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.

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) < 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].