Stiffness-Activated GEF-H1 Expression Exacerbates LPS-Induced Lung Inflammation
Figure 6
Effect of substrate stiffness and LOX inhibition on LPS-induced GEF-H1 expression.
A - Pulmonary EC were grown on polyacrylamide gels of different stiffness (1.5 kPa, and 40 kPa) and treated with LPS (200 ng/ml) for 6 hrs. GEF-H1 expression determined by western blot analysis. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. 1.5 kPa; n = 4. B – Pulmonary EC were cultured on 2.8 kPa substrates for 3 days in the presence of LPS (200 ng/ml) with or without BAPN (300 µM). After cell detachment, fresh EC were plated on deposited extracellular matrix and stimulated with LPS (200 ng/ml, 6 hrs). GEF-H1 expression was analyzed by western blot. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. LPS+BAPN treatment; n = 4. C – Pulmonary EC grown on 2.8 kPa substrates and treated with nonspecific or GEF-H1-specific siRNA for 72 hrs were stimulated with TNFα (2 ng/ml) or LPS (200 ng/ml) for 6 hrs, and ICAM-1 expression was analyzed by western blot. GEF-H1 depletion was confirmed by membrane reprobing with GEF-H1 antibody. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. non-specific RNA; n = 3.