Materials and methods

Materials and methods Materials Precut wafers (P/Boron<100>, SI-MAT) were purchased from Litcon AB (Sweden). PDDA (Poly(diallyldimethylammonium chloride)), PSS (Poly(sodium-4-styrenesulfonate)) and Octadecylmercaptan were purchased from Sigma. PAX-XL60 (polyaluminium chloride) was purchased from Kemira miljø (Denmark). Polystyrene colloidal particles, sulphate latex diameter 0.1µm, 0.2µm, 0.5µm and 1µm was from Invitrogen (US). Buffers were prepared with MQ-water (MilliQ Gradient, Millipore) and filtered through a 0.2µm pore filter prior to use. The buffers used was HEPES 10mM at ph7.4 for the PLL-g-PEG and TRIS 10mM (Tris hydroxylmethyl)aminomethane) 2.7mM KCl, 137mM NaCl ph7.4 for the protein. Bovine Serum Albumin (BSA) was purchased from Sigma-Aldrich (cell culture tested 96% purity). Fibronectin (Sigma- Aldrich, Denmark) was stored at 4 C and sterile filtered prior to use. Vitronectin (R&D systems, UK) was stored at -20 C until dissolved and used. PLL(20)-g[3.5]-PEG(2) (SurfaceSolutions, Switzerland) was dissolved in HEPES buffer to a concentration of 0.25 mg/ml and sterile filtered before use. For immunofluorescence, primary antibody for vinculin, rhodamine-labelled Phalloidin and DAPI (4,6-diamidino-2-phenylindole) were from Sigma and secondary antibody for vinculin (Alexa Flour 488 F(ab')2 fragment of goat anti-mouse IgG (H+L)) was from Invitrogen. Sample preparation Samples were prepared on precut oxidized silicon wafers and coated by 4nmTi and 30nm Au (RF magnetron sputtering (home made), 2x10-3 mbar argon pressure, Ti deposition rate 1nm/s (6.45 Watt/cm 2 ), Au deposition rate 2.2 nm/s (2.5 Watt/cm 2 ). Gold coated wafers were cleaned by UV/ozone for 1h prior to use followed by immersing in MQ-water for 1h after UV/ozone treatment to allow for the Au 2 O 3 formed 1 to be reduced back to Au 0. Thiolation (35mM) was performed in ethanol (p.a grade, Merck) for a minimum of 12 hours. After the assembly the surfaces were sonicated in ethanol and MQ-water and subsequently dried under a stream of nitrogen. Nanostructured samples were fabricated using the basic principles of hole mask lithography. 2 The modified procedure as follows: A triple layer of PDDA (2% in MQ), PSS (2% in MQ) and PAX- XL60 (5% in MQ) was deposited onto which colloidal assembly of polystyrene particles were made (0.2% in MQ for 0.1-0.3µm, 0.5% for 0.5-0.6µm, 1% for 0.8µm and 2% for 1-3µm particles). After particle deposition, the samples were carefully rinsed, transferred without dewetting to a pressure chamber with MQ-water in which they were heated to 120 C (130 C for 1µm particles and 140 C for 3µm particles). 2nm Ti and 11nm SiO 2 was evaporated coated onto the sample (3kW Multiple Crucible Linear e-gun, Port Townsend, US. Ti deposition rate 0.5-1Å/s, SiO 2 deposition rate 1-10Å/s). The particles were removed by tapestripping and sonication in ethanol and MQ-water. The samples were characterized using Scanning Electron Microscopy (NovaSEM 600 FEI company, the Netherlands) to determine hole size and spacing and Atomic Force Microscopy (Nanoscope III, Veeco Instruments) to determine the depth of the holes. Cell culture: The C2C12 myoblast cells (from American Type Culture Collection ) were maintained in Dulbecco s modified Eagle s Medium with Glutamax (DMEM) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin and 1 mm sodium pyrovate (all from Invitrogen). For cell adhesion experiments to substrates, cells were always used at the same passage number to reduce variability.

