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Supplementary file 1Platinum nanoparticles enhance exosome release in human lung epithelial adenocarcinoma cancer cells (A549): Oxidative stress and the ceramide pathway are key playersMaterials and methodsMaterialThe human lung adenocarcinoma cell line A549 was obtained from the Korean Cell Bank (Seoul, Korea). All cells were grown in 75-cm2 tissue culture flasks (Corning, New York, USA) at 37°C with 5% CO2 and 95% relative humidity. H2PtCl6.6H2O, N-acetylcysteine (NAC), and C6-ceramide were procured from Sigma-Aldrich (St. Louis, MO, USA). Penicillin–streptomycin, trypsin–EDTA, DMEM, fetal calf serum (FCS), and antibiotic–antimycotic reagents were obtained from Life Technologies/Gibco (Grand Island, NY, USA). The in vitro toxicology assay kit and the reagent kits used to measure oxidative stress markers, such as generation of reactive oxygen species, acetylcholinesterase assay kit, and neutral sphingomyelinase assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5-dodecanoylamino fluorescein (C12-FAM) and fluorescein were purchased from Thermo Fisher Scientific (USA) and Sigma–Aldrich (USA), respectively. Exosome-depleted fetal bovine serum (FBS), ExoQuick-TC exosome precipitation solution, and EXOCET exosome Quantitation kit were purchased from System Biosciences (Palo Alto, CA, USA). Until unless specified all the reagents and kits were obtained from Sigma-Aldrich.Synthesis and characterization of PtNPsPtNPs synthesis and characterization were performed as described previously PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5HdXJ1bmF0aGFuPC9BdXRob3I+PFllYXI+MjAxOTwvWWVh
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ADDIN EN.CITE.DATA 39. PtNPs were synthesized by the reduction of PtCl6 2- ions into PtNPs by mixing 10 mL of 1 mg/mL lutein with 90 mL of 1 mM aqueous H2PtCl6.6H2O (Sigma-Aldrich, St. Louis, MO, USA). The mixture was maintained at 100°C (on a hotplate) in a sealed flask to avoid evaporation, for 1 h, since temperature catalyzes the reduction process. For control experiments, equal amounts of platinum solution and lutein were maintained separately under the same reaction conditions. The reduced platinum solution was sonicated for 10 min to separate the platinum nanoparticles from biomolecules. After sonication, the solution was filtered using a 0.2-?m syringe filter. The reduced platinum metal was purified by repeated centrifugation at 5000 × g for 30 min, and the pellets were washed with distilled water to remove the impurities. Purified PtNPs were characterized using various analytical techniques, such as UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microcopy (TEM). Exosome size and zeta-potential were analyzed using the zetasizer nano ZS90 particle sizer at a 90° fixed angle (Malvern Instruments, Worcestershire, UK). Thermogravimetric analysis (TGA) was performed on lutein, and PtNPs-lutein using a Perkin-Elmer Diamond 500 (Waltham, MA, USA). The samples were heated from 50°C to 800°C at a heating rate of 10°C/min under an airflow of 100 mL/min.Cell culture and exposure to PtNPs, C6-cer, CSP and GW4869Human lung epithelial adenocarcinoma cells were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin in a humidified atmosphere with 5% CO2 at 37°C. DMEM was depleted from EVs by ultracentrifugation at 120,000 × g and 4°C for 70 min prior to preparing the medium. The medium was replaced thrice a week, and the cells were passaged at sub-confluent levels. Upon reaching 75% confluency, the cells were harvested with 0.25% trypsin and were sub-cultured into either 75-cm2 flasks, 6-well plates, or 96-well plates according to the requirements. Next, the cells were allowed to attach to the surface for 24 h prior to the treatment. A 100 μL aliquot of cells at a density of 1 × 105 cells/mL was then