SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: 1.138/nnano Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Zhou J. Deng, Mingtao Liang, Michael Monteiro, Istvan Toth and Rodney F. Minchin Supplementary Methods Protein separation and identification For 1-D gel electrophoresis, 2 μl of sample was separated on a 12% SDS-polyacrylamide (SDS-PAGE) gel. The gels were run at the constant voltage of 2 V for 35 min, and stained with Sypro Ruby Protein Stains (Bio-Rad). For 2-D gel electrophoresis, 4 μl of the prepared samples was mixed with 7 M urea, 2 M thiourea, 4% CHAPS,.4% DTT and 1% Bio-Lyte 3/1 ampholyte (Bio-Rad) in a final volume of 3 μl. The sample was then applied to a 17 cm ph 3-1 Readystrip IPG strip (Bio-Rad) and actively rehydrated at 5 V overnight using a Protean IEF cell. Isoelectric focusing was performed for a total of 45 kvh at 2 C. The IPG Strips were reduced with 1% DDT in equilibration buffer (1 mm Tris-HCl, 6 M urea, 3% glycerol, 2% SDS) for 15 min and then alkylated with 4% iodoacetamide in equilibration buffer for a further 15 min. Equilibrated IPG strips were transferred onto 17 cm 8-15% SDS-polyacrylamide gradient gels and sealed with 1% agarose. The gels were run using a Protean II xi cell apparatus at 15 W/gel until the dye front migrated to the bottom of the gel and silver-stained as described elsewhere 5. All gels were imaged with a ChemiDoc XRS system using Quantity One 4.5. software (Bio-Rad). Protein spots from 2-D gels were excised and in-gel digested with trypsin according to established procedures 5. The peptide extracts were analysed by LC-MS/MS on an Agilent 11 Nano HPLC (San Jose, CA, USA) coupled to a QStar Elite mass spectrometry (Applied Biosystems) equipped with a nanoelectrospray ion source. The spectra were acquired and processed using Analyst QS 2. software (Applied Biosystems). The database searching for protein identification was carried out using Protein Pilot 2..1 (Applied Biosystem). Nuclear extraction and electrophoretic mobility shift assay (EMSA) Extraction of THP-1 cell nuclei and procedures for electrophoretic mobility shift assays have been nature nanotechnology 1

2 supplementary information doi: 1.138/nnano described elsewhere 31. Briefly, THP-1 cells ( ) were harvested by centrifugation at 1, g for 5 min, washed once with ice-cold PBS and resuspended in 6 µl of ice-cold lysis buffer (1 mm Hepes, ph 7.9, 1.5 mm MgCl 2, 1 mm KCl,.5 mm DTT,.5 mm PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin A). On ice, the cells were disrupted by passage through a 27-gauge needle. The nuclei were collected by centrifugation at 15, g for 8 s. The supernatants were discarded and the cell pellets were resuspended in 7 µl of ice-cold extraction buffer (lysis buffer containing 2 mm Hepes, ph 7.9, 42 mm KCl,.2 mm EDTA and 25% glycerol). After 3 min on ice, 5 µl of storage buffer (2 mm Hepes, ph 7.9,.5 mm DTT,.2 mm EDTA, 2% glycerol,.5 mm PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin A) was added and the extracts centrifuged at 15 g for 1 min at 4 C. Protein concentrations (usually 3 6 µg/µl) were determined by the Bradford method, using BSA as a standard. The samples were snap-frozen in liquid nitrogen and stored in -8 o C until used. Nuclear extracts (5 µg of protein) were incubated for 3 min at 22 C in binding buffer (2% Ficoll 4, 5% glycerol, 2 mm Hepes ph 7.9, 5 mm KCl, 1 mm EDTA, 2.5 mm DTT) with.5 µg of poly[d(i-c)] and 45 nci 32 P-labelled double-stranded oligonucleotide containing the consensus NF-κB DNA binding motif (5'-AGT TGA GGG GAC TTT CCC AGG C-3', 22-mer, consensus sites are underlined; Geneworks) in a final volume of 1 µl. Double-stranded oligonucleotides were prepared by heating both sense and anti-sense strands at 65 C for 5 min before slowly cooling to 22 C. Oligonucleotides were labelled using [ - 32 P]ATP (3 Ci/mmol) and T4 polynucleotide kinase. Labeled probes were purified from unincorporated [ - 32 P]ATP using a MicroSpin G-25 column. For P65 supershift analysis, binding reactions were incubated with 1 µg of P65 antibody (Santa Cruz) for 1 min at 22 C before the addition of probe. Samples were then resolved by electrophoresis on 5% polyacrylamide gels at 12 V in.25 Tris/borate/EDTA buffer for 12 min at 4 C. Gels were dried and processed using a Fujifilm BAS-5 phosphorimager (FUJIFILM). Image integrity All gel images were enhanced in Adobe Photoshop by adjusting brightness and contrast to obtain a grey background. No other manipulations were employed. Processing was applied equally across each image. 2 nature nanotechnology

