restricted by the nanoscale spatial distribution of ICAM1

Transcription

1 1 Supporting information: Podosome formation and development in monocytes restricted by the nanoscale spatial distribution of ICAM Andreas S. Andersen 1, Hüsnü Aslan 1, Mingdong Dong 1, Xingyu Jiang 2, Duncan S. Sutherland 1 * : Interdisciplinary Nanoscience Center (inano), Århus University, Århus, Denmark 2: National Center for Nanoscience and Technology (NCNST), Chinese Academy of Sciences (CAS), Beijing, China

2 Nanopattern fabrication: This process is described in our previous work 1, 2. Briefly three layers of charged polymer are sequentially deposited onto a gold coated silica wafer. A solution of charged sulfate latex beads (Lifetechnologies), with diameters equal to the diameter of the desired holes is added, and through selfassembly they form a nanopattern on the surface. After incubation the samples are rinsed and heated before dried with nitrogen. 2nm Ti and 10nm SiO2 is deposited onto the surface and the latex beads are removed by tape-striping and sonication, resulting in the desired nanopattern, (S1). We have previously shown the possibility to fabricate ring and crescent structures 3, and here these principles are utilized. For the ring structures the latex beads were assembled on a silica wafer without an Au layer and a two-step deposition was performed. First 4nm Ti and 20nm Au was deposited from an angle (800/173Ring 37, 800/133Ring 23, 800/67Ring 11, 800/31Ring 6, 500/125Ring 65, 500/97Ring 30, 500/56Ring 15 ) to coat the area normally shadowed by the latex particles. Afterwards a deposition of 2nm Ti and 10nm SiO2 orthogonal to the surface covered the Au outside the shadowed area. The latex particles were removed by the previously mentioned process, and Au ring structures is obtained. The 950/10% structure is obtained by performing a standard deposition of 1800nm beads on an Au surface. At the SiO2 evaporation step (4nm Ti, 20nm SiO2) the angle of deposition is changed from orthogonal to 40 degrees which results in 950nm patches, with a larger nearest neighbor distance compared to the normal 800nm patterns. The same method is used for the 470/11%, 420/8% and 300/8%; 470/11%, 800nm starting particle, 43, 4nm Ti, 20nm SiO2; 420/8%, 800nm starting particle, 55, 4nm Ti, 20nm SiO2; 300/8%, 600nm starting particle, 65, 4nm Ti, 20nm SiO2. The crescent sample is fabricated starting from an 800nm particle on a silica surface. Au is then deposited from an angle like the ring samples, but without rotation, causing the Au to only coat underneath a single side of the 800nm particles. A 2nm Ti, 10nm SiO2 evaporation from an orthogonal

3 angle then coats all the Au not shadowed by the particle resulting in an Au crescent shape. The 300nm structures with a higher density than the normal 300nm/15% was prepared by adding salt (NaCl 2 ) to the particle solution before adding the solution to the Au surface. This causes a screening effect of the charged particle surfaces and results in a closer packing of the particles on the surface. For 300/22% 0.03mM NaCl 2 was added while 300/24% had 0.1mM NaCl 2 and 300/31% had 0.5mM NaCl 2. After this step the surfaces was made following the same protocol as the normal 300nm/15%. Protein patterning: All samples were made on 6*6mm precut 4 inch silica wafers (Crystal-GmbH). Thiolation with 2mM 1-octadecane thiol (Sigma-aldrich) in EtOH was allowed to incubate for 24h before being cleaned with 2 rounds of 3min sonication in EtOH. From this point on all work was done in a sterile flow bench. The samples were moved to a MilliQ bath, broken up and transferred to 48 well plates, with 600μL HEPES. Proteins were sequentially incubated with three washing steps between each incubation. 200μL of liquid were always left in the well to avoid dewetting of the samples. The sequential order of the proteins added, with the final concentration are: PLL-g-PEG (Poly-L-Lysine grafted Poly-Ethylene- Glycol, Surface Solutions) 0,25mg/mL in HEPES for 1h, streptavidin (Sigma-aldrich) 100ug/mL in Ringers for 2h, BSA (Bovine Serum Albumin, Sigma-aldrich) 2wt% in Ringers for 30min, protein-a (Sigma-aldrich) 20μg/mL in Ringers for 1h and finally ICAM1-FC (sinobiological) 5ug/mL in Ringers, o/n incubation. HEPES was made with 10mM HEPES and ph was adjusted to 7.4. Ringers was made with 10mM HEPES, 7,2mM KCL, 154mM NaCl and ph was adjusted to 7,4. Cell incubation: THP1 cells were cultured using RPMI 1640 with 10% heat inactivated FBS (fetal bovine serum) and 1% penicillin / streptomycin. THP1 cells were frozen after 10 initial passages and aliquoted out. Each batch were then passaged twice a week and kept running for up to three months after which a new

