AML-12 (CRL-2254) cells were maintained at 37°C in DMEM-F12 1:1 containing 10% fetal bovine serum, 1× ITS, 10 nM dexamethasone, and 1× penicillin/streptomycin in a 5% CO2 atmosphere. Cells were treated with GW3965 (Sigma-Aldrich) at a dose of 10 μM.
Sphingolipid analysis was performed as described before. In brief, cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and spiked with an internal standard mixture (C17-sphingosine, C17-sphinganine, C17-sphingosine-1-phosphate, C17-sphinganine-1-phosphate, C12-ceramide, C12-ceramide-1-phosphate, C12-glucosylceramide, C12-lactosylceramide, C12-sphingomyelin, and 14:0 phosphocholine from Avanti Polar Lipids). Thereafter, the cells were resuspended in 900 μL ice-cold chloroform-methanol (1:2) and incubated in ice for 15 min with vortexing every 5 min. 300 μL ice-cold distilled water (dH2O) and 300 μL ice-cold chloroform were added to the samples, which were then vortexed and centrifuged at 8,000 g for 2 min at 4°C. The lower organic phase was transferred into a clean microcentrifuge tube. A second extraction was performed by adding 300 μL ice-cold chloroform, and the lower organic phase was pooled with that of the first extraction. The collected samples were dried under a stream of nitrogen and stored at -80°C until ready for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
RNA isolation and qRT-PCR
Total RNA was isolated and qPCR performed using the QuantiTect SYBR Green PCR Kit (Qiagen, 204141) in accordance with the manufacturer’s instructions. Primers were purchased from Sigma-Aldrich (KiCqStart® SYBR® Green Predesigned Primers). Relative mRNA levels were assessed using 2-ddCt analysis.
Calculations and statistics
Results were expressed as mean ± SD. The statistical significance of differences (*p <0.05) was assessed by unpaired Student’s -t test when comparing different groups; t(DF) = 4.