3 doi: 1.138/nnano supplementary information Supplementary figures a Hydrodynamics diameter (nm) in water in PBS b b Sizes (nm) Time Time (min) kda kda c Number of washes kda d 1 7 ph Albumin Mol Wt (kd) Figure S1: : a, Hydrodynamic diameters of PAA-GNPs of 5 nm (triangle), 1 nm (square) and 2 nm (circles) in water and in PBS as measured by dynamic light scattering (at 25 o C and a concentration of 5 mg/ml, n=3). Nanoparticle agglomeration was not seen in either water or PBS. b, Human plasma protein adsorption on PAA-GNPs of 5 nm, 1 nm and 2 nm were examined. Sizes did not qualitatively affect the adsorption pattern. The adsorption patterns did not change qualitatively or quantitatively over 4 h of incubation, indicating the interaction saturated and reached equilibrium within the first few minutes of incubation. c, High affinity binding of fibrinogen to 5 nm PAA-GNPs. The adsorption pattern did not change after 4 time of washes. d, Representative two-dimensional gel of protein bound to the 5 nm PAA-GNP. Protein spots representing fibrinogen α, β and γ chains are shown and were confirmed by mass spectrometry. nature nanotechnology 3

4 supplementary information doi: 1.138/nnano Molar Ellipticity (deg.cm 2 dmol -1 [x 1 3 ]) g/ml PAA-GNP 1 g/ml + 4 g/ml PAA-GNP 1 g/ml Wavelength (nm) Figure S2: Circular dichroism of fibrinogen in the absence and presence 2 nm PAA-GNP. 1 Zeta-potential (mv) PAA content (%) Figure S3 Zeta-potentials of PAA-PDHA-GNPs 4 nature nanotechnology

5 doi: 1.138/nnano supplementary information a b 3 Albumin Cell count 2 1 THP-1 HL-6 THP-1 HL-6 Figure S4 a, Binding of THP-1 and HL-6 cells to albumin-coated surface (open bars) or fibrinogen-coated plastic surface (closed bars). Fifty thousands of cells were incubated on protein-precoated 96-well plate for 2 mins at 37 o C. Cells were then washed with PBS and resuspended in trypsin buffer. The total numbers of cells were estimated using a hemocytometer. b, Microscopic image of cells binding to protein-coated surface. Cells were stained by crystal violet. 8 8 IL-8 pg/ml TNF-alpha pg/ml PAA-GNP ( g/ml) PAA-GNP ( g/ml) Figure S5 IL-8 (a) and TNF-alpha (b) releases from THP-1 cells by increasing fibrinogen PAA-GNP complex (1:3 fibrinogen-nanoparticle weight ratio). Results are mean ± s.e.m, n = 3. nature nanotechnology 5

6 supplementary information doi: 1.138/nnano Table S1 Protein identification using mass spectrometry Protein ID* Unused Total %Cov %Cov(5) %Cov(95) Accession Name Albumin P2768 ALBU_HUMAN Serum albumin precursor - Homo sapiens (Human) α chain β chain P2671 FIBA_HUMAN alpha chain precursor - Homo sapiens (Human) P2675 FIBB_HUMAN beta chain precursor - Homo sapiens (Human) γ chain P2679 FIBG_HUMAN gamma chain precursor - Homo sapiens (Human) * Protein IDs correspond to the 2-D gels shown in supplementary figure 1d Table S2 Characterisation of PAA-PDHA hybrid GNPs 6 nature nanotechnology