4 batch were thawed. THP1 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, ACC 16). The cells were activated by adding a final concentration of 600ng/mL PMA to 5mL 2*10 5 cells/ml in 6 well plates and left for 15min at 37, 5% CO 2. The media was changed to RPMI 1640 without serum by spin down and resuspension. 500μL 2*10 5 cells/ml were added to each 48 well containing one sample and 500μL media each. The cells were incubated on the samples for 2h at 37, 5% CO 2 afterwards 500μL were slowly removed, 500μL 8% PFA was added and left o/n. Staining and imaging: The general staining procedure is as follows, with three washing steps between each incubation: 30min incubation with 1% triton X-100 in blocking buffer, 1.5h incubation with primary antibodies in blocking buffer, 1h incubation with secondary antibodies, DAPI and phalloidin in blocking buffer. Blocking buffer is PBS with 1% HAB (human antibody serum) and 2wt% BSA. Primary antibodies used are: Anti-vinculin mouse (abcam) 1:800, anti-icam1 mouse (R&D systems) 1:50, anti-mmp14 rabbit (abcam) 1:200, anti-paxillin rabbit (santa cruz biotechnology) 1:100, anti-cd11a m24 mouse (abcam) 1:100, anti-cd11a TS2/4 mouse FITC (biolegend) 1:100, anti-cd11a E-1 mouse (santa cruz biotechnology) 1:50, anti-cd11a EP1285Y rabbit (abcam) 1:100, anti-cd11b EP1345Y rabbit (abcam) 1:100, anti-cd11b M1/70 rat (abcam) 1:200, anti-cd11b EPR1344 rabbit (abcam) 1:250. Secondary antibodies used: Anti-mouse donkey AF488 (Invitrogen) 1:400, anti-rabbit donkey AF488 (Jackson immunoresearch) 1:200, anti-rat goat AF488 (abcam) 1:200, anti-rabbit donkey TRITC (Jackson immunoresearch) 1:100. Other stains used are: DAPI (4,6-Diamidino-2-phenylindole) 1mg/mL diluted 1:1000 (Sigma-Aldrich), phalloidin (Tetramethylrhodamine B isothiocyanate (TRITC) conjugated Phallodin) 0.5mg/mL in Methanol diluted 1:1000 (Sigma-Aldrich). 10x images for cell counts were taken on Leica DM6000B microscope (Leica microsystems) with a water immersion lens.

5 Confocal images were taken on LSM700 (Zeiss). Analyzes of the cell counts and confocal images were both done using the free software Fiji (NIH). For the quantification of cell numbers each 6x6mm sample where imaged at four preset locations and the summed cell number for these images were taken as the cell count for the sample. Six 6x6mm samples were imaged by this method giving n=6 for each sample type. Nanopattern characterization: SEM (scanning electron microscope) images were taken with Nova NanoSEM scanning electron microscope (FEI) and analyzed for nearest neighbor distances and area by Fiji (NIH). AFM (atomic force microscope) images were acquired using DimensionIcon AFM (Bruker, Santa Barbara, CA, USA) under ambient conditions (temperature: 21 C and humidity: 49%) on tapping mode in liquid (Fig. S3a) and in air (Fig. S3b). MPP , Tap150A, and ScanAsyst Fluid cantilevers with 5 N/m and 0.7 N/m spring constants and nominal resonant frequency 150 khz were used (Bruker, Santa Barbara, CA, USA). The image resolution was 512 by 512 and scan rate was 1 Hz. Operation parameters such as amplitude and gains were optimized to minimize the imaging force and achieve high resolution also to avoid the sample damagee. Acquired AFM raw data were further processed and analyzed with the SPIP software (Image Metrology ApS, Lyngby, Denmark). Analysis of confocal images: Confocal images was first enhanced with a standardized linear best fit option in the ZEN software (Blue edition, Zeiss) before any analysis were performed. All podosomes visible in the actin channel was measured for several cells and averaged. If a clear vinculin patch associated with the actin podosome the width of the vinculin patch was also measured and the offset between individual podosome / vinculin patch was measured as the center to center distance. For the ring samples if a clear

