Cell culture and treatments
The SW480, SW620, A549, KG1, KCL22 and KU812 cell lines were purchased from American Type Culture Collection and were maintained in DMEM (Gibco, 31,966–021) or RPMI 1640 medium (Gibco, 61,870–010) media supplemented with 10% fetal bovine serum (FBS) (Sigma, F75240) and penicillin-streptomycin solution (Corning, 30–002-CI). Cell cultures were maintained in the atmosphere of 5% CO2 and 37 °C. For all experiments cells were initially seeded and cultivated in normal media for 24 h. Then to induce metabolic stress media and/or growth conditions were respectively changed and cells were cultivated for additional 48 h under either one of the following condition: lipoprotein deficient medium (LPDS serum), low-serum (LS) medium (2% serum), hypoxia (2% O2), or hypoxia in combination with LS medium. For lipoprotein deficient conditions the media were supplemented with lipoprotein deficient serum (LPDS) that was purchased from Merck (LP4) and used according to manufacturer’s guidelines. For determining the cells number cells were stained with trypan blue and counted using Countess® automated cell counter (Invitrogen). Cell lines were commercially authenticated (Eurofins, Germany) and mycoplasma tested prior to submission of this manuscript.
Quantitative RT-PCR
For quantitative RT-PCR, total RNA was extracted from cell pellets using Quick-RNA™ MiniPrep Plus (Zymo Research). All RNA samples were reverse-transcribed into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Scientific, 18,080,093) and Oligo(dT)18 Primers (Thermo Scientific, SO131). Quantitative PCR was performed using a TaqMan™ Gene Expression Master Mix (4,369,016, Applied Biosystems) viaStepOne Real-Time PCR Systems (Applied Biosystems). The TaqMan Gene Expression assays used were Hs01005622_m1 (fatty acid synthase, FASN), Hs00168352_m1 (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR), Hs00996004_m1 (monoglyceride lipase, MGLL), Hs00173425_m1 (lipoprotein lipase, LPL) and Hs00354519_m1 (CD36). The expression of each gene was normalized to the expression of GADPH (Hs02786624_g1).
Lipidomic profiling and data processing
Dried sample extracts were reconstituted in 100 μL 2:1:1 v/v/v isopropanol/acetonitrile/water. 5 μL aliquots were injected into an ACQUITY I-class ultra-performance liquid chromatography (UPLC) system (Waters, Germany) coupled to an Impact II high-resolution quadrupole time-of-flight mass spectrometer (BrukerDaltonik GmbH, Germany). Chromatographic separation was achieved by gradient elution (%A: 0 min, 60; 1.2 min, 57; 1.26 min, 50; 7.2 min, 46; 7.26 min, 30; 10.8 min, 0; 12.96 min, 0; 13.02 min, 60; 14.4 min, 60) using a buffered solvent system (A: 60:40 v/v acetonitrile/water, B: 90:10 v/v isopropanol:water, both with 10 mM ammonium formate and 0.1% formic acid) and a 2.1 mm × 75 mm × 1.7 μm CSH-C18 column (Waters, Germany) equipped with an 0.2 μm pre-column in-line filter. The flow rate was 0.5 mL min− 1 and column temperature was 55 °C. Electrospray ionization (ESI) conditions were as follows: polarity (+), capillary voltage, 4500 V, end plate offset, 500 V, nebulizer pressure, 2.5 bar, dry gas (N2) flow, 8 L/min. Ion transfer parameters were set to: Funnel 1 RF, 200 Vpp, Funnel 2 RF, 200 Vpp, Hexapole RF, 50 Vpp, Quadrupole Ion Energy, 5 eV, Low Mass, 100 m/z, Collision Energy, 8.0 eV, Pre Pulse Storage, 6.0 μs, Stepping Mode, Basic, Collision RF, 500–1000 Vpp, Transfer Time, 60–100 μs, Timing, 50/50, Collision Energy, 100–250%. Alternating MS and MS/MS scans were acquired using a Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH) scheme (m/z 350–975, width 25 Da). For internal calibration, Na formate clusters were spiked into the LC effluent at the end of each run.
After data acquisition, files were converted to Analysis Base File (ABF) format using a publicly available converter (Reifycs, Japan) and imported into MS-DIAL (Tsugawa et al. 2015). MS-DIAL parameter settings were as follows: Soft Ionization, Data independent MS/MS, Centroid data, Positive ion mode, Lipidomics. Detailed analysis settings were left at default, except for Retention time end (10 min), Alignment Retention time tolerance (0.2 min), Identification Retention time tolerance (3 min) and Identification score cut off (60%). Identified peaks were exported to a text file and subjected to statistical analysis.
Saturation indices were calculated as a ratio of total saturated fatty acids to total unsaturated fatty acids [
27].The total level of saturated fatty acids in individual lipid class was calculated by summing up the intensities of each saturated fatty acid containing lipid multiplied by the number of saturated fatty acids present in that particular lipid moiety. The total level of un-saturated fatty acids in individual lipid class was calculated by summing up the intensities of each un-saturated fatty acid containing lipid multiplied by the number of un-saturated fatty acids present in that particular lipid moiety.