cell line: THP-1 treatment: 200 nM TPA followed by 50% TE-8CM
ESCC cell line, TE-8 was obtained from RIKEN BioResource Center (Tsukuba, Japan). The individuality of the TE series ESCC cell lines was confirmed by short tandem repeat analysis at RIKEN and at the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. THP-1 human acute monocytic leukemia cell line was purchased from the American Type Culture Collection (Mannasas, VA, USA).11 We routinely maintained ESCC cell lines in RPMI-1640 (Wako, Osaka, Japan) and THP-1 cells in DMEM (Wako) with 10% FBS (Sigma-Aldrich, Tokyo, Japan) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA). Conditioned media of TE series ESCC cell lines (TECMs) were prepared by plating 5 × 106 tumor cells in 10 mL complete medium in 100-mm dishes for 24 hr, thereafter changing the medium to complete DMEM supplemented with 10% human AB serum (Lonza, Walkersville, MD, USA) instead of fetal bovine serum. After 2–3 days, the supernatants were harvested, centrifuged and stored in aliquots at −80°C.12 To induce macrophage (MΦ)-like differentiation, 5 × 106 THP-1 cells were treated with 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA; Cell Signaling, Danvers, MA, USA) for 2 days (MΦ-like THP-1 cells). Then, MΦ-like THP-1 cells were exposed to 50% TECM for 2 days (TAM-like THP-1 cells).
Total cellular RNA was extracted using RNeasy Mini Kit
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1ug Total RNA using the Low Input Quick Amp Labeling Kit(Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60K array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.