Following a seven-day acclimatization period, mice were randomly assigned to four groups: Control (n=8), 1% Minoxidil (n=8), 1% Tieghemelin (n=8), and 1% Maslinic acid (n=8). Anesthesia was administered using isoflurane (Wako Pure Chemical Industries, Tokyo, Japan) before trimming the hair in the dorsal area. Subsequently, depilation was carried out using a depilatory cream to expose the pink skin on the mice's back. Each mouse received a daily topical application of Tieghemelin, Maslinic acid, and Minoxidil for 20 days.
Growth protocol
Seven-week-old C3H male mice (Charles River Laboratories, Japan Inc, Kanagawa, Japan) were used in the investigation of the hair growth effect of Tieghemelin and Maslinic acid.
Extracted molecule
total RNA
Extraction protocol
For RNA extraction, Isogen (Nippon Gene Co. Ltd., Toyama, Japan) was added and the cell suspensions were centrifuged for 5 minutes at 500 g and the pellet was stored at -80oC 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 Calriom_S_Mouse. GeneChips were washed and stained in the Gene Atlas Fluidics Station 400
Scan protocol
GeneChips were scanned using the GeneAtlas Imaging Station
Description
Gene expression data from water treated mice for 20 days
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.). 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). We also performed gene set enrichment analysis (GSEA) for the top DEGs. 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 (CHP files ). SST-RMA
