To induce differentiation, the same protocol described by Sasaki et al., 2019 was used. Briefly, hNSCs were seeded into 6 wells culture vessels coated with Geltrex® at a density of 2.5 x 104 cells/cm2. After 48 h, growth medium was replaced by differentiation medium: Knockout DMEM/F12 supplemented with 2% StemPro® neural supplement, 2 mM GlutaMAX-I supplement, 6 units/mL heparin and 200µM ascorbic acid supplemented or not with 1µM of Luteolin.
Growth protocol
The human neural stem cells (StemPro™ Neural Stem Cells) which are a cryopreserved Human fetal brain-derived neural stem cells derived from cortex male donor aged of 16 weeks, were purchased from Gibco, USA (Cat. no. A15654). It was cultured in T25 flasks (BD Falcon) or in 6-well plates (BD Falcon) with KnockOut™ DMEM/F-12 (Gibco, USA, cat.no. 12660012) supplemented with 2% StemPro™ Neural Supplement (Gibco, Cat. no. A1050801), 20 ng/mL of fibroblast growth factor (FGF) basic recombinant human, 20 ng/mL epidermal growth factor (EGF) recombinant human and 2 mM GlutaMAX™-I Supplement (Cat. no. 35050), 6 U/mL heparin (Sigma, Cat. no. H3149), and 200 µM ascorbic acid (Sigma, Cat. no. A8960). For adhesion, Geltrex® (Gibco, Cat. no. A14133) was used to coat the 6-well plate. The hNSCs were cultured at 37ºC in a 95% air / 5% CO2 humidified incubator. The medium was changed every two days.
Extracted molecule
total RNA
Extraction protocol
The total RNA was extracted from the cells using ISOGEN kit (Nippon Gene Co. Ltd., Japan) following the manufacturer’s instructions, as reported previously by Sasaki et al., 2019. Total RNA was purified using chloroform and Isopropanol (Wako, Japan) and quantified and assessed for quality with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 9.4 ug total RNA
Hybridization protocol
Following fragmentation, 100 ng of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome Array (HG-U219). 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 1µM treated control hNSCs after 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.). 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. Expression console signal intensity. For calculating average value, standard deviation, fold change, and p-value of signal intensity of replicates, we used freely available Transcriptome Analysis Console software V 4.0 (CHP files ). RMA
