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Insights Into the Cellular Response Triggered by Silver Nanoparticles

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Insights Into the Cellular Response Triggered by Silver Nanoparticles Insights into the Cellular Response Triggered by Silver Nanoparticles using Quantitative Proteomics Thiago Verano-Braga, † Rona Miethling-Graff, ‡ Katarzyna Wojdyla, † Adelina Rogowska- Wrzesinska, † Jonathan R. Brewer, †† Helmut Erdmann, ‡ Frank Kjeldsen †* †Protein Research Group and †† MEMPHYS Center for Biomembrane Physics – Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark ‡Biotechnology, University of Applied Science, Flensburg, Germany Supplementary Figure S1 – Proteomics reproducibility. In total, 3352 proteins were identified in this study using 3 independent biological replicates and 2524 proteins (75%) were identified in at least 2 replicates. Supplementary Figure S2 – Data normalization and significance analysis. (A) Boxplots for each of the 5 experimental conditions from the 3 independent biological replicates. Data was normalized by the median. (B) Histogram and (C) Q Q plot depicting the normal distribution of the dataset. (D) Volcano plot representing the p- values versus intensity changes for each experimental condition (20 nm AgNPs, 100 nm AgNPs, Ctrl 20 nm and Ctrl 100 nm). The p-values were corrected for multiple testing with Benjamini-Hochberg. Points above the dashed lines represent the proteins differentially regulated ( p < 0.01). Supplementary Figure S3 – Protein-protein interacting networks. Stronger interactions are represented by thicker lines. Over-represented biological functions based on gene ontology annotation are depicted in the figure. Confocal imaging – Confocal imaging was carried out on a Zeiss LSM 510 META. The samples were imaged using a 63 x 1.4 NA oil immersion objective (Carl Zeiss). For the 100 nm nanoparticles, the excitation laser was a 543 nm HeNe laser, they were imaged by detecting their reflection of the excitation laser. The reflection of the laser from the AgNPs was collected through a 500-550 nm band pass filter. The 20 nm nanoparticles were imaged using a femto second pulsed laser (Mai Tai broad band, Spectra Physics, Mountain View, CA ) operated at 800 nm. The emission was collected through a 685 nm short pass filter. Control measurements were performed to ensure that only the AgNPs were imaged in the channel designated for recording the nanoparticles. The fluorescent emission from the DiI was collected through a 560 nm long pass filter. The images shown are representative of more than 30 cells. The multiphoton excited emission of AgNPs has been previously demonstrated. Figure S4 shows spectrally resolved images of emission from 20 nm AgNPs excited by 800 nm femto second pulsed light. The data for the spectra were collected using a band width of about 43nm, using the Zeiss LSM 510 META. 1600 1500 1400 1300 1200 1100 Emission [arb. units] [arb. Emission 1000 900 800 400 450 500 550 600 650 Wavelength [nm] Supplementary Figure S4 – 20 nm AgNPs imaged using a Zeiss LSM 510 META, excited with 800 nm femto second pulsed light. Supplementary Table 1 - List of all proteins identified in the study AgNPs Ct rl ID GENE Control 20nm 