Preparation of protein nanopatterns: During all sample transfers, washing, cell seeding and the staining procedure precautions were taken to avoid dewetting of the samples. Immediately after thiolation (2mM octadecylmercaptan (Sigma) in ethanol, p.a. 12h and subsequent sonication in ethanol and water) and sterilization in 70% EtOH of the samples they were transferred to 48 wells plates containing 10mM HEPES ph 7,4 (buffer 1). After an initial incubation in 0,25mg/ml PLL-g-PEG in buffer 1 for 30 min the sample were washed once in buffer 1 and once in 10mM Tris, 2,7mM KCl, 137mM NaCl, ph 7,4 (buffer 2). Then samples were incubated with either bovine fibronectin (F-1141, Sigma) 20µg/ml (45nM); bovine vitronectin (2348-VN, R&D Systems) 5µg/ml (67nM) or human vitronectin (2349-VN, R&D Systems) 5µg/ml (67nM), all diluted in buffer 2. Human vitronectin was utilized in a limited set of experiments to visualize vitronectin via immunostaining. Good staining was achieved for human vitronectin but we could not identify a good commercial antibody that worked against bovine vitronectin. We did not observe differences in cellular response to bovine versus human vitronectin. Next day the samples were washed once in buffer 2, blocked with 2% BSA in buffer 2 for 30 min at room temperature and washed twice in buffer 2 before the C2C12 cells were seeded at a density of 8000 cells/cm 2 in DMEM with 50 U/ml penicillin, 50 µg/ml streptomycin, 1mM sodium pyrovate and 0.1% BSA (A 3803, Sigma). Immunofluorescence Without removing the media cells were first gently supplemented with percoll solution (73% percoll, 0,9% NaCl) for washing and next fixed in 6,4% para formaldehyde in 80% percoll for 30 min before washing in PBS. After permeabilization in 0.1% Triton X 100 in PBS (T-PBS) for 10 min and incubation with 2% BSA in T-PBS for 2 hours, the cells were incubed with primary antibodies for 1.5 hours. The cells were then washed 3 times in T-PBS followed by addition of the secondary antibodies and incubation for 1 hour. Simultaneously, DAPI (4,6-diamidino -2- phenylindole) (Sigma-Aldrich) was added for nuclear staining and when stated in the text rhodamine-labelled Phalloidin (p1951 Sigma-Aldrich) for staining of actin fibers. After washing three times in T-PBS the wafers were analyzed by automated fluorescence microscopy as described. Primary antibodies, all diluted in T-PBS: anti-zyxin (sc-6437, goat polyclonal IgG, Santa Cruz Biotechnology) diluted 1:400; anti-vinculin (Clone hvin-1 mouse Ascites fluid, Sigma-Aldrich) diluted 1: 800; anti-vitronectin (MAB 2349 mouse monoclonal, R&D Systems) diluted 1:50, antiintegrin αv (AB 1923, rabbit polyclonal, Millipore) diluted 1:500; anti-integrin α5 (AB 1928, rabbit polyclonal, Millipore) diluted 1:500; anti-fibronectin (F 3648, rabbit polyclonal, Sigma) diluted 1:500. Secondary antibodies, all diluted in T-PBS; Alexa Flour 488-Goat Anti Mouse IgG (1:400) (Molecular Probes, Invitrogen), Rhodamine (TRITC)-conjugated Donkey Anti Rabbit (1:200) (711-025-152, Jackson ImmunoResearch) and Rhodamine (TRITC)-conjugated Donkey Anti Goat (1:200) (705-025-147, Jackson ImmunoResearch). Microscopy and data analysis The stained samples were imaged in PBS using a motorized Leica DM6000B microscope with water immersion objectives. At least 3 pictures were taken for each magnification on each sample. The images with 10x magnification were randomly chosen on the sample using preset stage moves, whereas the 63x magnification images were manually chosen as representative (spread cells if available) of the population on the surface. The acquired 10x images, stored in the Leica IM500 database, were automatically analyzed by a Leica Qwin macro to determine the cell number and total cell area. Prior to the automatic analysis, a color threshold was set manually for the images.