plated in each well of the 96-well plates. After 24 h, the culture medium was replaced with a medium containing IC25 concentrations of PtNPs (10 ?M) or C6-cer (10 ?M), CSP (10 ?M) or GW4869 (20 ?M). A dose-dependent effect was observed upon using various concentrations of PtNPs, C6-cer, CSP and GW4869 for 24 h. unless otherwise indicated, the conditioned media were always collected 24 h after changing the media of cells. Cells were pre-incubated with GW4869 (20 ?M) or 1 mM NAC for 12 h prior to PtNPs or C6-cer or CSP exposure.Cell viability Cell viability was measured using the Cell Counting Kit-8 (CCK-8; CK04-01, Dojindo Laboratories, Kumamoto, Japan). Briefly, A549 cells were plated in 96-well flat-bottom culture plates containing the following substances, for 24 h: PtNPs (10 ?M) or C6-cer (10 ?M) or CSP (10 ?M) or GW4869 (20 ?M) or various concentrations of PtNPs (2.5-40 ?M) or C6-cer (2.5-40 ?M) or CSP (2.5-40 ?M) or GW4869 (2.5-40 ?M). After incubation for 24 h at 37°C and 5% CO2 in a humidified incubator, 10?μL of the CCK-8 solution was added to each well. The plates were then incubated for another 2?h at 37°C. Absorbance was measured at 450?nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific Inc., Waltham, MA, USA).Cell proliferation Cells were incubated with PtNPs (10 ?M) or C6-cer (10 ?M) or CSP (10 ?M) or GW4869 (20 ?M) or various concentrations of PtNPs (2.5-40 ?M) or C6-cer (2.5-40 ?M) or CSP (2.5-40 ?M) or GW4869 (2.5-40 ?M) for 24 h. The BrdU labeling solution was added to the culture medium 2 h before the incubation period ended. The cells were fixed and the level of incorporated BrdU was determined using the Cell Proliferation ELISA BrdU assay kit (Roche) following the manufacturer’s instructions. The proliferation activity of untreated cells at 0 h was considered as 100%. Cellular uptake of PtNPs and quantification of Pt ions To study the cellular uptake of PtNPs, cells were treated with PtNPs for 24 h, harvested, and fixed with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M PBS for 8 h at pH 7.2. After fixation, the cells were incubated with 1% osmium tetroxide in PBS for 2 h. The fixed cells were dehydrated in ascending concentrations of ethanol (70, 80, 90, 95, and 100%) and embedded in EMbed 812 resins (EMS, Warrington, PA, USA) via propylene oxide. Ultrathin sections were obtained using an ultramicrotome (Leica, IL, USA) and were double stained with uranyl acetate and lead citrate. The stained sections on the grids were then examined with a H7000 TEM (Hitachi, Chiyoda-ku, Japan) at 80 kV. Pt ion concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS) ICP-MS was performed to measure the Pt contents in the PtNPs exposed cells. Cells were treated with 10 ?M of PtNPs for 24 h, and then cells were collected by Trypsin/EDTA treatment. Silver ions in the cells by ICP-MS. Briefly, 1 x 106 A549 cells were cultured on the each 6-well cell culture plate, after treating PtNPs, the cells were collected by trypsin/EDTA, and silver ions were measured. Membrane integrity We evaluated the membrane integrity of A549 cells using the LDH Cytotoxicity Detection Kit. Briefly, the cells were exposed to PtNPs (10 ?M) or C6-cer (10 ?M) or CSP (10 ?M) or GW4869 (20 ?M) for 24?h. Subsequently, 100?μL of cell-free supernatant from each well was transferred into a 96-well plate and 100?μL of the LDH reaction mixture was added to each well. After 3 h of incubation under standard conditions, the optical density of the final solution was determined at a wavelength of 490?nm, using a microplate reader. This experiment was performed thrice.Estimation of ROS ROS production was estimated as described previously ADDIN EN.CITE <EndNote><Cite><Author>Gurunathan</Author><Year>2020</Year><RecNum>41</RecNum><DisplayText><style face="superscript">41</style></DisplayText><record><rec-number>41</rec-number><foreign-keys><key app="EN" db-id="5259vex9159zv7e0wda5erswrd9w5e0drvzt" timestamp="1604618615">41</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Gurunathan, S.