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

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: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A ) RNA-Seq heat map of gene expression changes in HUVECs treated or not (CTRL) for 24h in complete medium (conditioned for 2 days) with ALK1-Fc (1 μg/mL) or tacrolimus (FK-506, 0.3 μM) ( n = 6). Inset, cell extracts were analyzed by Western blotting (WB) using antibodies directed against the indicated proteins. ( B and C ) RNA-Seq Mean Average Plots (MA-Plots) displaying all differentially expressed transcripts in ALK1-Fc-treated (B) and FK506-treated (C) HUVECs. Genes with a FDR < 0.01 are shown in red. Genes unchanged by treatment are expected to lie on the horizontal dashed line at log2 fold change = 0. Low expression values reflect experimental measurement noise. Genes discussed in the text were annotated on the plots. ( D and E ) RNA-Seq scatter plots comparing gene expression changes (log fold change) between ALK1-Fc and FK-506 treatments for transcripts differentially expressed by either treatment (FDR < 0.01, D) and for all transcripts (E). Plot in (D) shows a clear inverse correlation and indicates that FK-506 treatment had the inverse effect of ALK1-Fc. ( F ) RNA-Seq UpSet plot showing the size of set intersections for transcripts differentially expressed by ALK1-Fc or FK506 treatments. Bars indicate how many transcripts were differentially expressed in each labeled condition. Lines connect dots to indicate set intersection. For instance, 572+187 transcripts were differentially expressed by FK-506 treatment, of which 187 were also differentially expressed when HUVECs were treated with ALK1-Fc. 64 genes were differentially expressed following ALK1-Fc treatment, but not in the other condition.

Article Snippet: For flow cytometry, antibody directed against ALK1 was from R&D Systems (AF370).

Techniques: RNA Sequencing, Gene Expression, Western Blot, Expressing, Labeling

Journal: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A ) HUVECs were treated or not (CTRL) for 24h in complete medium (conditioned for 2 days) with ALK1-Fc (1 μg/mL) or BMP9 (10 ng/mL). Cell extracts were analyzed by WB using antibodies directed against the indicated proteins. ( B ) Densitometric analyses and quantification of Dll4, pSmad1/5/8, and ID1 relative levels in n = 4 independent experiments, as in (A). Data represent mean ± s.e.m.; ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA, Dunnett’s multiple comparisons test.

Article Snippet: For flow cytometry, antibody directed against ALK1 was from R&D Systems (AF370).

Techniques:

Journal: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A ) Luciferase activity in C2C12BRA cells treated with different concentrations of BMP9 for 24h in depleted medium (0.1% FBS). Data represent mean ± s.e.m. (n = 5). ( B ) Luciferase activity in C2C12BRA cells treated or not (CTRL) for 24h in complete medium (conditioned for 2 days) with ALK1-Fc (1 μg/mL) and the indicated concentrations of tacrolimus (FK-506). Data in (A) and (B) represent mean ± s.e.m. (n = 4); * P < 0.05, *** P < 0.001, **** P < 0.0001; one-way ANOVA, Dunnett’s multiple comparisons test; # P < 0.05, ## P < 0.01 relative to non-ALK1-Fc-treated controls at the corresponding FK-506 concentration, Student’s t -test.

Article Snippet: For flow cytometry, antibody directed against ALK1 was from R&D Systems (AF370).

Techniques: Luciferase, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A ) NCC screen using Id1 transactivation luciferase assay in C2C12BRA cells. Drugs were tested in duplicate at 3 μM on cells maintained for 24h in depleted medium containing 0.1% FBS and 0.5 ng/mL BMP9 (BMP9 EC 50 ). Data (fold change mean ± range) are ranked by mean score and were normalized to DMSO control. ( B ) Luciferase activity in C2C12BRA cells treated with different concentrations of tacrolimus (FK-506) in depleted medium supplemented with BMP9 at EC 50, as in (A). Data represent mean ± s.e.m. ( n = 4); *** P < 0.001, **** P < 0.0001; one-way ANOVA, Bonferroni’s multiple comparisons test. ( C-E ) C2C12 cells (C) and HUVECs (D and E) were treated or not (CTRL) for 24h in complete medium (conditioned for 2 days) with ALK1-Fc (1 μg/mL, D) or tacrolimus at the indicated concentrations (C and D) or 0.3 μM (E). Cell extracts were analyzed by WB using antibodies directed against the indicated proteins. ( F ) Densitometric analyses and quantification of Dll4, pSmad1/5/8, and ID1 relative levels in n = 3 independent experiments, as in (E). Data represent mean ± s.e.m.; ** P < 0.01, **** P < 0.0001; Student’s t -test. ( G ) Flow cytometric analysis of surface ALK1 expression in HUVECs treated or not (CTRL) for 24h in complete medium (conditioned for 2 days) with BMP9 (10 ng/mL) or tacrolimus (0.3 μM).

Article Snippet: For flow cytometry, antibody directed against ALK1 was from R&D Systems (AF370).

Techniques: Luciferase, Control, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A ) HUVECs treated or not (CTRL) for 24h (in 0.05% FBS medium) with BMP9 (10 ng/mL) or tacrolimus (FK-506, 0.3 μM), were stimulated for 5 min with VEGF (25 ng/mL). Cell extracts were analyzed by WB using antibodies directed against the indicated proteins. ( B ) Densitometric analyses and quantification of the relative levels of the indicated proteins in n = 3 independent experiments, as in (A). Data represent mean ± s.e.m.; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA, Dunnett’s and Sidak’s multiple comparisons tests. ( C ) Schematic illustration of the proposed effect of tacrolimus on ALK1 and VEGFR2 signaling pathways.

Article Snippet: For flow cytometry, antibody directed against ALK1 was from R&D Systems (AF370).

Techniques: Protein-Protein interactions

Journal: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A and B ) C2C12 cells transfected with human WT ALK1 or ALK1 HHT mutants R411P (A) and T372fsX (B) were serum-starved for 3h (0.1% FBS medium) and then treated for 3h with the indicated concentrations of BMP9 or tacrolimus (FK-506). Asterisk denotes a nonspecific band. ( C ) Partial genomic DNA sequences of the HHT patient ACVRL1 gene. Arrow indicates the c.1112dup insertion (T372fsX mutation) in the mutated allele. ( D ) HHT BOECs were treated with the indicated concentrations of tacrolimus for 24h in depleted medium (0.1% FBS medium). Cell extracts in (A, B, and D) were analyzed by WB using antibodies directed against the indicated proteins.

Article Snippet: For flow cytometry, antibody directed against ALK1 was from R&D Systems (AF370).

Techniques: Transfection, Mutagenesis