Cells were plated at a cell density of 130 000 cells/well in 6-well plates. Following overnight incubation, cells were exposed to 1.8 mL of 1, 5, 10, and 25 µg/mL CuO NPs, CuO MPs or 7 µg/mL CuCl2. For all exposures, blank media exposed cells served as negative controls. For CuCl2 experiments, cells treated with 5 µg/mL NaCl were also used as additional control to rule out the possible contribution of Cl ions to the observed cellular toxicity in CuCl2 exposures. Following 2, 24, and 48 h of exposure, cell supernatant was harvested, cells were washed with PBS, trypsinized and suspended in fresh cell culture media. An aliquot of cell suspension was used for Trypan Blue dye exclusion staining and the rest of the cells were pelleted by centrifugation (8000 rpm, 10 minutes, 4°C) and frozen at -80°C for microarray analysis. Three biological replicates were used for each condition.
Growth protocol
FE1 immortalized alveolar lung epithelial cells were cultured as described in Decan et al 2015, in Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F-12HAM (DMEM F12HAM) supplemented with 2 % fetal bovine serum (FBS), 1 ng/mL epidermal growth factor (EGF), 100 U/mL penicillin G, and 100 µg/mL streptomycin and maintained at 37°C, with 5 % CO2.
Extracted molecule
total RNA
Extraction protocol
RNA extraction, quantification, and integrity analysis were conducted as in Decan et al., 2015. Following exposure and collection of cell pellets, TRIzol reagent was used to isolate RNA in conjunction with Direct-zol RNA Miniprep Kit (Zymo Research Corp, Irvine, USA) according to manufacturer’s protocol (with a 2 min incubation in water prior to elution). Total RNA concentration and purity was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc, Waltham, USA). RNA integrity and quality analysis was conducted using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Inc., Santa Clara, USA). RNA samples with an RNA Integrity Number (RIN) of ≥5 were used for subsequent microarray experiments.
Label
Cy5
Label protocol
In brief, 200 ng of total RNA per cell pellet sample or Universal Mouse Reference RNA (UMRR; Agilent Technologies) were used to synthesise labelled cDNAs and cRNAs using the Linear Amplification Kit (Agilent Technologies). T7 RNA polymerase-transcribed cRNA from experimental samples were labelled with Cyanine-5 and UMRR was labelled with Cyanine-3.
Cells were plated at a cell density of 130 000 cells/well in 6-well plates. Following overnight incubation, cells were exposed to 1.8 mL of 1, 5, 10, and 25 µg/mL CuO NPs, CuO MPs or 7 µg/mL CuCl2. For all exposures, blank media exposed cells served as negative controls. For CuCl2 experiments, cells treated with 5 µg/mL NaCl were also used as additional control to rule out the possible contribution of Cl ions to the observed cellular toxicity in CuCl2 exposures. Following 2, 24, and 48 h of exposure, cell supernatant was harvested, cells were washed with PBS, trypsinized and suspended in fresh cell culture media. An aliquot of cell suspension was used for Trypan Blue dye exclusion staining and the rest of the cells were pelleted by centrifugation (8000 rpm, 10 minutes, 4°C) and frozen at -80°C for microarray analysis. Three biological replicates were used for each condition.
Growth protocol
FE1 immortalized alveolar lung epithelial cells were cultured as described in Decan et al 2015, in Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F-12HAM (DMEM F12HAM) supplemented with 2 % fetal bovine serum (FBS), 1 ng/mL epidermal growth factor (EGF), 100 U/mL penicillin G, and 100 µg/mL streptomycin and maintained at 37°C, with 5 % CO2.
Extracted molecule
total RNA
Extraction protocol
RNA extraction, quantification, and integrity analysis were conducted as in Decan et al., 2015. Following exposure and collection of cell pellets, TRIzol reagent was used to isolate RNA in conjunction with Direct-zol RNA Miniprep Kit (Zymo Research Corp, Irvine, USA) according to manufacturer’s protocol (with a 2 min incubation in water prior to elution). Total RNA concentration and purity was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc, Waltham, USA). RNA integrity and quality analysis was conducted using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Inc., Santa Clara, USA). RNA samples with an RNA Integrity Number (RIN) of ≥5 were used for subsequent microarray experiments.
Label
Cy3
Label protocol
In brief, 200 ng of total RNA per cell pellet sample or Universal Mouse Reference RNA (UMRR; Agilent Technologies) were used to synthesise labelled cDNAs and cRNAs using the Linear Amplification Kit (Agilent Technologies). T7 RNA polymerase-transcribed cRNA from experimental samples were labelled with Cyanine-5 and UMRR was labelled with Cyanine-3.
Hybridization protocol
An equimolar amount of UMRR cRNA was mixed with each experimental cRNA and was hybridized to 8x60K Agilent SurePrint G3 Mouse Gene Expression v2 Microarray (Agilent Technologies, Inc., Santa Clara, USA) slides in a hybridization chamber for 17 h at 65°C rotating at 10 rpm.
Scan protocol
Microarray slides were washed according to manufacturer’s protocol and scanned on an Agilent G2505B scanner. Data was retrieved using the Feature Extraction 11.0.1.1 software (Agilent). Data analysis is described below.
Data processing
Images were quantified using Agilent Feature Extraction Software (version 11.0.1.1). Non background subtracted median signal intensities were LOWESS normalized using the maanova library in R. Probes with technical replicates were averaged using the median.
