged in cells without BMP-9 treatment. Immunofluorescence of (d) ALKI receptor (e) counterstaining with DAPI and (f) the two stains merged in cells treated with BMP-9 (100 ng/ml). Positive expression of ALK1 receptor is indicated by white arrows. BMP-9, bone morphogenetic protein-9; BMPR, BMP receptor; ALK1, anaplastic lymphoma kinase 1; OD, optical density.

Article Snippet: The cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, and were then incubated overnight at 4°C with the following primary antibody: Polyclonal goat anti-mouse ALK1 immunoglobulin (Ig) G (1:100; cat. no. 770-MA; R&D Systems, Inc., Minneapolis, MN, USA), quenched with glycine (0.1 M) for 1 h, incubated with secondary antibodies (polyclonal donkey anti-goat IgG; 1:200; cat. no. NL003; R&D Systems, Inc.) for 2 h at room temperature, and then washed three times with PBS.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Effects of BMP-9 and ALK1 receptor on the cell signal transduction pathway. (A) Western blot of the phosphorylation of ERK1/2 in cells treated with BMP-9 (100 ng/ml) and (B) quantification (*P<0.01, vs. 0 min). (C) Western blot of the phosphorylation of Smad2 in cells treated with BMP-9 (100 ng/ml) and (D) quantification (P>0.01, vs. 0 min). (E) Western blot of the phosphorylation of ERK1/2 in cells pre-treated with siRNA-ALK1 and (F) quantification (*P<0.01, vs. BMP-9). ERK1/2, extracellular signal-regulated kinase 1/2; p-ERK1/2, phosphorylated ERK1/2; Smad 2, small mothers against decapentaplegic 2; p-Smad2, phosphorylated Smad 2; BMP-9, bone morphogenetic protein-9; ALK1, anaplastic lymphoma kinase 1; siRNA, small interfering RNA.

Journal: Molecular Medicine Reports

Article Title: Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

doi: 10.3892/mmr.2016.5803

Figure Lengend Snippet: Effects of BMP-9 and ALK1 receptor on the cell signal transduction pathway. (A) Western blot of the phosphorylation of ERK1/2 in cells treated with BMP-9 (100 ng/ml) and (B) quantification (*P<0.01, vs. 0 min). (C) Western blot of the phosphorylation of Smad2 in cells treated with BMP-9 (100 ng/ml) and (D) quantification (P>0.01, vs. 0 min). (E) Western blot of the phosphorylation of ERK1/2 in cells pre-treated with siRNA-ALK1 and (F) quantification (*P<0.01, vs. BMP-9). ERK1/2, extracellular signal-regulated kinase 1/2; p-ERK1/2, phosphorylated ERK1/2; Smad 2, small mothers against decapentaplegic 2; p-Smad2, phosphorylated Smad 2; BMP-9, bone morphogenetic protein-9; ALK1, anaplastic lymphoma kinase 1; siRNA, small interfering RNA.

Techniques: Transduction, Western Blot, Phospho-proteomics, Small Interfering RNA

Effects of the ALK1 receptor and ERK1/2 pathways on BMP-9-induced osteoclast differentiation (A) TRAP staining of cells (a) induced by BMP-9 (100 ng/ml) and RANKL (100 ng/ml), and (b) pre-transfected with siRNA-ALK1 or (c) cultured with U0126 (1,000 nmol/l) prior to BMP-9+RANKL. (Phase contrast microscope; magnification, ×20). White arrows indicate TRAP-positive cells. (B) Protein expression of CTR, determined using an enzyme-linked immunosorbent assay, in cells treated with BMP-9 (100 ng/ml) and RANKL (100 ng/ml), transfectecd with siRNA-ALK1 or treated with U0126 (1,000 nmol/l). *P<0.01, vs. BMP-9+RANKL group. BMP-9, bone morphogenetic protein-9; ALK1, anaplastic lymphoma kinase 1; RANKL, receptor activator for nuclear factor-κb ligand; CTR, calcitonin receptor; siRNA, small interfering RNA; OD, optical density; TRAP, tartrate-resistant acid phosphatase.

