independent of the loading force. Moreover, contact elastic models for flat tips predict a linear relationship between loading force and resulting sample deformation that holds in both compression and extension, thereby facilitating data analysis [14,15]. In addition, FE-AFM tips provide unique versatility since the tip diameter, the characteristics of the exogenous force –including directionality, time-dependence and amplitude–, and the biochemical nature of the tip surface coating can be controlled independently. However, the application of FE-AFM tips to study cell-ECM mechanical interactions is still very scarce. The goal of this work was to study how lung parenchymal and stromal cells respond mechanically to local bidirectional forces of compression and extension by nanofabricated FE-AFM cylindrical tips. To apply mechanical stimuli to integrins specifically, tips were coated with a synthetic peptide containing the tri-aminoacid Arg- Gly-Asp (RGD) sequence, which is an integrin-specific binding domain present in fibronectin and other ECM components [16] widely used in studies of cell-ECM mechanical interactions [10,17]. Bare tips or tips coated with non-integrin specific molecules were used as negative controls. To mimic key geometrical aspects of physiological cell-ECM adhesions, tips were nanofabricated with a cross-section area of ,1 mm2 and brought into contact with the cell surface for ,30 s, thereby enabling the formation of focal adhesion (FA) precursors [7,18]. A protocol including a bidirectional loading regime was used to apply compression and extension to the surface of single parenchymal or stromal lung cells, and to probe cell resistance to these deforming forces by measuring the Young’s elastic modulus (E). This protocol was previously validated on synthetic + positively charged polyacrylamide (PAA ) gels known to exhibit linear elasticity [19]. Materials and Methods Cell culture and treatments All experiments were carried out with stromal CCD-19Lu human lung fibroblasts and parenchymal A549 human alveolar epithelial cells (ATCC, Manassas, VA). Both cell lines were cultured as described elsewhere [14,20]. Two days before the experiments, cells were plated on 25-mm diameter glass coverslips in serum-containing culture media for 24 h, and in serum-free media one day before measurements. In some experiments, cells were treated with either latrunculin-A (latA) (2 mM), nocodazole (10 mM), EGTA (5 mM), BAPTA/AM (10 mM), phenylarsine oxide (PAO) (2 mM), or aminoguanidine (AMG) (1 mM), (Calbio- chem, La Jolla, CA). All treatments or the corresponding vehicle were added to the culture medium 30 min before measurements, and were present throughout the experiment. PAA+ gels Gel samples were prepared as previously described [21] (see details in Text S1). To render the gel positively charged, the acrylamide solution contained 70% of 40%-acrylamide (Bio-Rad) and 30% of 3-acrylamidopropyl triethylammonium-acrylamide (Sigma). The concentration of the bisacrylamide crosslinker was chosen to elicit a gel stiffness comparable to lung tissue (,10 kPa) [22]. Nanofabrication of cylindrical FE-AFM tips by Focused Ion Beam (FIB) The pyramidal tip of silicon cantilevers (MikroMasch, Tallinn, Estonia) was shaped into flat-ended cylinders by FIB following a slightly modified two-step procedure reported elsewhere [14]. First, a cylinder was obtained by milling the tip applying a ring-like Lung Cell Mechanoresponse to Force Directionality ion beam (1 mm inner radius, 2.5 mm outer radius) centered on the tip apex with a tilt angle of 11u, dwell time of 10 s, and beam intensity of ,3000 pA. This step was repeated by reducing progressively the outer diameter. Second, a flat ended tip was obtained by milling a straight line pattern (,1000 pA) perpen- dicular to the cylinder axis. Finally, Scanning Electron Microscopy (SEM) images of the tips (Figure 1A) were acquired to assess the actual radius, and to measure the height and circularity of the cross-section with SEM software navigation tools. Modification of FE-AFM tips For measurements with PAA+ gels, tips were rendered negatively charged by cleaning with piranha solution (30 min), rinsing in distilled water, and cleaning with low power oxygen plasma in a plasma cleaner-sterilizer (PDC-002, Harrick Scientific Corporation, Ossining, NY) for 1 min. For peptide or protein coatings, tips were first cleaned using piranha solution (30 min), acetone (5 min) and UV irradiation (15 min). Second, the cantilevers were soaked in a solution of 5% 3-aminopropyl- triethoxysilane (Fluka, Buchs, Switzerland) in acetone, rinsed with distilled water and shaken for 30 min in a solution of 0.5% glutaraldehyde (Sigma), washed with distilled water and air dried. Finally, the cantilevers were immersed overnight at 4uC in 0.1 mg/ml solutions containing either RGD (GRGDSP-peptide, Calbiochem, Giessen, Germany), an RGD mutant containing the tri aminoacid Arg-Gly-Glu (RGE) (GRGES-peptide, Peptides International, Louisville, KY) or bovine serum albumin (BSA) (Sigma). AFM setup The details of AFM setup are given elsewhere [14]. In brief, the setup was provided with a long range linearized piezoelectric translator to control the tip position with respect to the sample. The deflection (d) of the cantilever was measured with the optical beam deflection method. A control code written in LABVIEW (National Instruments, Austin, TX) was used to generate the driving signal for the piezoelectric position and to acquire both the actual piezoelectric vertical displacement (z) and d signals. For each cantilever, the actual spring constant (k) was calibrated using the thermal fluctuations method [14], and used to calculate the force (F) as F = kd. AFM measurements For each cell, we first recorded three standard force-displace- ment (F-z) curves on the perinuclear region of the cell surface (Figure S1), and used these curves to assess the tip-sample contact point (zc) and the deflection offset at zero-force (d0) as previously described [23]. Second, the piezotranslator was lifted 13.5 mm above zc, and F was recorded as a function of time (t) while this four-step protocol was applied (Figure 1B, E): (step I) the tip was brought back in contact with the cell by moving the piezo- translator forward with a 14.5 mm amplitude ramp at 3 mm/s, eliciting a maximum indentation (d)#1 mm; (step II) the piezo- translator position was held for 30 s to enable the formation of FA precursors; (step III) the piezotranslator was moved forward further with a 0.5 mm amplitude ramp at 3 mm/s, and (step IV) backward at the same speed with a 15 mm amplitude ramp. The data collected during step III and IV were used to assess E in compression (EC) and in extension (ET). In these conditions, the hydrodynamic viscous drag force on the cantilever was negligible [24]. For each tip surface modification, AFM measurements were repeated on at least two independent cell samples (n = 12). The same protocol was applied on PAA+ gels. In some experiments, the four-step protocol was slightly modified to control the force during PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32261