The 4-day post-confluent Porcine intramuscular preadipocyte (PIP) cells were fed with differentiation medium for another 4 more days to yield the differentiated adipocyte. The differentiation medium was prepared by DMEM with 10% FBS, 50 ng/ml insulin (swine, Sigma), 0.25 μM dexamethasone (Sigma), 2 mM octanoate (Wako), 200 μM oleate (Ardorich, Milwaukee, WI, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. The differentiated pMA cells were cultured at density of 2.5×104/cm2 in 6 well or 12 well plates (BD Falcon, Tokyo, Japan). The 4-day post-confluent pMA cells were stimulated with 5-HT @100 μM/ml for 12 hours and TNF-α @ 2.5 µg/ml for 12 hours.
Growth protocol
The porcine intramuscular pre-adipocyte (PIP) cell lines (between the 26th and 35th passages) were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, Paisley, Scotland, UK) supplemented with 10% fetal calf serum (FCS), 100 mg/ml penicillin, and 100 U/ml streptomycin as a growth medium. PIP cells were plated at density of 2.5×104/cm2 in 6-well cell culture plates (BD Falcon, Tokyo, Japan) and incubated at 37°C in a humidified atmosphere of 5% CO2. The 4-day post-confluent PIP cells were fed with differentiation medium for another 4 more days to yield the differentiated adipocyte. The differentiation medium was DMEM containing 10% FBS, 50 ng/ml insulin (swine, Sigma-Aldrich), 0.25 μM dexamethasone (Sigma-Aldrich), 2 mM octanoate (Wako, Osaka, Japan), 200 μM oleate (Ardorich, Milwaukee, WI, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the ligand-treated and control PMA cells using PureLink RNA Mini Kit (Life Technology Inc., USA) along with on-column DNase treatment. RNA integrity, quality and quantity were evaluated with microcapillary electrophoresis (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA) using the RNA 6000 Nano kit.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy mini spin column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
The Gene Expression Hybridization kit (Agilent Technologies) was used for hybridization. In brief, 1650 ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Porcine (V2) Gene Expression Microarray, 4x44K (G2519F) 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 by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