Every picture was manually inspected to correct for cases were the nuclei of cells were so close that the software could not separate them. 3 Statistics Quantitative data is displayed showing average and standard deviations. Significant differences were judged using students T-test with t<0.05 representing a significant difference. Significant differences are only indicated for each data point compared to the homogenous surfaces for FN and VN and for the significance between FN and VN on the same sample type. Preparation of cells for SEM After fixing, immunoflorescence staining and microscopy, the cells were dehydrated in a graded series of ethanol (25%, 50%, 70%, 85%, 95% and 2 x 100%) for 5 min each. Dehydrated samples were dried under a stream of nitrogen and imaged by SEM. Supplementary results Table S1: Characteristic properties of patterned substrates Sample Measured diameter (nm) Estimated number of integrins per protein patch Measured characteristic distance center to center (nm) 0.1µm 110 3 221±115 110 0.2µm 190 9 380±190 190 0.3µm 260 16 535±220 280 0.5µm 450 50 772±300 320 0.6µm 590 80 927±340 340 0.8µm 780 140 1204±380 420 1µm 960 220 1248±360 290 3µm 3100 2300 3403±1200 300 Calculated characteristic distance edge to edge (nm) The characteristic spacing of the holes on each sample type was determined by analysis of 4 SEM images from one sample of each type. The images were used to identify the center of each hole which was then used to calculate the hole radial distribution functions for each image and the average peak position marks the characteristic spacing, whereas the average full width at half maximum of the peak gives the error bars presented above. The estimate for the number of integrins per protein patch is based on the areas of each patch and from an estimate of 300 integrins per square micron from reference 4. We have quantified the binding of fibronectin and vitronectin to homogeneous surfaces under the same conditions utilising the Quartz Crystal Microbalance with Dissipation. We obtain an estimated minimum of 1400 fibronectin molecules or 12000 vitronectin molecules per micron squared. We use the Sauerbrey equation 4 to calculate the wet mass, and subsequently calculate the dry mass by assuming the density of the dry protein to be 1.3 g/cm 3 and a protein film density of 1.1 g/cm 3 (which is an estimate in line with literature values). 5-7 These results indicate that there are roughly 4 times more fibronectins or 40 times more vitronectins per patch than the number of integrins that can fit over the patch. So we believe that the number of ligands does not play a significant role in determining the cell binding properties.

Figure S1: A range of sizes of FN patterns. Size of pattern indicated in picture (nm), scalebar 20µm. 100 90 80 70 cell number cells/mm 2 60 50 40 30 FN VN BSA 20 10 0 100 200 300 500 600 800 1000 3000 Au SiO2 evap

Area/cell of spread cells 2000 1500 µm 2 /spread cell 1000 FN VN 500 0 100 200 300 500 600 800 1000 3000 Au Figure S2: Quantitative data from full repeat showing cell number and area of spread cells for each sample type. The cell area was averaged only from cells interacting substantially with the substrate with round cells below a threshold size excluded. Standard error means are displayed. The data is derived from 4 images per sample using a 10x objective, and 4 samples per condition. Significant differences (t<0.05) between each sample and the flat control (Au) are denoted with a star, and between FN and VN on the same sample type with a cross.

Figure S3: Zoom in to regions of interest in pictures displayed in figure 3 (main paper). Red stains actin and green vinculin. Scalbar 10µm. Figure continued from previous page.

Figure S4: Fluorescent microscopy images using 40x objective with a 1.6x optical zoom from the repeat experiment. High magnification images of 0.1µm patterns not included here. Blue staining the nucleus, red the actin cytoskeleton and green vinculin. The patch size is displayed for each pair of images (FN and VN). Scale bar 30µm

Figure S5: Fluorescent microscopy images using 40x objective with a 1.6x optical zoom from the experiment presented in main paper. Blue staining the nucleus, red staining zyxin and green vinculin. The patch size is displayed for each pair of images (FN and VN). Scale bar 30µm

Figure S6: Cells on 0.5µm surfaces coated with FN or VN. Stained against zyxin. Scale bar 20µm

Figure S7: Anti-integrin α 5 or α v on FN and VN patterns and homogenous controls. The size of pattern (200, 500, 800nm or Au=homogenous control) is indicated top left of each figure panel. Scale bar 20µm.