100nm 20nm 100nm Description Q9NRG9 AAAS 0.1 0.0 -0.1 0.0 -0.1 aladin isoform 1 Q13685 AAMP -0.1 0.3 0.1 -0.1 -0.2 angio -associated migratory cell protein P49588 AARS -0.4 0.3 0.2 -0.1 0.0 alanine--tRNA ligase, cytoplasmic Q5JTZ9 AARS2 0.2 -0.1 -0.1 0.1 -0.1 Alanine--tRNA ligase, mitochondrial Q9NRN7 AASDHPPT -0.2 0.1 0.1 -0.1 0.1 L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase Q9UDR5 AASS 0.3 -0.1 -0.2 0.1 -0.1 alpha-aminoadipic semialdehyde synthase, mitochondrial Q9NY61 AATF 0.1 0.0 -0.1 0.1 0.0 protein AATF P08183 ABCB1 0.3 -0.1 -0.2 0.1 -0.1 multidrug resistance protein 1 Q9NRK6 ABCB10 0.3 0.0 -0.2 0.0 -0.1 ATP-binding cassette sub-family B member 10, mitochondrial O75027 ABCB7 0.1 -0.1 -0.1 0.0 0.0 ATP-binding cassette sub-family B member 7, mitochondrial Q9NUT2 ABCB8 0.1 0.3 -0.5 0.0 0.0 ATP-binding cassette sub-family B member 8, mitochondrial P33527 ABCC1 0.0 -0.1 0.0 0.1 0.0 multidrug resistance-associated protein 1 O15439 ABCC4 0.2 -0.1 -0.2 0.1 0.0 multidrug resistance-associated protein 4 isoform 1 P28288 ABCD3 0.1 -0.1 -0.1 0.0 0.0 ATP -binding cassette sub -family D member 3 isoform a P61221 ABCE1 -0.2 0.1 0.2 -0.2 0.0 ATP-binding cassette sub-family E member 1 Q8NE71 ABCF1 -0.2 0.1 0.0 -0.1 0.2 ATP -binding cassette sub -family F member 1 isoform a Q9UG63 ABCF2 0.2 0.0 0.1 -0.3 0.0 ATP-binding cassette sub-family F member 2 isoform a Q9UNQ0 ABCG2 0.5 -0.1 -0.3 0.1 -0.1 ATP -binding cassette sub -family G member 2 isoform 1 Q9NUJ1 ABHD10 0.0 0.1 -0.1 0.1 0.0 abhydrolase domain-containing protein 10, mitochondrial precursor Q8NFV4 ABHD11 0.1 0.0 -0.1 0.1 -0.1 abhydrolase domain -containing protein 11 isoform 1 Q8N2K0 ABHD12 -0.2 0.0 -0.1 0.1 0.1 monoacylglycerol lipase ABHD12 isoform a O95870 ABHD16A 0.2 0.0 -0.2 0.1 -0.1 abhydrolase domain-containing protein 16A isoform a Q9ULW3 ABT1 0.3 0.1 0.1 -0.4 0.0 activator of basal transcription 1 P09110 ACAA1 0.2 -0.2 -0.1 0.1 -0.1 3-ketoacyl-CoA thiolase, peroxisomal isoform a P42765 ACAA2 -0.1 0.0 0.0 0.1 0.0 3-ketoacyl-CoA thiolase, mitochondrial Q13085 ACACA -0.1 0.1 0.1 -0.1 0.0 acetyl-CoA carboxylase 1 isoform 2 Q6JQN1 ACAD10 -0.3 0.2 -0.1 0.3 -0.1 acyl-CoA dehydrogenase family member 10 isoform b Q9UKU7 ACAD8 0.2 0.1 -0.1 -0.2 -0.1 isobutyryl-CoA dehydrogenase, mitochondrial Q9H845 ACAD9 0.2 0.0 -0.1 -0.1 0.0 acyl-CoA dehydrogenase family member 9, mitochondrial P11310 ACADM 0.3 -0.1 -0.2 0.1 -0.1 medium-chain specific acyl-CoA dehydrogenase, mitochondrial isoform a precursor P16219 ACADS 0.3 0.0 -0.2 0.2 -0.3 short-chain specific acyl-CoA dehydrogenase, mitochondrial precursor P45954 ACADSB 0.2 -0.1 -0.2 0.1 0.0 short/branched chain specific acyl-CoA dehydrogenase, mitochondrial precursor P49748 ACADVL 0.1 0.0 -0.1 0.1 0.0 very long-chain specific acyl-CoA dehydrogenase, mitochondrial isoform 1 precursor P24752 ACAT1 0.1 -0.1 -0.1 0.1 -0.1 acetyl -CoA acetyltransferase, mitochondrial precursor Q9BWD1 ACAT2 -0.1 0.0 0.1 0.0 -0.1 acetyl -CoA acetyltransferase, cytosolic Q9H3P7 ACBD3 -0.2 -0.1 0.1 0.2 0.0 Golgi resident protein GCP60 Q9UKV3 ACIN1 0.0 0.0 0.0 0.1 -0.1 Apoptotic chromatin condensation inducer in the nucleus P53396 ACLY -0.2 0.1 0.2 -0.2 0.1 ATP-citrate synthase isoform 1 Q99798 ACO2 0.3 -0.1 -0.1 0.0 0.0 aconitate hydratase, mitochondrial