Journal: Scientific Reports

Article Title: LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells

doi: 10.1038/s41598-022-16667-5

Figure Lengend Snippet: LATS1 and LATS2 phosphorylate SLUG on T208. ( A ) SAS and SAS-δ cells were co-transfected with siRNAs against LATS1 and LATS2 (siLATS1/2), and then cultured with TGF-β1 for 48 h, followed by WB with the indicated antibodies. ( B ) SAS cells transfected with GL2 (as a negative control) or LATS2 siRNA duplex were treated with cycloheximide (CHX) for the indicated periods, and then subjected to subcellular fractionation. Protein levels of SLUG and LATS2 were determined by WB. Lamin A/C and α-tubulin are nuclear and cytoplasmic fraction markers, respectively. The relative levels of cytoplasmic and nuclear SLUG normalized to the corresponding band intensities of α-tubulin and Lamin A/C, respectively, are shown below top panel. The normalized band intensities of SLUG at 0 h after CHX treatment were defined as 1.0 (lanes 1, 5, 9, and 13). ( C ) WB of parental SAS and SAS-δ, which were cultured in medium containing 10% FBS in the presence (+) or absence (–) of TGF-β1 (48 h), was performed with the indicated antibodies. The relative levels of the indicated proteins normalized to the corresponding band intensity of actin, SLUG, or SMAD2, are shown below panels. ( D ) Immunofluorescence staining with anti-SLUG-pT208 (red) and α-tubulin (green) antibodies. Cells were cultured as in ( C ). DNA was visualized by Hoechst 33258 staining (blue). The cell size were comparable between SAS and SAS-δ in the presence or absence of TGF-β1.

Article Snippet: Recombinant human LATS1 or LATS2 kinase (Carna Biosciences, Hyogo, Japan) was incubated at 30 °C for 30 min with GST-fused mouse Slug-WT, -T183A, -T209A, and all-A (S101A, S139A, S161A, T183A, S196A, T209A, S215A, S248A, and S252A) mutants in LATS1/2-kinase buffer (20 mM PIPES [pH 6.8], 4 mM MnCl 2 , 1 mM DTT, 1 mM NaF, 1 mM Na 3 VO 4 ) containing 20 μM ATP and 10 μCi [γ- 32 P] ATP.

Techniques: Transfection, Cell Culture, Negative Control, Fractionation, Immunofluorescence, Staining

Journal: Translational stroke research

Article Title: Induction of Brain Arteriovenous Malformation through CRISPR/Cas9 Mediated Somatic Alk1 Gene Mutations in Adult Mice

doi: 10.1007/s12975-018-0676-1

Figure Lengend Snippet: A. Structure of vectors. Mecp2 pr: the promoter of a neural specific gene, Mecp2; SYN pr: the promoter of a neural specific gene, synapsin. B. Surveyor assay image. Indel mutations in Alk1 gene were detected in Neuro2A cells transfected with plasmids expressing Cas9 with e4sgRNA or e5sgRNA. For exon 4, wild-type band (Ctrl) is 761 base pairs (bp); mutant bands are 557 and 204 bps (arrows). For exon 5, wild-type band (Ctrl) is 830 bp; mutant bands are 480 and 350 bps (arrows).

Article Snippet: For analysis of Alk1 positive endothelial cells: two sections adjacent to the sections used for vessel quantification were co-stained with anti-CD31 antibody and anti-Alk1 antibody (1:50, AF770, R&D Systems, Minneapolis, MN).