</author><author>Jeyaraj, M.</author><author>Kang, M. H.</author><author>Kim, J. H.</author></authors></contributors><auth-address>Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul 05029, Korea.</auth-address><titles><title>Anticancer Properties of Platinum Nanoparticles and Retinoic Acid: Combination Therapy for the Treatment of Human Neuroblastoma Cancer</title><secondary-title>Int J Mol Sci</secondary-title><alt-title>International journal of molecular sciences</alt-title></titles><periodical><full-title>Int J Mol Sci</full-title><abbr-1>International journal of molecular sciences</abbr-1></periodical><alt-periodical><full-title>Int J Mol Sci</full-title><abbr-1>International journal of molecular sciences</abbr-1></alt-periodical><volume>21</volume><number>18</number><edition>2020/09/20</edition><keywords><keyword>anticancer activity</keyword><keyword>apoptosis</keyword><keyword>cytotoxicity</keyword><keyword>endoplasmic reticulum stress</keyword><keyword>mitochondrial dysfunctions</keyword><keyword>oxidative stress</keyword><keyword>platinum nanoparticle</keyword><keyword>retinoic acid</keyword></keywords><dates><year>2020</year><pub-dates><date>Sep 16</date></pub-dates></dates><isbn>1422-0067</isbn><accession-num>32947930</accession-num><urls></urls><custom2>PMC7554966</custom2><electronic-resource-num>10.3390/ijms21186792</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>41. 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ADDIN EN.CITE.DATA 42. The cells were treated with PtNPs (10 ?M) or C6-cer (10 ?M) or CSP (10 ?M) or GW4869 (20 ?M) for 24 h, and subsequently the activity of caspase-3 was measured using a kit from Sigma-Aldrich Co., according to the manufacturer’s instructions.Exosome isolation by ultracentrifugationThe cells were treated with PtNPs (10 ?M) or C6-cer (10 ?M) or CSP (10 ?M) or GW4869 (20 ?M) for 24 h. Exosomes were prepared from culture supernatants of A549 cells by differential centrifugation according to a previously described method PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5HdXJ1bmF0aGFuPC9BdXRob3I+PFllYXI+MjAwMjwvWWVh
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ADDIN EN.CITE.DATA 43, 44. Briefly, the cell culture medium was collected and centrifuged at 300 × g for 15 min, followed by centrifugation at 2000 × g for 30 min to remove cells and cell debris. The cell-free culture medium was centrifuged at 20 000 × g for 70 min and then ultracentrifuged at 170 000 × g for 1.5 h to pellet the exosomes. The pelleted exosomes were resuspended in 20 nm-filtered PBS (Whatman, USA). All centrifugation steps were performed at 4°C. Finally, the pellets were resuspended in 100 ?L of PBS and stored at -80°C until subsequent analyses.Exosome isolation by commercial extraction kitsExosomes were isolated and purified using ExoQuick (EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, the reagents were added to concentrated culture medium and the mixture was vortexed and centrifuged at 4°C as described in the manufacturer’s protocol. The pellet containing exosomes was resuspended in PBS or ultrapure water. Subsequently, the exosome pellet was diluted in M-PER reagent (Thermo Fisher Scientific, Waltham, MA, USA). Morphological analysis of exosomes using scanning electron microscopy (SEM)The SEM analysis was carried out according to a previously described method PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5XdTwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJlY051
