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alk1 (R&D Systems)

Name: alk1
Brand: R&D Systems
Rating: 94 (30 reviews)

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R&D Systems alk1

BMPR2 is highly expressed in patients with AS. (A) Two healthy control (HC)-OPs and two AS-OPs conducted RNA sequencing. Heatmap with the BMP-related genes were selected and shown. (B) BMPR1A, BMPR1B, BMPR2, <t>ALK1,</t> and ALK2 expressions were validated by RT-qPCR (HC-OPs: n=8; AS-OPs: n=8). BMPR2 and ALK1 expressions were validated by (C) immunoblotting (HC-OPs: n=2, AS-OPs: n=6) and (D) immunohistochemistry (HC: n=6; AS: n=6). BMPR2-positive cells in bone-lining cells were counted. Representative images are shown from two individual samples per group. (D) The scale bar showed is 200 μm. AS: ankylosing spondylitis, OP: osteoprogenitor. Values are the mean±standard error of the mean. Statistical significance was determined with *p<0.05, **p<0.01, by Mann–Whitney U test.

Alk1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/alk1/product/R&D Systems
Average 94 stars, based on 30 article reviews

alk1 - by Bioz Stars, 2026-02

94/100 stars

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1) Product Images from "Elevated BMPR2 expression amplifies osteoblast differentiation in ankylosing spondylitis"

Article Title: Elevated BMPR2 expression amplifies osteoblast differentiation in ankylosing spondylitis

Journal: Journal of Rheumatic Diseases

doi: 10.4078/jrd.2023.0024

Figure Legend Snippet: BMPR2 is highly expressed in patients with AS. (A) Two healthy control (HC)-OPs and two AS-OPs conducted RNA sequencing. Heatmap with the BMP-related genes were selected and shown. (B) BMPR1A, BMPR1B, BMPR2, ALK1, and ALK2 expressions were validated by RT-qPCR (HC-OPs: n=8; AS-OPs: n=8). BMPR2 and ALK1 expressions were validated by (C) immunoblotting (HC-OPs: n=2, AS-OPs: n=6) and (D) immunohistochemistry (HC: n=6; AS: n=6). BMPR2-positive cells in bone-lining cells were counted. Representative images are shown from two individual samples per group. (D) The scale bar showed is 200 μm. AS: ankylosing spondylitis, OP: osteoprogenitor. Values are the mean±standard error of the mean. Statistical significance was determined with *p<0.05, **p<0.01, by Mann–Whitney U test.

Techniques Used: Control, RNA Sequencing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, MANN-WHITNEY

Image Search Results

Journal: Journal of Rheumatic Diseases

Article Title: Elevated BMPR2 expression amplifies osteoblast differentiation in ankylosing spondylitis

doi: 10.4078/jrd.2023.0024

Figure Lengend Snippet: BMPR2 is highly expressed in patients with AS. (A) Two healthy control (HC)-OPs and two AS-OPs conducted RNA sequencing. Heatmap with the BMP-related genes were selected and shown. (B) BMPR1A, BMPR1B, BMPR2, ALK1, and ALK2 expressions were validated by RT-qPCR (HC-OPs: n=8; AS-OPs: n=8). BMPR2 and ALK1 expressions were validated by (C) immunoblotting (HC-OPs: n=2, AS-OPs: n=6) and (D) immunohistochemistry (HC: n=6; AS: n=6). BMPR2-positive cells in bone-lining cells were counted. Representative images are shown from two individual samples per group. (D) The scale bar showed is 200 μm. AS: ankylosing spondylitis, OP: osteoprogenitor. Values are the mean±standard error of the mean. Statistical significance was determined with *p<0.05, **p<0.01, by Mann–Whitney U test.

Article Snippet: The antibodies used for immunoblotting and immunofluorescence were as follows: RUNX2 (12556; Cell Signaling Technology), BMPR2 (sc-393304; Santa Cruz Biotechnology, Dallas, TX, USA), ALK1 (AF370; R&D Systems, Minneapolis, MN, USA), phos-smad1/5/8 (sc-12353; Santa Cruz Biotechnology), total-smad1/5/8 (sc-6031-R; Santa Cruz Biotechnology), OPG (sc-390518; Santa Cruz Biotechnology), phos-ERK (5683; Cell Signaling Technology), phos-p38 (9215; Cell Signaling Technology), and GAPDH (2118; Cell Signaling Technology).

