step II by manually adjusting the position of the piezotranslator with small steps to keep the position of the laser reflected off the surface of the cantilever on the photodetector within 5% of its position at the beginning of step II for 5 min (referred to as force control protocol). AFM data processing The contact elastic theory for a flat-ended rigid cylinder deforming an incompressible elastic half space predicts a constant contact area (A) given by A = pa2, and a F(d) equation given by F = kd0+2Ead/(12n2) [14,25], where E is the Young’s elastic modulus of the sample, a is the cylindrical tip radius, and n is the sample’s Poisson ratio (assumed to be 0.5). The F(d) equation applies to both compression and extension, and was used to analyze all AFM measurements. Sample deformation (referred to as indentation in compression) was computed as d = z2zc2(d2d0). Time recordings exhibiting sudden (,1 s) force variations (.0.1 nN) in step II were discarded, since these behaviors depart from the assumptions of the model. Discarded recordings were ,30% for cells and 0% for PAA+ gels. EC was calculated by fitting the F(d) equation to (F,d) data in step III corresponding to a range in z of the loading ramp defined by 1–1.5 mm below zc (Figure 1F, G). ET was calculated by fitting the F(d) equation to (F,d) data in step IV corresponding to the range in z of the unloading ramp between 0.2 mm above zc and z of the first detectable tip-sample detachment. For gel measurements, ET was assessed using the range in z defined by 1–1.5 mm above zc. The different z range used to assess ET in cells and in gels was due to the fact that z corresponding to the first tip-sample detachment was smaller than 0.7 mm above zc in cells, whereas it was larger than 1.5 mm in PAA+ gels. To assess the asymmetry between EC and ET, we computed the ratio EC/ET. F data recorded during step II were analyzed in terms of stress relaxation by fitting a power-law F,t2a to the last 20 s to avoid any transient artifact. The tip-sample detachment force (F*) (Figure 1C, D) was computed as previously described [14]. The linearity of E in compression within the z range covered in step III (0.5–1.5 mm) was confirmed in both cells and gels as reported elsewhere [14] (Figure S2). Statistics Paired comparisons between EC and ET were carried out using paired Student’s t-test. For a given loading condition, the effect of peptide or protein coating versus bare was assessed by unpaired Student’s t-test. Statistical analyses were performed with SigmaStat software (Systat Software, Richmond, CA). Statistical significance was assumed at p,0.05, where p is the probability of the hypothesis [26]. Unless otherwise stated, data are given as mean 6 SE. Results Nanofabrication of flat-ended cylindrical tips by FIB Our nanofabrication protocol yielded highly reproducible FE- AFM tips, with an average radius a = 0.5360.26 mm, circularity C = 0.8660.05, and height h = 1163 mm (mean 6 SD). SEM images of a representative FE-AFM tip at different magnifications are shown in Figure 1A. The smooth edge and flatness of the tip ensured that the tip-sample contact area remained constant throughout the measurements both in compression and extension. PAA+ gel stiffness is independent of the directionality of the applied force The 4-step protocol based on FE-AFM tips was first validated on PAA+ gels with EC comparable to human lung tissue. A Lung Cell Mechanoresponse to Force Directionality representative time-recording of F on a PAA+ gel measured with a negatively charged FE-AFM tip driven in z according to Figure 1B is shown in Figure 1C. The sudden changes observed in F at ,5 s and ,35 s correspond to the short-term compressive loadings applied after the first contact or after holding z position for 30 s, respectively. Due to unspecific electrostatic interactions between the charged surfaces of the tip and the PAA+ gel, a net attractive force was developed between them, resulting in large adhesion forces as the F*,220 nN displayed in the unloading ramp beyond zc in Figure 1C. This large adhesion enabled calculating ET around an operating point (,1.5 mm above zc) symmetric to that used to calculate EC. Figure 1F shows an F versus z plot corresponding to Figure 1B–C. The average E data obtained from this protocol are given in Figure 2A. These measurements clearly show several hallmarks of linear elastic materials. First, a linear relationship between F and z as predicted by the F(d) equation (Figure 1F). Second, the overlap between the loading and unloading curves, which indicate lack of hysteresis due to viscous dissipation, (Figure 1F). Third, the absence of stress relaxation in step II (Figure 1C), indicating the time-independence of E at least in the time window of our experiments. Finally, we did not find any statistically significant difference among E measured in compression and extension (Figure 2A). Consistently, the average ratio EC/ET was equal to 1 (Figure 2B), indicating that E was symmetric, i.e. independent of the force directionality. Compara- ble results were observed using RGD-coated FE-AFM tips (Figure S3), confirming that the linear elasticity measured in PAA+ gels was not compromised by the coating of the FE-AFM tip. Local cell stiffness depends on the directionality of the applied force Unlike PAA+ gels, all cell types displayed several hallmarks of viscoelastic behavior. First, F decayed markedly and smoothly in step II (Figure 1D), indicating that cells exhibit stress relaxation when subjected to sustained compression. Similar decays were observed with either uncoated and coated tips. Likewise, the time- dependence of the decay in F for all tip coatings was well fitted to a power-law with a small exponent a,0.14–0.20 (r2$0.9), in agreement with previous findings in A549 and other cell types [23]. The stress relaxation of the A549 cell seen in Figure 1D is displayed as a sudden drop in F in Figure 1G (step II); accordingly, this drop should not be mistakenly interpreted as the tip penetrating the cell. Second, Figure 1G showed hysteresis, indicating dissipation of mechanical energy due to internal viscous friction. Unlike in PAA+ gels, tip detachment from the cell was not abrupt, but rather smooth (Figure 1G). To examine how lung cells respond to force directionality, we compared EC and ET measured at both integrin-specific and non- specific adhesion sites. Unlike PAA+ gels, the mechanoresponse of all cell types to compression and extension was markedly non- symmetric (Figure 2C–F). EC in CCD-19Lu fibroblasts were six- fold larger than ET regardless the tip-coating (Figure 2D), and their difference was statistically significant in all conditions (Figure 2C). Similar results were found in A549 cells, although the ratio EC/ET was smaller than in CCD-19Lu cells (Figure 2D, F). Likewise, all E data were at least four-fold smaller in A549 (Figure 2E) compared to CCD-19Lu cells (Figure 2C), which is unsurprising given the well established force generating role of fibroblasts [20]. These data indicate that the mechanical asymmetric response between compression and extension is not restricted to a single cell type and is independent of the biochemical nature of the coating. To examine whether the asymmetric resistance to force directionality observed with RGD-coated tips was due to insufficient contact time to form mature FAs, which are typically PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32261