Figure S8: Examples of Anti-FN (red) and anti-vinculin (green) staining of cells on FN and VN patterns and homogenous controls. The size of pattern (200, 500, 1000nm or Au=homogenous control) is indicated top left of each figure panel. Scale bar 20µm. Figure S9: Florescence microscopy image showing vinculin (green) and the underlying pattern of 1µm in diameter FN patches (red). Table S2 Bridging focal adhesion analysis: Length and aspect ratio estimates for vinculin stained bridging focal adhesions in adherent cells Sample type Length SEM Width SEM Aspect SEM (µm) (µm) ratio VN Homogeneous 7.04 1.36 0.98 0.19 7.9 1.3 FN Homogeneous 9.63 3.52 0.74 0.09 11.9 2.8 VN200 3.93 1.78 0.55 0.04 6.1 2.9 FN200 1.24 0.61 0.51 0.04 2.5 1.1 VN800 4.68 1.34 0.64 0.05 6.0 1.9 FN800 4.44 1.29 0.60 0.12 7.3 1.8 The cells were in general characterised by having a broad distribution of focal adhesions. For the nanopatterned surfaces the majority of the vinculin stained domains corresponded to a single focal adhesion limited by the patch size. In addition we observe bridging events where vinculin (and zxyin) stained adhesion bridge several patches. The characteristics of bridging focal adhesions were quantified by measuring the length and aspect ratio of the six longest focal adhesions per field of view (from images taken at 63 times magnification - 40 times magnification with a 1.6 optical zoom typically 2-4 cells). The results thus are biased to long focal adhesions. The data are measured from 4 images from each of 4 samples and displayed as an average and a standard error mean of all images quantifying the variation from image to image. The results show a large variation which is not so surprising given the diversity of cellular response. There is a clear result that the length of the longest focal adhesions on the FN200 is around 1.2 microns in length with a low aspect ratio and

substantially lower that those at 800nm and homogeneous patterns, The nanopatterned surfaces show shorter adhesions than homogeneous VN or FN samples. These adhesions also show lower aspect ratio. Cells at the VN200 sample have very similar adhesions compared to at VN800 and FN800 patterns and are substantially longer with higher aspect ratio than for cells at FN200. These results support the idea that bridging events are occurring differently at VN200 patterns compared to FN200 patterns. The width of the adhesions shows a trend of narrower adhesions for smaller pattern sizes. However the resolution of our microscope (~500nm) precludes any further analysis. References 1. Krozer, A.; Rodahl, M. Journal of Vacuum Science and Technology a-vacuum Surfaces and Films 1997, 15, (3), 1704-1709. 2. Fredriksson, H.; Alaverdyan, Y.; Dmitriev, A.; Langhammer, C.; Sutherland, D. S.; Zaech, M.; Kasemo, B. Advanced Materials 2007, 19, (23), 4297-+. 3. Lovmand, J.; Justesen, E.; Foss, M.; Lauridsen, R. H.; Lovmand, M.; Modin, C.; Besenbacher, F.; Pedersen, F. S.; Duch, M. Biomaterials 2009, 30, (11), 2015-2022. 4. Sauerbrey, G. Zeitschrift Fur Physik 1959, 155, (2), 206-222. 5. Hovgaard, M. B.; Rechendorff, K.; Chevallier, J.; Foss, M.; Besenbacher, F. Journal of Physical Chemistry B 2008, 112, (28), 8241-8249. 6. Reimhult, E.; Larsson, C.; Kasemo, B.; Hook, F. Analytical Chemistry 2004, 76, (24), 7211-7220. 7. Voros, J. Biophysical Journal 2004, 87, (1), 553-561.