Techniques: Control, RNA Sequencing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, 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

Twenty-Seven Predicted miR-31-5p Target Genes That Were Down-Regulated Significantly in Colonic Mucosa of High-miR-31-5p CD Patients Compared With NIBD Controls

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: Twenty-Seven Predicted miR-31-5p Target Genes That Were Down-Regulated Significantly in Colonic Mucosa of High-miR-31-5p CD Patients Compared With NIBD Controls

Article Snippet: Rabbit anti-human ALK1 antibody, rabbit anti-human CA1 antibody, rabbit anti-human OLFM4 antibody, and mouse/rabbit IgG VisUCyte horseradish-peroxidase polymer antibody were purchased from Sigma-Aldrich (St. Louis, MO) (HPA007041), Novus Biologicals (Littleton, CO) (NBP1-88191), Cell Signaling Technology (Beverly, MA) (14369S), and R&D Systems (VC002-025).

Techniques:

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 = 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 = 27). Gene expression was quantified by qPCR and samples were split into 3 equally sized groups (N = 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 = 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 = 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 = 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: 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 = 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 = 27). Gene expression was quantified by qPCR and samples were split into 3 equally sized groups (N = 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 = 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 = 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 = 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.

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

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 intracellular domain (NICD) protein expression between NIBD and CD patients ( right ). ( B ) BMP9 concentration in the serum of NIBD controls (N = 17) and CD patients (N = 23). ( C ) NOTCH target gene expression in colonic epithelial cells from CD patients (N = 15) and NIBD controls (N = 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 = 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: 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 intracellular domain (NICD) protein expression between NIBD and CD patients ( right ). ( B ) BMP9 concentration in the serum of NIBD controls (N = 17) and CD patients (N = 23). ( C ) NOTCH target gene expression in colonic epithelial cells from CD patients (N = 15) and NIBD controls (N = 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 = 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, Concentration Assay, Derivative Assay, Cell Culture, MANN-WHITNEY

Expression of miR-31-5p and ALK1 in primary-cultured colonic epithelial monolayers derived from NIBD controls and CD patients. N = 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: Expression of miR-31-5p and ALK1 in primary-cultured colonic epithelial monolayers derived from NIBD controls and CD patients. N = 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, MANN-WHITNEY

BMP9–ALK1 signaling restricts the stemness of human colonic IECs. ( A ) EdU assay in NIBD patient-derived colonic epithelial cell monolayers (N = 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 = 6 per group). ( C ) Proliferation- and stemness-related gene expression in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 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 = 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: BMP9–ALK1 signaling restricts the stemness of human colonic IECs. ( A ) EdU assay in NIBD patient-derived colonic epithelial cell monolayers (N = 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 = 6 per group). ( C ) Proliferation- and stemness-related gene expression in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 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 = 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, Expressing, Isolation, Immunohistochemical staining, Staining, MANN-WHITNEY

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 = 6 per group). ( B ) CA1 protein expression in NIBD patient-derived colonic epithelial monolayers (N = 4 per group). ( C ) Colonocyte marker expression in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 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 = 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: 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 = 6 per group). ( B ) CA1 protein expression in NIBD patient-derived colonic epithelial monolayers (N = 4 per group). ( C ) Colonocyte marker expression in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 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 = 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, Expressing, Derivative Assay, Cell Culture, Marker, Isolation, Immunohistochemistry, MANN-WHITNEY

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 = 6 per group). ( B ) Gene expression of tight junction proteins in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 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 = 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 ΔTEER% (N = 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: 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 = 6 per group). ( B ) Gene expression of tight junction proteins in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 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 = 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 ΔTEER% (N = 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: Expressing, Derivative Assay, Cell Culture, Isolation, Permeability, MANN-WHITNEY

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 = 10) and CD patients (N = 28) by qPCR. ( B ) ALK1 expression in the colonic mucosa of CD patients at the time of surgery and after surgery (N = 5 per group). ( C ) Percentages of CD patients who were diagnosed as CD before and after age 40 years in low-ALK1 (N = 15) and hi-ALK1 (N = 13) CD subsets. ( D ) Kaplan–Meier survival analysis to evaluate the impact of colonic ALK1 expression on endoscopic relapse in patients with CD (N = 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: 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 = 10) and CD patients (N = 28) by qPCR. ( B ) ALK1 expression in the colonic mucosa of CD patients at the time of surgery and after surgery (N = 5 per group). ( C ) Percentages of CD patients who were diagnosed as CD before and after age 40 years in low-ALK1 (N = 15) and hi-ALK1 (N = 13) CD subsets. ( D ) Kaplan–Meier survival analysis to evaluate the impact of colonic ALK1 expression on endoscopic relapse in patients with CD (N = 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

Clinical Characteristics at the Time of Sample Collection

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: Clinical Characteristics at the Time of Sample Collection

Techniques: Sampling, Activity Assay

Correlations Between Explanatory Variables for the Risk of Surgery

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: Correlations Between Explanatory Variables for the Risk of Surgery

Techniques:

Binomial Logistic Regression Analysis for Surgery

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: Binomial Logistic Regression Analysis for Surgery

Techniques:

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: Primer Sequences for RT-qPCR

Techniques:

Distribution of immunohistochemical scores with the 3 different clones (ALK1, 5A4 and D5F3).