Techniques: Transfection, Expressing, Mutagenesis

Journal: Translational stroke research

Article Title: Induction of Brain Arteriovenous Malformation through CRISPR/Cas9 Mediated Somatic Alk1 Gene Mutations in Adult Mice

doi: 10.1007/s12975-018-0676-1

Figure Lengend Snippet: A. Structure of pAd-Cas9 and pAd-Alk1e4sgRNA+e5sgRNA-Cas9 vectors. pr: promoter. PGK: phosphoglycerate kinase. ITR: inverted terminal repeats. B. Location of e4sgRNA and e5sgRNA sites (arrows at the bottom) and primers F and R (purple arrows) for detection of deletions between e4sgRNA and e5sgRNA sites. SacI (green arrow at the top) is a restriction enzyme that cleaves wild-type PCR product. C. Deletion mutation in pAd-Alk1e4sgRNA+e5sgRNA-Cas9 transfected Neuro2A cells. A 1479 bp (top blue and purple arrows) fragment was amplified from pAd-Cas9 trensfected cells (Ctrl). SacI cut this fragment into 591 and 888 bp (bottom two blue two arrows). Two additional bands (1300 and 1000 bps, bottom two purple arrows) containing frameshift and deletion mutations were amplified from pAd-Alk1e4sgRNA+e5sgRNA-Cas9 transfected cells (pAd). D. A representative image of western blot using protein isolated from bEnd3 cells. CTRL: pAd-Cas9 transfect bEnd.3 cells; pAd-CRISPR: pAd-Alk1e4sgRNA+e5sgRNA-Cas9 transfected bENd.3 cells. E. Quantitative analysis of Alk1 expression in bEnd3 cells. N=3.

Article Snippet: For analysis of Alk1 positive endothelial cells: two sections adjacent to the sections used for vessel quantification were co-stained with anti-CD31 antibody and anti-Alk1 antibody (1:50, AF770, R&D Systems, Minneapolis, MN).

Techniques: Mutagenesis, Transfection, Amplification, Western Blot, Isolation, CRISPR, Expressing

Journal: Translational stroke research

Article Title: Induction of Brain Arteriovenous Malformation through CRISPR/Cas9 Mediated Somatic Alk1 Gene Mutations in Adult Mice

doi: 10.1007/s12975-018-0676-1

Figure Lengend Snippet: A. Design for in vivo studies. Ad-Alk1e4sgRNA+e5sgRNA-Cas9 and AAV-VEGF were co-injected into basal ganglia of mouse brain. Eight weeks after virus injection, brain vessels were casted by latex to detect AVM phenotypes. Through intra-cardiac ventricle perfusion, latex dye enters vein only in the presence of arteriovenous shunt. Due to the particle size, latex cannot pass capillaries. Dysplasia vessels were quantified on immunostained sections. Alk1 gene mutation was analyzed by qPCR. Alk1 expression was analyzed by western blot. B. Schematic of mouse Alk1 exons 4 to 8 (e4-e8). Blue boxes are exons. The red lines are e4sgRNA and e5sgRNA target sites. The primers used for detecting deletion mutations between e4sgRNA and e5sgRNA sites and to amply sequence between exons 7 and 8 are indicated by arrows. C. Quantification of deletion mutations. N=6 for Ad-GFP group and N=12 for Ad-Alk1e4sgRNA+e5RNA-Cas9 (Ad-CRISPR) group. D. A western blot image. E. Quantification of Alk1 protein expression. WT: brain tissues of mice injected with Ad-GFP (N=7). Ad-CRISPR: brain tissues of mice injected with Ad-Alk1e4sgRNA+e5sgRNA-Cas9 (N=6).

Article Snippet: For analysis of Alk1 positive endothelial cells: two sections adjacent to the sections used for vessel quantification were co-stained with anti-CD31 antibody and anti-Alk1 antibody (1:50, AF770, R&D Systems, Minneapolis, MN).

