DOI: 10.5061/dryad.2bvq83c4h
title: Lipidomic dataset of MDA-MB-231 human breast cancer cells treated with diacylglycerol acyltransferase (DGAT) inhibitors and docosahexaenoic acid (DHA)

description: Lipid droplets (LDs) are dynamic fat storage organelles involved in fatty
 acid metabolism. By storing polyunsaturated fatty acids (PUFAs) in the
 form of neutral lipids, LDs can either mitigate or exacerbate lipotoxic
 damage. However, their role in regulating cellular fatty acid
 distribution, membrane unsaturation, and ferroptosis susceptibility
 remains poorly understood. To explore how LD turnover modulates membrane
 lipid composition and ferroptosis sensitivity in cancer cells exposed to
 exogenous PUFAs, MDA-MB-231 human triple-negative breast cancer cells were
 supplemented with non-lethal concentrations of exogenous docosahexaenoic
 acid (DHA) and treated with inhibitors of diacylglycerol acyltransferases
 (DGAT) 1 and 2. Untargeted lipidomic analysis revealed substantial
 lipidome remodeling with 302 significantly altered lipids in response to
 DGAT inhibition, DHA supplementation, or combined treatment.
description: Sample Type: Total cell lysates of MDA-MB-231
 cells. Cell Culture and Sample Preparation:
 MDA-MB-231 cells were seeded in 6-well plates at 3 × 10^5
 cells/well and left to attach for 24 h. After 24 h, the cells were treated
 with DGAT inhibitors and 25 μM DHA in RPMI-1640 media supplemented with
 10% FBS. After 1, 4, and 24 h, cell lysates were collected. Total protein
 content was determined in each sample using the Pierce 660 nm Protein
 Assay Kit following the manufacturer’s instructions and used to normalize
 lipid signals. Lipid Extraction for Mass
 Spectrometry: Lipids were extracted using the LIMeX
 LC–MS workflow, including biphasic solvent system consisting of cold
 methanol, methyl tert-butyl ether, and 10% methanol. An aliquot of the
 upper organic phase was collected, evaporated, and resuspended in methanol
 containing the internal standard
 [12-[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA).
 LC-MS Data Acquisition and Lipid Annotation:
 For untargeted lipidomics, the LC–MS systems consisted of a
 Vanquish UHPLC System (Thermo Fisher Scientific, Bremen, Germany) coupled
 to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen,
 Germany). We separated the lipids on an ACQUITY Premier BEH C18 column (50
 × 2.1 mm; 1.7 μm) with VanGuard FIT (5 × 2.1 mm; 1.7 μm) and detected them
 using positive and negative electrospray ionization (ESI). Raw data were
 processed using MS-DIAL 4.94 with an in-house retention time–m/z library
 and MS/MS libraries from public and commercial sources (MassBank, MoNA,
 NIST23). Metabolites were annotated according to the LIPID MAPS
 classification system and the shorthand notation for lipid structures. All
 analytes in MS-DIAL were manually curated. Data
 Processing and Quality Control: Data were filtered
 using blank samples, serial dilution samples, and quality control (QC)
 pool samples with a relative standard deviation (RSD) &lt; 30%.
 Normalization was performed using the LOESS approach, based on QC pool
 samples injected regularly between every 10 actual samples. Samples were
 randomized across the platform run. Signal intensities were normalized to
 protein content  (arb. units/µg protein). Outliers within biological
 triplicates were identified by calculating the coefficient of variation
 (CV); values with a CV greater than 30% were considered outliers and
 removed. Missing values were imputed by averaging the remaining two
 biological replicates. Lipids in individual samples that still exhibited a
 CV greater than 30% after this step were excluded from further analysis.
 Data are reported as signal intensities normalized to total protein levels
 [arb. units/μg protein].
description: # Lipidomic dataset of MDA-MB-231 human breast cancer cells treated with
 diacylglycerol acyltransferase (DGAT) inhibitors and docosahexaenoic acid
 (DHA) Dataset DOI:
 [10.5061/dryad.2bvq83c4h](https://doi.org/10.5061/dryad.2bvq83c4h) ##
 Description of the data and file structure Human MDA-MB-231 cancer cells
 were treated with a mixture of DGAT1 and DGAT2 inhibitors, supplemented
 with exogenous DHA, harvested at three time points, and analyzed by LC/MS
 using the LIMex platform. Untargeted lipidomic analysis revealed
 substantial lipidome remodeling with 302 significantly altered lipids in
 response to DGAT inhibition, DHA supplementation, or combined treatment.