Journal: Pathologica

Article Title: Immunohistochemistry with 3 different clones in anaplastic lymphoma kinase fluorescence in situ hybridization positive non-small-cell lung cancer with thymidylate synthase expression analysis: a multicentre, retrospective, Italian study

doi: 10.32074/1591-951X-756

Figure Lengend Snippet: Distribution of immunohistochemical scores with the 3 different clones (ALK1, 5A4 and D5F3).

Article Snippet: ALK IHC assay was performed using 3 different clones: Novocastra mouse monoclonal antibody p80 ALK (clone 5A4, Leica Biosystems, Newcastle Upon Tyne, United Kingdom); Companion Diagnostic Kit Ventana anti-ALK rabbit monoclonal primary antibody (clone D5F3, Cell Signaling Technology); mouse monoclonal anti-human CD246 (clone ALK1, Dako/Agilent, Carpentaria, CA).

Techniques: Immunohistochemical staining, Clone Assay

Detailed distribution of immunohistochemical staining score along the entire series of 37 ALK FISH-positive cases according to the 3 different clones (ALK1, 5A4, D5F3).

Journal: Pathologica

doi: 10.32074/1591-951X-756

Figure Lengend Snippet: Detailed distribution of immunohistochemical staining score along the entire series of 37 ALK FISH-positive cases according to the 3 different clones (ALK1, 5A4, D5F3).

Techniques: Immunohistochemical staining, Staining, Clone Assay

Crosstabulation considering 2+ and 3+ cases as positive.

Journal: Pathologica

doi: 10.32074/1591-951X-756

Figure Lengend Snippet: Crosstabulation considering 2+ and 3+ cases as positive.

Techniques:

Example of invasive lung adenocarcinoma with acinar pattern on surgical specimen (A, haematoxylin-eosin staining) showing weak positivity (score 1+) (B, immunohistochemistry) with clone ALK1, moderate positivity (score 2+) (C, immunohistochemistry) with clone 5A4 and strong positivity (score 3+) (D, immunohistochemistry) with clone D5F3.

Journal: Pathologica

doi: 10.32074/1591-951X-756

Figure Lengend Snippet: Example of invasive lung adenocarcinoma with acinar pattern on surgical specimen (A, haematoxylin-eosin staining) showing weak positivity (score 1+) (B, immunohistochemistry) with clone ALK1, moderate positivity (score 2+) (C, immunohistochemistry) with clone 5A4 and strong positivity (score 3+) (D, immunohistochemistry) with clone D5F3.

Techniques: Staining, Immunohistochemistry

Example of metastatic adenocarcinoma on cell-block from pleural effusion (A, haematoxylin-eosin staining) showing weak positivity (score 1+) (B, immunohistochemistry) with clone ALK1, moderate positivity (score 2+) (C, immunohistochemistry) with clone 5A4 and strong positivity (score 3+) (D, immunohistochemistry) with clone D5F3.

Journal: Pathologica

doi: 10.32074/1591-951X-756

Figure Lengend Snippet: Example of metastatic adenocarcinoma on cell-block from pleural effusion (A, haematoxylin-eosin staining) showing weak positivity (score 1+) (B, immunohistochemistry) with clone ALK1, moderate positivity (score 2+) (C, immunohistochemistry) with clone 5A4 and strong positivity (score 3+) (D, immunohistochemistry) with clone D5F3.

Techniques: Blocking Assay, Staining, Immunohistochemistry

Crosstabulation considering only 3+ cases as positive.

Journal: Pathologica

doi: 10.32074/1591-951X-756

Figure Lengend Snippet: Crosstabulation considering only 3+ cases as positive.

Techniques:

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

(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).

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

(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

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

Techniques: Injection, Staining, Expressing

(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).

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).

Techniques: Injection, Staining, Labeling

(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)

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)

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

(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).

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).

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

(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).

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).

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

(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)

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)

Techniques: Staining

(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.

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.

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

(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)

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)

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

(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)

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)

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

(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

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

Techniques: Staining, Transfection

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