Techniques: In Vivo, Injection, Virus, Mutagenesis, Expressing, Western Blot, Sequencing, CRISPR

Journal: Translational stroke research

Article Title: Induction of Brain Arteriovenous Malformation through CRISPR/Cas9 Mediated Somatic Alk1 Gene Mutations in Adult Mice

doi: 10.1007/s12975-018-0676-1

Figure Lengend Snippet: A. Images show ALK1 null endothelial cells (arrows). Endothelial cells were stained by an anti-CD31 antibody (green). ALK1 expression was detected using an anti-ALK1 antibody (red). Scale bar: 20 μm. B. Representative images of latex perfused brains. Scale bar: 100 μm. The bottom pictures are zoom in images of the viral vector injected regions. Arrows indicate latex perfused veins.

Article Snippet: For analysis of Alk1 positive endothelial cells: two sections adjacent to the sections used for vessel quantification were co-stained with anti-CD31 antibody and anti-Alk1 antibody (1:50, AF770, R&D Systems, Minneapolis, MN).

Techniques: Staining, Expressing, Plasmid Preparation, Injection

Journal: Molecular Oncology

Article Title: Hepsin promotes breast tumor growth signaling via the TGFβ‐EGFR axis

doi: 10.1002/1878-0261.13545

Figure Lengend Snippet: Hepsin regulates EGFR signaling in a TGFβ‐dependent manner. (A) Western blot comparison of total‐EGFR (tEGFR) levels in control MCF10A pL6‐Ctrl ( N = 3) and MCF10A pL6‐TGFβ1V5, V5‐tagged TGFβ1 overexpressing cells ( N = 3), which secrete around 80–100 pg·mL −1 of TGFβ1. Data are presented as mean ± SD. Significance was tested using the student's t‐test. (B) Western blot analysis of tEGFR in control doxycycline‐ (DOX − ) ( N = 3) and hepsin overexpressing (DOX + ; N = 3) MCF10A‐ pIND20‐HPN pL6‐ TGFβ1V5 cells. Data are presented as mean ± SD. Significance was tested with the student's t ‐test. (C) Western blot analysis of tEGFR, phospho‐SMAD2/3 (pSMAD2/3), and total‐SMAD2 (tSMAD2) in MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cells. The cells were treated with ALK5 inhibitor (i) (10 μ m RepSox, N = 4) and ALK4/5/7 inhibitor (5 μ m A‐83‐01, N = 3) or DMSO (control, N = 4) for 48 h with (DOX + ; 1 μg·mL −1 ) or without (DOX − , control) hepsin overexpression (the numbers below the tEGFR blot indicate loading normalized values for tEGFR band intensity fold leftmost control lane). The histogram data are presented as mean ± SD. Significance was tested using unpaired t ‐test. (D) Western blot analysis of phospho‐EGFR (pEGFR), tEGFR, total‐MAPK (tMAPK) and phospho‐MAPK (pMAPK), and vinculin (loading control) in MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cells. The cells were treated with the ALK1/2/3/6 inhibitor (i) (10 μ m K02288), EGFR inhibitor (i) (10 μ m Erlotinib), and DMSO (control) for 48 h. Data are presented as mean ± SD. Significance was tested with the student's t ‐test ( N = 3). (E) Phase‐contrast microscopy images of MCF10A‐pIND20‐HPN cells and MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 (overexpressing TGFβ1) cells grown in 3D culture for 2 weeks with (DOX + ; 1 μg·mL −1 ) or without (DOX − ) induction of hepsin overexpression. Cells were treated with 10 μ m EGFR or ALK1/2/3/6 inhibitor for 2 weeks as indicated in the figure. Scale bar represents 100 μm. (F) Phase‐contrast microscopy images of MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cells grown in 3D culture for 2 weeks in the presence of ALK1 inhibitory antibody (25 μg·mL −1 ). Scale bar represents 50 μm. For E and F, the experiments were repeated three times, with at least 100 epithelial structures counted per group in each repeat (one dot represents one structure), and black lines denote the mean values of each group. Significance was tested using the student's t ‐test. (G) Western blot analysis of the mitotic marker phospho histone H3 (pH3 S10) MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cell line grown in 3D culture in the presence of the EGFRi (1 μ m , 24 h) or DMSO. The quantification of blots of three independent experiments is shown in the graph (on the right), where the black lines denote the mean of each group. Significance was tested using the student's t ‐test. ns, not significant.

