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. 2014 Apr 16;9(4):e92670. doi: 10.1371/journal.pone.0092670

Figure 3. Effect of substrate stiffness and LOX inhibition on LPS-induced EC activation.

Figure 3

A - Pulmonary EC were grown on polyacrylamide gels of different stiffness (1.5 kPa, and 40 kPa) and treated with TNFα (2 ng/ml) or LPS (200 ng/ml) for 6 hrs. Left panel: Expression of ICAM-1 and VCAM-1 was determined by western blot analysis. 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. 1.5 kPa; n = 4. Right panel: Analysis of fibronectin mRNA levels after 12-hr treatment with LPS or TNFα was performed by RT-PCR; *p<0.05. 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 TNFα (2 ng/ml, 6 hrs). ICAM-1 expression and MYPT phosphorylation was analyzed by western blot. 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. LPS+BAPN treatment; n = 4. C - IL-8 production in response to TNFα (2 ng/ml, 6 hrs) was evaluated by ELISA assay. *P<0.05.