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ADDIN EN.CITE.DATA 46. Freshly isolated exosomes from cells were resuspended in cold PBS. Exosome samples were prepared for TEM analysis, using the exosome-TEM-easy kit (101Bio, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, resuspended exosomes were mounted on Formvar carbon-coated EM mesh 400 grids and incubated for 10 min. The resulting grids were rinsed twice with wash buffer and deposited on the EM solution for 10 min. After washing and dehydration steps, exosomes were subjected to TEM at an accelerating voltage of 300 kV.Determination of total protein concentration of exosomesTotal protein concentration of exosomes was determined using the bicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. In addition, we followed a previously described protocol PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MaW08L0F1dGhvcj48WWVhcj4yMDE5PC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA 47. This method is based on the measurement of acetylcholinesterase-enriched activity in exosomes. For each preparation condition, 10 μL of exosomal preparation in PBS was diluted in 90 μL of lysis buffer. The mixture was then incubated at 37°C for 5 min, vortexed, and centrifuged at 1500 × g at room temperature for 5 min. The subsequent steps of exosome particle quantitation were performed according to the manufacturer's instructions, and absorbance was measured at 405 nm using the Infinite M200 plate reader. Quantification of exosomes by fluorescence polarization (FP)Quantification of exosomes by fluorescence polarization (FP) was performed as described previously ADDIN EN.CITE <EndNote><Cite><Author>Kalimuthu</Author><Year>2019</Year><RecNum>48</RecNum><DisplayText><style face="superscript">48</style></DisplayText><record><rec-number>48</rec-number><foreign-keys><key app="EN" db-id="5259vex9159zv7e0wda5erswrd9w5e0drvzt" timestamp="1604620254">48</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kalimuthu, K.</author><author>Kwon, W. Y.</author><author>Park, K. S.</author></authors></contributors><auth-address>Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029 Republic of Korea. ISNI: 0000 0004 0532 8339. GRID: grid.258676.8</auth-address><titles><title>A simple approach for rapid and cost-effective quantification of extracellular vesicles using a fluorescence polarization technique</title><secondary-title>J Biol Eng</secondary-title><alt-title>Journal of biological engineering</alt-title></titles><periodical><full-title>J Biol Eng</full-title><abbr-1>Journal of biological engineering</abbr-1></periodical><alt-periodical><full-title>J Biol Eng</full-title><abbr-1>Journal of biological engineering</abbr-1></alt-periodical><pages>31</pages><volume>13</volume><edition>2019/04/25</edition><keywords><keyword>Biosensor</keyword><keyword>Extracellular vesicles</keyword><keyword>Fluorescence polarization</keyword><keyword>Quantification</keyword><keyword>interests.Springer Nature remains neutral with regard to jurisdictional claims in</keyword><keyword>published maps and institutional affiliations.</keyword></keywords><dates><year>2019</year></dates><isbn>1754-1611 (Print)
1754-1611</isbn><accession-num>31015861</accession-num><urls></urls><custom2>PMC6469078</custom2><electronic-resource-num>10.1186/s13036-019-0160-9</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>48. Briefly, the exosomes were mixed with 1.6 μM C12-FAM in a reaction buffer composed of 1 mM HEPES (pH 8) and 1.6 mM NaCl in a total reaction volume of 160 μL. After incubation of the reaction mixture for 20 min at room temperature, the fluorescence polarization values were measured at excitation and emission wavelengths of 485 and 528 nm, respectively.Particle size and concentration analysesThe exosomes were resuspended in PBS, and the particle size and concentration were measured using the Nanoparticle Tracking Analysis System 300 (NTA300, Malvern Instruments, UK). The prepared exosomes were diluted and loaded into the sample chamber. The