Article Snippet: The inhibitors used in this study were K02288 (Selleckchem, S7359, Houston, TX, USA), Galunisertib/LY2157299 (Selleckchem, S2230), RepSox (Sigma‐Aldrich/Merck, R0158), A‐83‐01 (MedChem Express, HY‐10432, Monmouth Junction, NJ, USA), Erlotinib (EGFRi, Selleckchem, S1023), and human ALK1 inhibitory antibody (R&D Systems, MAB3701).

Techniques: Western Blot, Comparison, Control, Over Expression, Microscopy, Marker

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

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. .

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: Transfection, MANN-WHITNEY, Western Blot, Expressing

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).

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, Injection

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.

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

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβR1 mediates TGFβ-induced Smad1/5 phosphorylation in mPASMC. SB505124 (SB), an ALK4/5/7 inhibitor, prevented TGFβ-induced Smad1/5 phosphorylation, while dorsomorphin (DM), an ALK1/2/3/6 inhibitor, partly decreased TGFβ-induced Smad1/5 phosphorylation. Serum-starved cells were treated with 1 μM SB, 10 μM DM, or DMSO vehicle for 1 h before additional treatment with 2.5 ng/ml TGFβ1 or 20 ng/ml BMP4 for 1 h. Cell lysates were collected, and the protein expression level of the indicated Smads and GAPDH were detected using immunoblotting. Immunoblot images shown are representative of at least three independent experiments. For the densitometry analysis, n = 4 in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK1 is expressed in mPASMC, mFibroblasts, and the mouse pup lung. A: ALK1 mRNA expression in the indicated adult and P10 mouse tissues and cells detected using RT-PCR. B: ALK1 protein expression in the indicated tissues and cell lysates detected using immunoblotting. Equal amounts of proteins were resolved using PAGE; effective protein transfer to the immunoblot membrane was demonstrated using Ponceau S staining. ALK1 protein expression data shown are representative of at least three independent experiments. C: ALK1 immunoreactivity (red) was detected in smooth muscle cells in pulmonary arteries (arrows), epithelial cells (arrowheads), interstitial cells (double arrows), and macrophages (*) in P10 mouse pup lungs, counterstained with hematoxylin. Representative images from three pups; scale bar is 25 µm long.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Membrane, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK1 regulates TGFβ-stimulated pSmad1/5 nuclear localization in mPASMC. A: ALK1 immunoreactivity is detected on the surface of mPASMC unless the antibody is preadsorbed with solubilized extracellular ALK1 domain (mALK1-Fc). Live cells were reacted with an anti-mALK1 antibody, an IgG2b monoclonal rat antibody that was generated against an extracellular fragment of mouse ALK1, without and with previous exposure to mALK1-Fc, or rat IgG2b, washed, fixed, and then reacted with a fluorescently labeled secondary antibody and DAPI. Subsequently, epifluorescence microscopy was performed. Typical images of two or three independent studies are shown. B: pretreatment with an anti-mALK1 antibody inhibits TGFβ-mediated BMP R-Smad phosphorylation. Cells were treated with 0 or 15 µg/ml anti-mALK1 and then incubated with 0 or 2.5 ng/ml TGFβ1 for 1 h. Subsequently, the cells were fixed and pSmad1/5 immunoreactivity, and DAPI reactivity was assessed. In additional immunoreactivity control studies, the cells were reacted with an isotype control instead of the anti-pSmad1/5 antibody. The nuclear immunoreactivity intensity signal in a region of interest identified by the DAPI reactivity was quantified and normalized with the mean level detected in the control cells. n = 45 per group; *P < 0.05. Scale bars = 50 µm.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Generated, Labeling, Epifluorescence Microscopy, Incubation