To evaluate the cytotoxicity induced by Ludox® NPs, the cells were plated and allowed to attach for 24 h. Then, NPs were diluted to appropriate concentrations and immediately applied to the cells. We used a short incubation for 2 h in serum-free medium, followed by a post-treatment recovery of 3 or 22 hours in complete medium (10 % FCS). NPs concentrations (0.005–0.6 mg/mL) were chosen to evaluate the dose/survival according to the treatment conditions. Control cells underwent the same steps of treated cells except for NP exposure.
Growth protocol
The human cell lines A549 (lung adenocarcinoma), were obtained from American Type Culture Collection (ATCC, Rockville, USA) and cultured in monolayer. A549 cells were maintained in F12-K medium supplemented with 10 % heat-inactivated Fetal Calf Serum (FCS), 38 units/ml streptomycin, and 100 units/ml penicillin G in standard culture conditions and during the post-treatment recovery (complete medium). Cells were kept at 37 °C in a humidified atmosphere containing 5 % CO2.
Extracted molecule
total RNA
Extraction protocol
RNA extraction was performed using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. All samples were quantitated using a NanoDrop ND-1000 spectrophotometer; RNA quality was then analyzed using the Agilent Bioanalyser 2100 (Agilent, Santa Clara, CA, USA) (Agilent RNA 6000 nano kit; RIN at least 7 accepted).
Label
Cy3-dCTP
Label protocol
1 μg of total RNA was labeled with “Agilent One-Color Microarray-Based Gene Expression protocol” according to the manufacturer's protocol. The synthesized cDNA was transcribed into aRNA and labeled with Cy3-dCTP. Labeled aRNA was purified with RNeasy Mini columns (Qiagen, Valecia, CA, USA). The quality of each aRNA sample was verified by total yield and specificity calculated with NanoDrop ND-1000 spectrophotometer measurements (Nanodrop, Wilmington, DE, USA).
Hybridization protocol
1.65 μg of labeled aRNA was used in each reaction and hybridization was carried out at 65°C for 17 hours in a hybridization oven rotator (Agilent, Santa Clara, CA, USA). The arrays were washed using Agilent Gene expression washing buffers and Stabilization and Drying Solution, as suggested by the supplier.
Scan protocol
Slides were scanned on an Agilent microarray scanner (model G2565CA) and Agilent Feature Extraction software version 10.5.1.1 was used for image analysis. Gene expression data were performed on three biological replicates for each condition.
Description
C-2+3h-B-SM30 After 24 h, the culture medium was removed, and the cells were incubated for 2 hours with 150 μL of medium, without serum,and SM30 NPs. After recovery time (3 hours in complete medium with 10% of serum) total RNA was extracted.
Data processing
Inter-array normalization of expression levels (gProcessedSignal) was performed with quantile method to correct possible experimental distortions. Normalization function was applied to the expression data of all the experiments and the values for within-arrays replicate spots were then averaged (according the same ProbeName ID). Feature Extraction Software, which provided spot quality measures, was used to evaluate the quality and reliability of the hybridization. In particular, the flag “positive and significative” (set to 1 if the spot had an intensity value significantly different from the local background and to 0 when otherwise) was used to filter out unreliable probes: the flag equal to 0 was to be noted as “not available (NA)” the spot intensity and therfore not used in the average calculation. Probes with a high proportion of NA values were removed from the dataset in order to carry out a more solid and unbiased statistical analyses. Thirty-three percent of NA was used as the threshold in the filtering process, and a total of 32.096 transcripts of 41.093 were used in the subsequent analysis. Differentially expressed genes identification (One-way ANOVA), hierarchical clustering analysis (Sperman Rank Correlation or Euclidean distance) using average linkage method were performed using the TMev suite. Gene Ontology analysis of differentially expressed genes was performed on web tool GeneCodis3. Cytoscape tool in association with the Agilent Literature Search plug-in (http://apps.cytoscape.org/apps/agilentliteraturesearch (accessed on 19 December)) was used to produce network of differentially expressed genes. Network was analyzed using Network Analyzer and CentiScaPe plug-in. Supervised pathway analysis was performed by GSEA implemented in the Graphite web tool.