light scatter mode of the tracking system used a camera filter 1. Data were analyzed using the NTA 3.0 software (Malvern Instruments, UK).Size analysis of exosomes by DLSThe exosome size/diameter was estimated by dynamic light scattering (DLS) as described previously ADDIN EN.CITE <EndNote><Cite><Author>Genneb?ck</Author><Year>2013</Year><RecNum>49</RecNum><DisplayText><style face="superscript">49</style></DisplayText><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="5259vex9159zv7e0wda5erswrd9w5e0drvzt" timestamp="1604620287">49</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Genneb?ck, N.</author><author>Hellman, U.</author><author>Malm, L.</author><author>Larsson, G.</author><author>Ronquist, G.</author><author>Waldenstr?m, A.</author><author>M?rner, S.</author></authors></contributors><auth-address>Department of Public Health and Clinical Medicine, Ume? University, Ume?, Sweden.</auth-address><titles><title>Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes</title><secondary-title>J Extracell Vesicles</secondary-title><alt-title>Journal of extracellular vesicles</alt-title></titles><periodical><full-title>J Extracell Vesicles</full-title><abbr-1>Journal of extracellular vesicles</abbr-1></periodical><alt-periodical><full-title>J Extracell Vesicles</full-title><abbr-1>Journal of extracellular vesicles</abbr-1></alt-periodical><volume>2</volume><edition>2013/09/07</edition><keywords><keyword>cardiomyocytes</keyword><keyword>exosomes</keyword><keyword>extracellular vesicles</keyword><keyword>gene expression</keyword><keyword>growth factors</keyword></keywords><dates><year>2013</year></dates><isbn>2001-3078 (Print)
2001-3078</isbn><accession-num>24009898</accession-num><urls></urls><custom2>PMC3760655</custom2><electronic-resource-num>10.3402/jev.v2i0.20167</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>49. The mean hydrodynamic diameter of exosomes was calculated by fitting a Gaussian function to the measured size distribution. DLS measurements were conducted at 25°C using a Nano Zetasizer (Malvern Instruments, UK) operating at 633 nm. Measurement of acetylcholinesterase (AChE) activityAChE activity was determined according to a previously described method PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Fc3NhbmRvaDwvQXV0aG9yPjxZZWFyPjIwMTU8L1llYXI+
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ADDIN EN.CITE.DATA 51, using a kit and following the manufacturer’s protocol. The Amplex Red sphingomyelinase assay kit (Molecular Probes Inc., Eugene, OR, USA) was used to quantify neutral sphingomyelinase activity. The exosomes were incubated for 30 min at 37°C in 100 ?L of a working solution that consisted of 2 U/mL HRP, 0.2 U/mL choline oxidase, 8 U/mL of alkaline phosphatase, and 0.5 mM sphingomyelin. Neutral sphingomyelinase activity was determined in a working buffer of pH 7.4. The reaction product was measured using a fluorescent plate reader (absorption at 571 nm and emission at 585 nm; SpectraMax M2e, Molecular Devices, CA, USA). RNA isolation, and mRNA expression analysis using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Exosomes were prepared from culture supernatants of A549 cells by differential centrifugation according to a previously described method ADDIN EN.CITE <EndNote><Cite><Author>Cheng</Author><Year>2019</Year><RecNum>44</RecNum><DisplayText><style face="superscript">44</style></DisplayText><record><rec-number>44</rec-number><foreign-keys><key app="EN" db-id="5259vex9159zv7e0wda5erswrd9w5e0drvzt" timestamp="1604619669">44</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Cheng, Y.</author><author>Dai, X.</author><author>Yang, T.</author><author>Zhang, N.</author><author>Liu, Z.</author><author>Jiang, Y.</author></authors></contributors><auth-address>State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, 510120, China.
