Differentiated 3T3-L1 cells were incubated with 1 mM final concentrations of fatty acids. All fatty acid stocks were initially reconstituted in absolute ethanol. Sub-stocks of fatty acids at 25 mM were prepared in filter-sterilised KOH at 70 mM. Complete DMEM supplemented with fatty acid-free BSA (Sigma-Aldrich, St. Louis, MO) were filter sterilised, and incubated for 30 min with fatty acids to complex BSA to fatty acids. Ratio of BSA to fatty acids were approximately 1:2. Vehicle control treatments only contained equivalent mixture of ethanol and KOH.
Growth protocol
3T3-L1 pre-adipocytes (# CL-173) were obtained from American Type Culture Collection (ATCC; Manassas, VA) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 1000 µg/mL streptomycin (Lonza, Verviers, Belgium) in a humidified chamber at 37oC with 5% CO2. Cells were seeded in 6-well plates. Two-days post-confluence, the cell culture media was changed to induction medium (0.5 mM IBMX, 5 µg/mL insulin, 1 µM dexamethasone) for 2 days. Induction medium was then replaced with insulin medium (5 µg/mL insulin) for 3 days, after which the fully differentiated cells were treated with fatty acids.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from samples using the RNeasy kit (Qiagen) with a DNase digestion step according to the manufacturer’s protocol. RNA quality was verified on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands) using 6000 Nano Chips according to manufacturer’s instructions. RNA was considered suitable for array hybridization only if RNA integrity number exceeded 8.0. RNA from 3 samples per group was pooled for microarray analysis.
Label
biotin
Label protocol
Total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 2.1 ST peg arrays were performed on a GeneTitan Instrument according to the manufacturer's recommendations (Affymetrix, Santa Clara, CA, USA).
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA, USA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.46.0).