6 vinculin ring could be observed in association with an actin podosome core the total diameter of the vinculin ring was measured. The data shown in figure 3 is pooled from two repeat experiments. Local coverage calculations: Local coverages were calculated for different sized overlay circles and rings. For 100nm, 160nm, 200nm and 300nm/15% the maximum number of particles within the overlay was estimated based on SEM images and the coverage was calculated as the number of particles multiplied by the average size pr. patch divided by the total area of the overlay. For the 300nm/22%, 300nm/24% and 300nm/31% the coverage was calculated by thresholding a SEM image in FIJI and thereby converting it into a binary image with the Au area marked. A ring was placed at different spots manually chosen to optimize the amount of Au underneath and a measurement was made that calculated the area present under the overlay. Several measurements were made pr. sample and the highest measurement was chosen. For the ring samples and the 500nm sample the local coverage was calculated theoretically based on the average values obtained in SEM analysis of the surfaces, shown in fig. S2. The overlay was either calculated for a directly centered overlay or as being between two patches. When the overlay was calculated between patches the NND was used as the patch to patch distance, while an equation (1) for overlapping circles was used to calculate the theoretical overlap between the overlay and Au patch and multiplied by 2 to account for the overlap on either side of the overlay. A = r 2 cos 1 ( d2 + r 2 R 2 ) + R 2 cos 1 ( d2 + R 2 r 2 ) 2dr 2dR 1 (1) 2 ( d + r + R)(d + r R)(d r + R)(d + r + R) Here A is the area of the overlap, r and R is the radius of the two circles respectively and d is the center to center distance between the two circles. For ring samples the SiO 2 inner circle overlap with the

7 overlay was subtracted if the overlay was reaching that far into the ring sample e.g. the 500/125Ring sample. Flow cytometry: Cells were activated as described, spun down and resuspended in 50μL HAB for at least 1 min, before 50μL primary antibody (anti-cd11a m24 or anti-cd11b CBRM1/5) was added for 10min at 37, 5% CO 2, then 50μL secondary antibody (anti-mouse AF488) was added for 10min at 37, 5% CO 2. Directly after the staining the cells were analyzed with a Gallios flow cytometer (Beckman Coulter) cells were analyzed for each measurement. A gate was used to ensure that the analyzed cells were single cells by gating for cells only in the low end of FS TOF / FS PEAK graph References: 1. Kristensen, S. H.; Pedersen, G. A.; Ogaki, R.; Bochenkov, V.; Nejsum, L. N.; Sutherland, D. S. Acta Biomaterialia 2013, 9, (4), Agheli, H.; Malmström, J.; Larsson, E. M.; Textor, M.; Sutherland, D. S. Nano Lett. 2006, 6, (6), Bochenkov, V. E.; Sutherland, D. S. Nano Lett. 2013, 13, (3),

8 Supporting figure 1: SEM images of the nanostructures. The bright circular spots are Au surrounded by the darker SiO 2. Scalebar 2μm.

9 Supporting figure 2: Quantified data for the nanostructures. Au patch size is the average diameter for a single patch or outer ring for ring samples. The value for the crescent sample is the thickest dimension. Inner ring size is only for the ring samples. Nearest neighbor distances (NND) is the average distance from one particle to the closest particle to it. This was measured by converting a SEM image into a binary image and analyzing this with a NND plugin for imagej, which gives a NND for each particle. These values were then averaged across all particles measured. Global coverage is estimated from a wide field SEM image while the local coverage is the Au coverage inside a 600nm in diameter overlay circle, for some samples the local coverage is shown both if the overlay is placed centered on the patch (Center) and if the overlay is placed between two patches (Between). St.D. is the standard deviation. Local coverages of different overlays are shown in S15.

10 Supporting figure 3: Protein is specifically adsorbed to the Au regions in the ring nanopattern, shown by AFM measurements. a) ring functionalized with ICAM1. b) ring structure without functionalization. A linescan shows the dimensions of 800/187Ring a) and b) respectively. c) Overlay of the linescans from ring structures before b) and after a) biofunctionalization. The increase in height illustrates the biofunctionalization specifically located to the Au regions. 162

11 Supporting figure 4: Flow cytometry of activated THP1 cells. a) FSC/SSC plots of all sample types used. b) Histogram of the single cell fluorescence intensity shown on a log scale. THP1 cells were activated with 60ng/mL PMA for 15min and stained for high affinity LFA1 or high affinity MAC1.

12 No antibody shows the auto fluorescence of the cells. No primary, shows the unspecific binding of the secondary antibody. Isotype control shows the unspecific binding of the primary antibody. Media is showing the expression level of high affinity LFA1 or MAC1 in THP1 cells that have not been exposed to PMA cells were counted for each measurement and are plotted as the number of cells with a certain fluorescent intensity on a log scale. The fluorescence intensity is directly correlated with the amount of high affinity LFA1 or MAC1 on each cell. c) Quantified data: High affinity LFA1 or MAC1 expression is set to 1 for THP1 cells without activation (media). Data is shown as the median of the fluorescence distribution. 175

13 Supporting figure 5: Representative 10x images of THP1 cells adhering to different sized patterns, homogenous and PEG control. The nukleus was stained with DAPI and shows up as blue dots in the images. Scalebar 200μm. 180

