Journal: eLife

Article Title: Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling

doi: 10.7554/eLife.72723

Figure Lengend Snippet:

Article Snippet: Antibody , anti-pSmad1/5/9 (Rabbit monoclonal) , Cell Signaling Technology , Cat#CST13820; RRID: AB_2493181 , IF(1:500).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Software, Microscopy, Staining

Journal: Thorax

Article Title: Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant

doi: 10.1136/thoraxjnl-2021-218083

Figure Lengend Snippet: Significant reduction in BMP signalling at the earliest stage of lung vascular pathology is accompanied by microvessel loss in postnatal lung injury. (A) Preclinical mouse model of bronchopulmonary dysplasia (BPD) with induction of lung injury by exposure of neonatal mice to mechanical ventilation (MV) and/or hyperoxia (FiO 2 =0.4) for 8 hours or 24 hours. (B) Immunoblot analysis reveals a significant decrease in pSMAD 1-5-9 and Id-1 protein levels in WT mice in the early course of postnatal lung injury provoked by short-term MV-O 2 and O 2 exposure. (C) Representative images of immunofluorescence (IF) staining for cleaved caspase-3 (green) and VE-cadherin positive cells (red) in lung tissue sections (4 µM, 400X, upper panel; nuclei (DAPI, blue) from 5 to 8 day old pups show a significant increase in EC apoptosis, confirmed by quantitative analysis (lower-left panel). Histological analysis reveals a significant decrease in the number of micro vessels (20–100 µm diameter) in the lungs of mice undergoing only 8 hours of O 2 and/or MV when compared with room air (RA) controls (lower-right panel). (D) In BMPR2 deficient mice, immunoblot analysis demonstrates significantly lower VE-cadherin protein expression in the neonatal lung at baseline (RA) when compared with WT pups (left panel). Postnatal exposure to moderate O 2 further decreases VE-cadherin protein expression in BMPR2 deficient mice (right panel). (E) Representative images of immunofluorescence (IF) staining for BMPR2 (green) and VE-cadherin positive cells (red) in lung tissue sections (4 µM, 400X, upper panel; nuclei (DAPI, blue) following 24 hours MV show a prolonged and significant decrease in BMPR2 expression, confirmed by quantitative analysis (right panel). (F) ISH demonstrates a persistent decrease in BMPR2 transcription in neonatal mouse PCLS exposed to O 2 for 24 hours when compared with RA control samples. Data are mean±SD *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3–4 mice/group compared with RA controls. Quantification of if in 10 fields of view (FOV) per section in two sections per animal, normalisation of positive cells to 100 nuclei; arrows point to positive cells. ISH quantification in 212.55 µm x 212.55 µm FOV. BMP, bone morphogenetic protein; BMPR2, BMP receptor 2; ISH, RNA in situ hybridization; PCLS, precision cut lung slices; VE, vascular endothelial; WT, wild type.

Article Snippet: Immunoblotting on Bis-Tris or Tris-Acetate gels (Life Technologies, Germany, #NP0321BOX, #EA0375BOX) included detection of CYP1A1 (A-9, Santa Cruz Biotechnology #SC-393979), ID1 (Abcam #ab168256), SMAD2/3 (Cell Signalling Technologies #3102), pSMAD2 (Cell Signalling Technologies #3101), SMAD 1 (Cell Signalling Technology, #6944), pSMAD 1-5-9 (Cell Signalling Technology, #9511), SMAD4 (Santa Cruz Biotechnology, #7966), SPARC (D10F10, Cell Signalling Technologies #8725), VE-Cadherin (H-72, Santa Cruz Biotechnology #28644), VEGF-A (147, Santa Cruz Biotechnology #507), VEGFR2 (R and D system, #AF644) and and β-actin (Santa Cruz Biotechnology, #sc-81178) (secondary antibodies goat anti-rabbit IgG (Santa Cruz Biotechnology, #2301) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-81178)).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Control, RNA In Situ Hybridization

Journal: Thorax

Article Title: Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant

doi: 10.1136/thoraxjnl-2021-218083

Figure Lengend Snippet: Impaired BMPR2 signalling is ameliorated on treatment with FK506 in vitro and in vivo. (A) Exposure of human pulmonary microvascular endothelial cells (HPMEC) to cyclic stretch or O 2 (FiO 2 =0.4) for 24 hours in vitro mimics postnatal injury. (B) Immunoblot analysis shows a significant downregulation of pSMAD 1-5-9 protein expression in HPMECs after exposure to O 2 and/or stretch, (C) enhanced by TGF-β (D) and translated into decreased ID-1 and (E) Smad 4 protein expression. (F) Immunoblot analysis shows significant upregulation of pSMAD 1-5-9 protein levels in HPMECs on treatment with a single dose of 2 ng/mL FK506 in the presence of O 2 in combination with stretch and/or TGF-β incubation. (G) The significant reduction in BMPR2 mRNA levels after O 2 treatment is reversed by FK506 in HPMECs. (H) In vivo, a single intraperitoneal dose of 2 ng/mL FK506 in neonatal mice at the onset of O 2 or MV-O 2 exposure for 2 hours resulted in an upregulation of pulmonary CD31 mRNA expression levels in neonatal mice when compared with PBS treated control mice. Data are mean±SD *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3–4 mice/group. BMPR2, bone morphogenetic protein receptor 2; ns, not significant; PBS, phosphate buffered saline.

Article Snippet: Immunoblotting on Bis-Tris or Tris-Acetate gels (Life Technologies, Germany, #NP0321BOX, #EA0375BOX) included detection of CYP1A1 (A-9, Santa Cruz Biotechnology #SC-393979), ID1 (Abcam #ab168256), SMAD2/3 (Cell Signalling Technologies #3102), pSMAD2 (Cell Signalling Technologies #3101), SMAD 1 (Cell Signalling Technology, #6944), pSMAD 1-5-9 (Cell Signalling Technology, #9511), SMAD4 (Santa Cruz Biotechnology, #7966), SPARC (D10F10, Cell Signalling Technologies #8725), VE-Cadherin (H-72, Santa Cruz Biotechnology #28644), VEGF-A (147, Santa Cruz Biotechnology #507), VEGFR2 (R and D system, #AF644) and and β-actin (Santa Cruz Biotechnology, #sc-81178) (secondary antibodies goat anti-rabbit IgG (Santa Cruz Biotechnology, #2301) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-81178)).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Incubation, Control, Saline