Journal: Molecular Medicine Reports

Article Title: Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

doi: 10.3892/mmr.2016.5803

Figure Lengend Snippet: Effects of the ALK1 receptor and ERK1/2 pathways on BMP-9-induced osteoclast differentiation (A) TRAP staining of cells (a) induced by BMP-9 (100 ng/ml) and RANKL (100 ng/ml), and (b) pre-transfected with siRNA-ALK1 or (c) cultured with U0126 (1,000 nmol/l) prior to BMP-9+RANKL. (Phase contrast microscope; magnification, ×20). White arrows indicate TRAP-positive cells. (B) Protein expression of CTR, determined using an enzyme-linked immunosorbent assay, in cells treated with BMP-9 (100 ng/ml) and RANKL (100 ng/ml), transfectecd with siRNA-ALK1 or treated with U0126 (1,000 nmol/l). *P<0.01, vs. BMP-9+RANKL group. BMP-9, bone morphogenetic protein-9; ALK1, anaplastic lymphoma kinase 1; RANKL, receptor activator for nuclear factor-κb ligand; CTR, calcitonin receptor; siRNA, small interfering RNA; OD, optical density; TRAP, tartrate-resistant acid phosphatase.

Techniques: Staining, Transfection, Cell Culture, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, Small Interfering RNA

A. qRT-PCR analysis of P17 retinas from pups subjected to OIR revealed the expression of transcripts corresponding to components of the canonical BMP9 signaling ( n = 4 control mice and 4 OIR mice). B. Alk1 immunofluorescence staining of OIR retinas at P17 shows specific expression of Alk1 in blood vessels. Arrowheads show vascular tufts. Scale Bar: 20 μm. C. BMP9 ELISA of plasma from mice subjected to OIR collected at P12 (after vaso-obliteration) ( n = 3 control and n = 3 OIR) and P17 (neovascularization phase) ( n = 3 control and n = 4 OIR). D. qRT-PCR of choroid-sclera complexes subjected to laser-CNV of genes involved in BMP9 signaling ( n = 4 mice per group). All histograms represent mean ± standard error of the mean. * P < 0.05.

Journal: Oncotarget

Article Title: BMP9/ALK1 inhibits neovascularization in mouse models of age-related macular degeneration

doi: 10.18632/oncotarget.11182

Figure Lengend Snippet: A. qRT-PCR analysis of P17 retinas from pups subjected to OIR revealed the expression of transcripts corresponding to components of the canonical BMP9 signaling ( n = 4 control mice and 4 OIR mice). B. Alk1 immunofluorescence staining of OIR retinas at P17 shows specific expression of Alk1 in blood vessels. Arrowheads show vascular tufts. Scale Bar: 20 μm. C. BMP9 ELISA of plasma from mice subjected to OIR collected at P12 (after vaso-obliteration) ( n = 3 control and n = 3 OIR) and P17 (neovascularization phase) ( n = 3 control and n = 4 OIR). D. qRT-PCR of choroid-sclera complexes subjected to laser-CNV of genes involved in BMP9 signaling ( n = 4 mice per group). All histograms represent mean ± standard error of the mean. * P < 0.05.

Article Snippet: Staining with either FITC-labeled isolectin GS IB4 (Life technologies corporation), rhodamine phalloidin (Cedarlane Laboratories) or goat anti-mouse Alk1 primary (R&D systems) and anti-goat secondary (Life technologies) antibodies were performed on whole and/or sectioned retinas/choroids.

Techniques: Quantitative RT-PCR, Expressing, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

A. IsoB4 staining of P17 retinas from Alk1-flox and Cdh5Cre-Alk1 flox mice subjected to OIR. Injections of tamoxifen were performed at P12, at the onset of neovascularization. Scale bar: 500 μm. B. High magnification of the retinal vasculature shown enlarged vessels accompanied by neovascularization following deletion of Alk1. Scale bar: 50 μm. C. Quantification of neovascular and vaso-obliterated areas in P16 retinas subjected to OIR using ImageJ/Swift ( n = 4 Alk1 fl/fl, n = 4 Cdh5CreERT2-Alk1f/f).

Journal: Oncotarget

Article Title: BMP9/ALK1 inhibits neovascularization in mouse models of age-related macular degeneration

doi: 10.18632/oncotarget.11182

Figure Lengend Snippet: A. IsoB4 staining of P17 retinas from Alk1-flox and Cdh5Cre-Alk1 flox mice subjected to OIR. Injections of tamoxifen were performed at P12, at the onset of neovascularization. Scale bar: 500 μm. B. High magnification of the retinal vasculature shown enlarged vessels accompanied by neovascularization following deletion of Alk1. Scale bar: 50 μm. C. Quantification of neovascular and vaso-obliterated areas in P16 retinas subjected to OIR using ImageJ/Swift ( n = 4 Alk1 fl/fl, n = 4 Cdh5CreERT2-Alk1f/f).

Techniques: Staining

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Structured Review

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1) Product Images from "Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage"

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