Institute for Chemical Carcinogenesis, Guangzhou Medical University, Guangzhou 511436, China.</auth-address><titles><title>Low Long Noncoding RNA Growth Arrest-Specific Transcript 5 Expression in the Exosomes of Lung Cancer Cells Promotes Tumor Angiogenesis</title><secondary-title>J Oncol</secondary-title><alt-title>Journal of oncology</alt-title></titles><periodical><full-title>J Oncol</full-title><abbr-1>Journal of oncology</abbr-1></periodical><alt-periodical><full-title>J Oncol</full-title><abbr-1>Journal of oncology</abbr-1></alt-periodical><pages>2476175</pages><volume>2019</volume><edition>2019/06/13</edition><dates><year>2019</year></dates><isbn>1687-8450 (Print)
1687-8450</isbn><accession-num>31186629</accession-num><urls></urls><custom2>PMC6521541</custom2><electronic-resource-num>10.1155/2019/2476175</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>44. Briefly, the culture medium was collected and centrifuged at 300 × g for 15 min, followed by centrifugation at 2000 × g for 30 min to remove cells and cell debris. The cell-free culture medium was centrifuged at 20 000 × g for 70 min and then ultracentrifuged at 170 000 × g for 1.5 h to pellet the exosomes. The pelleted exosomes were lysed using the TRI reagent (Ambion, Austin, TX, USA). Total RNA was isolated using the TRI reagent protocol, and cDNA synthesis was performed using oligo (dT) primers. qRT-PCR was performed using the SYBR Green Master Mix (Life Technologies) according to the manufacturer’s instructions on Applied Biosystems 7300 Sequence Detection System. The expression levels of target genes were quantified and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ELISA assay The expression of TSG101, CD9, CD63, and CD81 was determined with ELISA, as described previously PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MaW08L0F1dGhvcj48WWVhcj4yMDE5PC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA 46 and also we followed according to manufacturer instructions (Thermo Fisher Scientific, System Biosciences and R&D systems). Briefly, 100 μL of primary antibody (1 μg/100 μL) was coated onto a 96-well plate and incubated at 4°C overnight. The plate was then blocked with 1% BSA in PBS buffer at 37°C for 1 h. After washing with 0.1% BSA-PBS buffer thrice, the plate was incubated with the exosome solution in PBS buffer (100 μL) at 37°C for 1 h. Upon removing the solution, the plate was washed twice with 0.1% BSA-PBS buffer, and detection antibodies (anti- TSG101, CD9, CD63, and CD81) in PBS buffer (100 μL; 500 ng/mL) were added, followed by incubation at room temperature for 1 h. After washing thrice with 0.1% BSA-PBS buffer, the plate was incubated again with a solution of HRP-conjugated streptavidin in PBS buffer (100 μL; 1:1000) at room temperature for 30 min and then washed thrice with 0.1% BSA-PBS buffer. The TMB Ready Solution (Thermo Fisher Scientific) was then added to the plate, which was then incubated at room temperature for 15 min, followed by the addition of 50 μL of stop solution to each well. The absorbance was read using a UV/VIS spectrophotometer at a wavelength of 450 nm.Statistical analysis All experiments were repeated at least thrice (triplicate). Data were analyzed with the Student’s t-test or ANOVA, as required. To assess the differences between two groups, Student’s t-test (paired or unpaired, 2-tailed) was performed, as applicable. Differences were considered as significant when the p value was less than 0.05 (p < 0.05).GeneList of primersCD63F: ATG ATC ACG TTT GCC ATC TT R: AGG GAT TTT CTC CCA ATC TGCD9F: TCT TGG TGA TAT TCG CCA TTR: TTC GAG TAC GTC CTT CTT GGCD81F: CTG TAT CTG GAG CTG GGA GAR: GAA CTG CTT CAC ATC CTT GGTSG101F: CTC TCA TCT CTG CGG TCA GTR: TCA ACC TCG GCT ACT TCT TGGAPDHF:AGGTCGGTGTGAACGGATTTGR:TGTAGACCATGTAGTTGAGGTCASupplementary Table. 1Legends for supplementary table and figures Supplementary Table 1. List of primers used in this studySupplementary Figure 1. Representative Thermogravimetric analysis (TGA) curve of lutein, and PtNPs functionalized with lutein.Supplementary Figure 2. Effect of various concentrations of lutein on A549 cells. A549 cells were treated with various concentrations of lutein (0.2 mg -1.0 mg/mL) for 24 h and measured the viability using CCK-8. ................
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