alterations compared to their values after 30 s of tip-cell contact. These results suggest that, unlike EC, both ET and F* reached plateau values after ,30 s of contact time. Local cell stiffness depends on the integrin-specificity of the applied force A common feature observed in both compression and extension measurements was that RGD-coated tips elicited E values that were two-fold larger than bare or RGE-coated tips in all cell types (Figure 2C, E), indicating that this integrin-specific stiffening was not restricted to a single cell type. Likewise, F* was at least two- fold higher when probed with RGD- than with bare or RGE- coated tips (Figure 3A, B). Plotting F* against ET from all conditions and cell types (Figure 3C) revealed that the positive correlation between ET and F* was not specific for RGD-coated tips. This correlation was well captured by fitting a linear function (F* = 0.899?ET, r2 = 0.89), as illustrated by the solid line in Figure 3C. Unexpectedly, BSA-coating, elicited a raise in ET and F* comparable to RGD in A549 cells only, although their absolute values were slightly smaller than those obtained with RGD (Figure 2E, Figure 3B). Likewise, BSA induced a raise in ET Lung Cell Mechanoresponse to Force Directionality in these cells, although this raise was not as statistically robust as that observed with RGD. Role of cytoskeleton (CSK) integrity, and Ca2+ and tyrosine phosphorylation-dependent signaling in lung cell mechanoresponses Given the prominent role of the CSK in the mechanical properties of cells [29], we examined RGD-induced cell mechan- oresponses upon modification of essential cytoskeletal components with selective drugs. CCD-19Lu fibroblasts were treated with either the actin polymerization inhibitor latrunculin-A (latA), or the microtubule polymerization inhibitor nocodazole. LatA not only prevented RGD-induced stiffening in both compression and extension, but it induced a dramatic softening in all conditions, although the ratio EC/ET was dramatically reduced (Figure 4A– C). These data reveal that actin polymerization is required to elicit the largest EC, ET and F*. In contrast, compromising the integrity of the microtubules had no detectable effects in E or F* values probed with RGD, suggesting that an intact microtubule network was not required for driving none of the integrin-specific mechanoresponses examined here (Figure 4A–C). Similar latA- Figure 3. RGD-induced strengthening of lung cell-tip adhesion. (A, B) F* data obtained on (A) CCD-19Lu fibroblasts and on (B) A549 cells. Bar filling patterns shown on the right apply to plots A–B. (C) Plot of F* vs ET average data collected on all cell types with all tip coatings. Solid line corresponds to a least-squares fitting of a linear polynomial to the data. +P,0.05, ++P,0.01 and +++P,0.005 were determined by unpaired Student’s t-test w.r.t. bare. doi:10.1371/journal.pone.0032261.g003 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32261