14 Supporting figure 6: Quantified cell counts of three repeat experiments. Six 6x6mm sample pr sample type were imaged at four preset locations and the total number of cells were counted for each sample. The 300nm surface was not included in one of the experiments, while the 950/10% and 800/187Ring where part of one of the experiments. Error bars is the standard deviation between samples

15 Supporting figure 7: Quantified cell counts of two repeat experiments. Six 6x6mm sample pr sample type were imaged at four preset locations and the total number of cells were counted for each sample. The values shown here are normalized against the Homogen. value for each experiment. The blue graph is also shown unnormalized in fig. 2. It should be noted that the red graph have a cell number for the Homogen. of 2932 ±194, which is significantly higher than the value for the other experiment, but the comparable binding levels for all the samples were also higher as can be seen by comparing the normalised data. Error bars is the standard deviation between samples. 196

16 Supporting figure 8: Statistical analysis of cell binding experiment. The cell binding of the experiment

17 shown in fig. 2a were tested for variance and normality, which after a square transformation was achieved. The transformed data were then analyzed with an anova and Turkeys multiplecomparison test to look for significant differences at the 0.05 level. The data was then backtransformed, which is the data shown in fig. 2a. Compared samples indicate which sample types are being tested. Prob. is the probability two samples are statistically different. If the probability is less than 0,001 it is written as <0.001 under Prob.. The probability is compared against the 0.05 probability level where a probability below this level shows the samples to be statistically different indicated by a 1 under S while a 0 show statistically identical samples at the 0.05 level

18 Cell count pr sample Normal Anti CD11a Anti CD11b Anti CD18 No ICAM nm surface Supporting figure 9: THP1 blocking experiment. All tests were done on 800nm surfaces. Normal corresponds to THP1 cells treated as a standard cell experiment. Another set of THP1 cells were treated with either anti CD11a (LFA1 subunit), anti CD11b (MAC1 subunit) or anti CD18 (LFA1 and MAC1 subunit), before being seeded onto the ICAM1 coated 800nm surface. No ICAM1 represents 800nm surfaces that have not been incubated with ICAM1, but instead a mock antibody were added to protein- A. THP1 cells for this surface have been treated in the normal fashion. Each 6x6mm sample were imaged at four preset locations and the total number of cells were counted, with six samples pr surface type. Error bars is the standard deviation between samples. 218

19 Supporting figure 10: Confocal images of THP1 cells on homogeneous surfaces. Not all cells show rosette formation. Top row, from left to right: Paxilin (green), actin (red) and overlay of the two. Bottom row, from left to right: Vinculin (green), actin (red) and overlay of the two. Arrows show individual podosomes in the rosette. Scalebar 10μm

20 Supporting figure 11: Confocal images of THP1 cells on structured and homogeneous surfaces. A) Cells were stained for paxillin (green) and actin (red). The merged are the overlay images. Arrows show individual podosomes in the rosette. Scalebar 5μm. 230

21 Supporting figure 12: Confocal images of THP1 cells on surfaces. THP1 cells were stained for vinculin (green) and actin (red). The merged images show the merge of vinculin and actin. Scalebar 5μm. A squrare box shows the zoom in areas shown in figure

22 Supporting figure 13: Podosomes imaged for another experimental repeat compared to the ones shown in fig. 3. Vinculin is shown in green and actin is shown in red. Merged image illustrates the overlap between the vinculin and actin. Scale bar 2µm. 240

23 Supporting figure 14: Local coverage of different overlays. Au coverage inside a 650nm in diameter overlay circle and 550nm in diameter overlay circle is shown. The local coverage of an overlay ring 600nm outer ring diameter and 400nm inner ring diameter is also shown for some of the samples. For some samples the local coverage is shown both if the overlay is placed centered on the patch (Center) and if the overlay is placed between two patches (Between). St.D. is the standard deviation

24 Supporting figure S15: Close-up SEM images with a 600nm ring overlay to illustrate podosome size on the different nanopatterns. The ring has been placed to both show centered and between patches. The red percentages show the average local coverage calculated to be present within the red ring. Each image measures 2x2µm with a 500nm scalebar. Original images can be seen in S1. 254

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26 Supporting figure S16: THP1 cells adhering to 800nm samples stained for cd11a (LFA1) or cd11b (MAC1). Each row represents a different antibody against cd11a or cd11b (green channel), with clone name written to the side. The actin cytoskeleton is shown in red and the merge of the green and red is shown as merged. For some of the antibody stains the image of actin and cd11a/b is very similar causing suspicion of fluorescent bleed-through, even though each channel is imaged sequentially. To test this, the actin stain was bleached with a 555nm laser and the image was reacquired under the same settings. The merged image of the red and green channel after bleaching is shown as merged + bleached. Scalebar 5µm. 264