After the 24 hour seeding in 6-well plates, cells were treated with 0 (Control), 200nM α-MSH (Positive control), and 5µM of OL, and OC for 24 hours
Growth protocol
B16F10 cells were purchaced from RIKEN and were maintained in RPMI1640
Extracted molecule
total RNA
Extraction protocol
For RNA extraction, Isogen (Nippon Gene Co. Ltd., Toyama, Japan) was added to each sample well and then the total RNA sample in 70% ethanol was stored at -20℃ until use.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 9.4 ug total RNA
Hybridization protocol
Following fragmentation, 250 ng of cRNA were hybridized for 16 hr at 45C on Clariom_S_Mouse Array. GeneChips were washed and stained in the GeneAtlas® Fluidics Station
Scan protocol
GeneChips were scanned using the GeneAtlas Imaging Station
Description
Gene expression data from 5 µM of OL treated for 24 hours
Data processing
The raw data were normalized using Expression Console Software provided by the Affymetrix following robust multichip average (RMA) algorithm (http://www.affymetrix.com). Subsequent analysis of the gene expression data was carried out in the freely available Transcriptome Analysis Console (TAC) version 4 (Thermofisher inc.). Genes with a linear fold change >1.5 and p-value < 0.05(one-way between-subjects ANOVA) were considered as differentially expressed genes(DEGs). Further analysis was conducted using an online data mining tool DAVID (Database for Annotation, Visualization and Integrated Discovery, ver. 6.8). We used 'Functional Annotation' tool of DAVID to identify the most relevant biological terms, including gene ontology (GO) terms, biological pathways, tissue expression, and disease associations (Huang da et al., 2009). Expression console signal intensity. For calculating average value, standard deviation, fold change, and Anova p-value of signal intensity of replicates, we used freely available Transcriptome Analysis Console software. algorithm: RMA
