sponses. On the other hand, our inhibitor experiments were consistent with a previous requirement of phosphatases in force- dependent strengthening of integrin-CSK connections during early adhesion in fibroblasts [6]. Furthermore, these inhibitor experiments further support that extra- and intra-cellular Ca2+ are likely necessary to regulate integrin-specific mechanoresponses through distinct processes. Extracellular Ca2+ is required for integrin binding to their ligands [41], and may be needed to facilitate alterations in Ca2+ influx through mechanosensitive ion channels [2,17]. In contrast, inside-out integrin activation requires alterations in intracellular Ca2+ and downstream signaling [45]. Potential mechanisms underlying BSA-induced mechanoresponses in A549 cells Because BSA is thought to absorb non-specifically to the cell surface, force probes coated with BSA are commonly used as a negative control in studies of cell-ECM mechanical interactions [5,10]. Accordingly, it is unsurprising that BSA-coated tips elicited mechanoresponses similar to bare or RGE-coating in lung fibroblasts (Figure 2 and Figure 3). In contrast, BSA-coated tips induced stiffening in both compression and extension in A549 cells comparable to RGD-coating (Figure 2E), although cell adhesion to BSA-coated tips was smaller than to RGD but larger than bare or RGE-coated tips (Figure 3B). A straightforward explanation for these unexpected findings could be that A549 cells do have receptors for BSA. In this context, a former study showed that A549 cells express receptors for BSA modified as advanced glycation end products (AGE) [46]. AGE formation occurs when glucose or other reducing sugars react non-enzymatically with free residues of proteins and modify them by reversible glycosylation, which may further develop into irreversibly glycated proteins or AGE [46]. To examine the possibility that BSA-induced mechanoresponses were due to BSA glycosylation driven by the glucose present in the culture medium, we measured attachment of A549 cells to BSA-coated substrata in the presence of the AGE formation inhibitor AMG [47]. Likewise, we assessed EC, ET and F* in A549 cells with BSA-coated tips in the presence of AMG (Figure S6). We observed a modest reduction in cell attachment, ET and F* in the presence of AMG compared to vehicle, which did not achieve statistical significance (p = 0.15 for cell attachment, p = 0.07 for ET, and p = 0.06 for F*). Based on these findings, it is possible that mechanisms other than BSA glycosylation underlie BSA-dependent mechanoresponses. We can envision other mechanisms based on known properties of BSA. First, the surface charge of BSA could trigger mechanoresponses by inducing alterations in the membrane potential of A549 cells [48,49], since changes in membrane potential can drive local alterations in actin polymerization large enough to modify cell stiffness according to a recent study [50]. Second, serum albumin is known to act as a carrier of aminoacids and other biomolecules in the blood by facilitating their binding owing to the presence of surface-charged groups and ionic/hydrophobic sites [48]. Accordingly, it is conceivable that BSA-induced mechanoresponses are due to signaling driven by endogenous extracellular stimulatory factors expressed by A549 cells and bound to BSA during the course of an AFM experiment. However, testing these or other possible mechanisms will require further investigations. Biological implications of lung cell mechanoresponses We can envision functional advantages of the cell mechan- oresponses observed here based on their fitness to well-established Lung Cell Mechanoresponse to Force Directionality physiological processes. First, the integrin-specific stiffening and adhesion strengthening are consistent with a general need of cells to interrogate the mechanochemical properties of the insoluble local microenvironment. These integrin-specific mechanore- sponses may be particularly useful in guiding cell migration driven by a gradient in ECM stiffness (i.e. durotaxis) by either facilitating ECM mechanosensing, stabilizing cell-ECM adhesive sites, or generating migratory forces [2,6,10]. Second, the asymmetric response to bidirectional forces demonstrates that, at least in the timescale of 30 s, force transmission through the cell is not symmetric, unlike previously suggested [13]. This asymmetric mechanoresponse could be part of a ‘built-in’ sensing mechanism for force directionality, which might provide important cues for the maintenance of tissue architecture or for driving collective cell migration during lung development and repair. Supporting Information Text S1 Supplemental Methods including the protocol to prepare PAA+ gels, to perform qRT-PCR analysis, and the Relative binding assay. (PDF) Figure S1 Illustrative bright field images of a FE-AFM tip in contact with the perinuclear region of an A549 cell or a CCD- 19Lu fibroblast. (PDF) Figure S2 Linearity of the Young’s modulus (E) in compression in the indentation range used in the 4-step protocol. (PDF) Figure S3 E of PAA gels probed with RGD-coated FE-AFM tips in compression and extension. (PDF) Figure S4 Illustrative example of F versus t recorded on a CCD19-Lu fibroblast after holding an RGD-coated FE-AFM tip in contact with the cell for 5 min using a force control protocol. (PDF) Figure S5 Role of the CSK integrity in RGD-induced stiffening and adhesion strengthening in A549 cells probed with RGD- coated FE-AFM tips. (PDF) Mechanoresponses of A549 cells to BSA-coated FE- Figure S6 AFM tips. (PDF) Acknowledgments We thank F Rico (Inst. Curie) for critical reading of the manuscript, X Trepat (IBEC) and X Gasull (UB) for helpful discussions, R Nieto and MA Rodr´ıguez (Unitat de Biof´ısica i Bioenginyeria –UBB-, UB), and MJ Lo´pez (Plataforma de Nanotec., UB) for technical assistance, and other members of the UBB for support. Author Contributions Conceived and designed the experiments: IA TL AG MP DN JA. Performed the experiments: IA TL AG MP. Analyzed the data: IA TL AG MP DN JA. Contributed reagents/materials/analysis tools: NR RF. Wrote the paper: IA JA. PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e32261