precursor Q86TX2 ACOT1 0.1 0.0 -0.1 0.0 0.0 acyl -coenzyme A thioesterase 1 Q9NPJ3 ACOT13 -0.1 0.1 0.0 0.2 -0.1 acyl-coenzyme A thioesterase 13 isoform 1 O00154 ACOT7 -0.3 0.1 0.2 0.0 0.0 cytosolic acyl coenzyme A thioester hydrolase isoform hBACHb Q9Y305 ACOT9 0.0 -0.1 -0.1 0.3 0.0 acyl-coenzyme A thioesterase 9, mitochondrial isoform b precursor Q15067 ACOX1 0.1 -0.1 -0.2 0.1 0.0 peroxisomal acyl -coenzyme A oxidase 1 isoform b O15254 ACOX3 0.1 0.1 -0.2 0.1 0.0 peroxisomal acyl-coenzyme A oxidase 3 isoform a P24666 ACP1 -0.3 0.1 0.1 0.1 0.0 low molecular weight phosphotyrosine protein phosphatase isoform c Q96CM8 ACSF2 0.1 -0.1 -0.1 0.0 0.0 acyl-CoA synthetase family member 2, mitochondrial precursor P33121 ACSL1 0.0 -0.1 0.0 0.1 0.0 long -chain -fatty -acid -- CoA ligase 1 O95573 ACSL3 0.1 -0.1 -0.1 0.1 0.0 long-chain-fatty-acid--CoA ligase 3 O60488 ACSL4 0.2 -0.1 -0.2 0.0 0.0 long -chain -fatty -acid -- CoA ligase 4 isoform 2 Q9ULC5 ACSL5 0.2 -0.2 -0.2 0.2 0.0 long-chain-fatty-acid--CoA ligase 5 isoform b Q9NUB1 ACSS1 0.3 -0.2 -0.2 0.1 0.0 acetyl -coenzyme A synthetase 2 -like, mitochondrial isoform 1 precursor P60709 ACTB 0.1 0.0 -0.1 0.0 0.0 actin, cytoplasmic 1 Q562R1 ACTBL2 -0.2 0.1 0.3 -0.3 0.0 beta -actin -like protein 2 P68032 ACTC1 0.2 0.0 -0.2 0.0 -0.1 actin, alpha cardiac muscle 1 proprotein O96019 ACTL6A 0.0 0.0 0.0 0.0 0.0 actin -like protein 6A isoform 1 P12814 ACTN1 -0.2 0.1 0.1 0.0 0.0 alpha-actinin-1 isoform b Q08043 ACTN3 -0.3 -0.2 0.1 -0.3 0.7 Alpha -actinin -3 O43707 ACTN4 -0.2 0.1 0.0 0.0 0.1 alpha-actinin-4 P61163 ACTR1A 0.0 0.0 0.2 -0.3 0.1 alpha -centractin P61160 ACTR2 -0.1 0.0 0.1 -0.1 0.0 actin-related protein 2 isoform b P61158 ACTR3 0.0 0.0 0.2 -0.2 0.0 actin -related protein 3 O14672 ADAM10 0.1 -0.1 -0.1 0.2 -0.1 disintegrin and metalloproteinase domain-containing protein 10 precursor P55265 ADAR -0.1 0.0 0.0 0.1 0.0 Double-stranded RNA-specific adenosine deaminase Q8NI60 ADCK3 -0.1 -0.1 0.1 0.4 -0.3 chaperone activity of bc1 complex-like, mitochondrial P35611 ADD1 0.0 0.0 0.0 0.0 -0.1 alpha -adducin isoform a Q9UEY8 ADD3 0.2 0.0 0.0 -0.1 -0.1 gamma-adducin isoform a P11766 ADH5 -0.2 0.1 0.2 -0.1 0.0 alcohol dehydrogenase class -3 P55263 ADK -0.2 0.3 0.3 -0.2 -0.2 adenosine kinase isoform b Q9H2P0 ADNP 0.1 0.0 -0.1 0.0 0.0 activity -dependent neuroprotector homeobox protein Q9BRR6 ADPGK 0.2 -0.1 -0.1 0.1 0.0 ADP-dependent glucokinase Q16186 ADRM1 -0.3 0.2 0.3 -0.2 0.0 proteasomal ubiquitin receptor ADRM1 precursor P30566 ADSL -0.2 0.1 0.3 -0.2 0.1 adenylosuccinate lyase isoform a P30520 ADSS -0.1 0.1 0.0 0.0 0.0 adenylosuccinate synthetase isozyme 2 Q9Y4W6 AFG3L2 0.0 -0.1 -0.1 0.1 0.0 AFG3-like protein 2 P52594 AGFG1 -0.2 0.3 0.4 -0.2 -0.3 arf-GAP domain and FG repeat-containing protein 1 isoform 2 Q53H12 AGK 0.1 -0.1 -0.1 0.1 0.0 acylglycerol kinase, mitochondrial precursor P35573 AGL 0.0 0.1 0.2 -0.5 0.2 glycogen debranching enzyme isoform 1 Q9BSE5 AGMAT -0.1 -0.1 -0.1 0.3 0.0 agmatinase, mitochondrial precursor Q99943 AGPAT1 0.1 -0.1 0.1 0.0 -0.1 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha O15120 AGPAT2 0.0 0.1 0.0 -0.1 0.0 1-acyl-sn-glycerol-3-phosphate acyltransferase beta isoform a precursor Q9NRZ7 AGPAT3 -0.1 0.0
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