1, 2, 4-benzenetriol (BT) exposure: For control or BT exposure sample, HL-60 cells suspended in RPMI 1640 supplemented with 10% heat-inactivated FBS at 4×10^5/mL were exposed to BT (0 or 50μM) at 37℃ in 5% CO2 for 1 hour or 4 hours. For 4-aminobenzoic acid hydrazine (ABAH, MPO inhibitor) treatment sample, HL-60 cells suspended in RPMI 1640 supplemented with 10% heat-inactivated FBS at 4×10^5/mL were treated with ABAH (100μM) and preincubated at 37℃ in 5% CO2 for 24 hours. After the pretreatment of ABAH, media was then replaced with new media containing the reagent plus 50μM of BT.
Growth protocol
HL-60, a human promyelocytic cell line, was cultured in RPMI-1640 medium (SIGMA-Aldrich, St. Louis, MO) with 10 % of heat-inactivated fetal bovine serum (FBS) (Nichirei Bioscience, Tokyo, Japan) at 37℃ in a humidified atmosphere with 5 % CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cells using TRIzol Reagent (nitrogen) and purified using SV Total RNA Isolation System (Promega)
Label
Cy3
Label protocol
cRNA was amplified and labelled using a Low input Quick Amp Labelling Kit (Agilent Technologies).
Hybridization protocol
cRNA was hybridized to a 44K 60-mer oligomicroarray (SurePrint G3 Human Gene Expression Microarray 8×60K v2; Agilent Technologies) according to the manufacturer's instructions.
Scan protocol
The hybridized microarray slides were scanned using an Agilent scanner. The relative hybridization intensities and background hybridization values were calculated using Feature Extraction Software version 9.5.1.1 (Agilent Technologies).
Data processing
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. The raw signal intensities and flags for each probe were calculated from the hybridization intensities and spot information according to the procedures recommended by Agilent Technologies using the Flag criteria in the GeneSpring Software. In addition, the raw signal intensities were normalized by the quantile algorithm with the Bioconductor.
