Analyzing Lung Explant Branching Morphogenesis

Ex vivo cultures of normal lung explants were complemented daily with distinct doses of recombinant rat ROBO1 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 1749-RB-050), recombinant human ROBO2 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 3147-RB-050), or recombinant human IgG1 Fc (used as control at higher dose, R&D; Cat No. 110-HG-100). These doses were selected according to the literature [41 (link), 42 (link)]. Lung explants were obtained in three independent experiments (n ≥ 9 for each dose tested). After 4 days in culture, control, ROBO1 and ROBO2 inhibited lung explants were analyzed for branching morphogenesis in terms of area, external and internal epithelial perimeter, and the number of peripheral airway buds. In addition, SOX2, SOX9, BMP4, total and non-phospho (active) β-Catenin were quantified by Western blot at day 4 in all experimental groups.

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Gonçalves A.N., Correia-Pinto J, & Nogueira-Silva C. (2020). ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia. Respiratory Research, 21, 302.

Publication 2020

recombinant human ROBO2 Fc Chimera recombinant human IgG1 Fc

Bmp4 Chimera Human Igg1 Lung Morphogenesis Perimeter Rat robo1 Sox2 Sox9 Western blot β catenin

Corresponding Organization :

Other organizations : University of Minho

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Variable analysis

independent variables

Recombinant rat ROBO1 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL)
Recombinant human ROBO2 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL)
Recombinant human IgG1 Fc (used as control at higher dose)

dependent variables

Branching morphogenesis in terms of area, external and internal epithelial perimeter, and the number of peripheral airway buds
Expression of SOX2, SOX9, BMP4, total and non-phospho (active) β-Catenin (quantified by Western blot)

control variables

Ex vivo cultures of normal lung explants

positive control

Recombinant human IgG1 Fc (used as control at higher dose)

negative control

Not explicitly mentioned

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