 ### Files and variables #### File: MDA231_DGATi_lipidomics_data_FINAL.csv
 **Description:** The file shows lipidomic data from MDA-MB-231 cells
 treated with DGAT inhibitors, 20 μM T863 for DGAT1 and 20 μM PF-06424439
 for DGAT2, and 25 μM DHA. Total cell lysates were collected at 1, 4, and
 24 h following treatment. ##### Variables * Sample numbers: 2_1, 3_1, ....
 * Conditions: control (untreated cells), DGATi (DGAT inhibitors- treated
 cells), DHA (DHA-treated cells), DGATi+DHA (DGATi and DHA-treated cells).
 * 0 h (before treatment), 1 h (1 h following treatment), 4 h (4 h
 following treatment), 24 h (24 h following treatment) * Metabolite name
 (full) * Lipid Class: Lipid class nomenclature | Abbreviation | Lipid
 class | Example: chain summed | Example: chain separated | |
 :------------------------------------------------------- | :---------- |
 :-------------------- | :------------------------ | | Acylcarnitine | CAR
 | CAR 18:1 | CAR 18:1 | | Cholesteryl ester | CE | CE 18:1 | CE 18:1 | |
 Cardiolipin | CL | CL 70:5 | CL 16:0\_18:1\_18:2\_18:2 | | Ceramide
 non-hydroxyfatty acid-dihydrosphingosine | Cer-NDS | Cer 44:0;2O | Cer
 18:0;2O/26:0 | | Ceramide non-hydroxyfatty acid-sphingosine | Cer-NS | Cer
 42:1;2O | Cer 18:1;2O/24:0 | | Cholesterol | Cholesterol | Cholesterol |
 Cholesterol | | Coenzyme Q | CoQ | CoQ10 | CoQ10 | | Diacylglycerol | DG |
 DG 38:4 | DG 18:0\_20:4 | | Free fatty acid | FA | FA 18:1 | FA 18:1 | |
 Ganglioside GM3 | GM3 | GM3 36:1;2O | GM3 18:1;2O/18:0 | |
 Dihexosylceramide | Hex2Cer | Hex2Cer 34:1;2O | Hex2Cer 18:1;2O/16:0 | |
 Trihexosylceramide | Hex3Cer | Hex3Cer 34:1;2O | Hex3Cer 18:1;2O/16:0 | |
 Hexosylceramide non-hydroxyfatty acid-dihydrosphingosine | HexCer-NDS |
 HexCer 40:0;2O | HexCer 18:0;2O/22:0 | | Hexosylceramide non-hydroxyfatty
 acid-sphingosine | HexCer-NS | HexCer 38:1;2O | HexCer 18:1;2O/20:0 | |
 Lysophophatidylcholine | LPC | LPC 18:1 | LPC 18:1 | |
 Lysophosphatidylethanolamine | LPE | LPE 18:1 | LPE 18:1 | |
 Monogalactosyldiacylglycerol | MGDG | MGDG 34:1 | MGDG 16:0\_18:1 | |
 N-acyl ethanolamines | NAE | NAE 20:4 | NAE 20:4 | | Phosphatidylcholine |
 PC | PC 38:4 | PC 18:0\_20:4 | | Ether-linked phosphatidylcholine |
 EtherPC | PC O-38:4 | PC O-18:0\_20:4 | | Phosphatidylethanolamine | PE |
 PE 38:4 | PE 18:0\_20:4 | | Ether-linked phosphatidylethanolamine |
 EtherPE | PE O-38:4 | PE O-18:0\_20:4 | | Oxidized
 phosphatidylethanolamine | OxPE | PE 38:4;4O | PE 18:0\_20:4;4O | |
 Phosphatidylglycerol | PG | PG 38:4 | PG 18:0\_20:4 | |
 Phosphatidylinositol | PI | PI 38:4 | PI 18:0\_20:4 | | Ether-linked
 phosphatidylinositol | EtherPI | PI O-36:4 | PI O-16:0\_20:4 | |
 Phosphatidylserine | PS | PS 38:4 | PS 18:0\_20:4 | | Sphingomyelin | SM |
 SM 38:1;2O | SM 18:1;2O/20:0 | | Sterol sulfate | SSulfate | ST 27:1;O;S |
 ST 27:1;O;S | | Triacylglycerol | TG | TG 56:8 | TG 16:0\_18:2\_22:6 | |
 Ether-linked triacylglycerol | EtherTG | TG O-56:7 | TG O-16:0\_18:1\_22:6
 | **Units of measurement:** AU/μg total protein