Lipidomics analysis of MDA-MB-231 cells upon WMK-1 treatment

MDA-MB-231 cells were treated with compounds, including DMSO (Vehicle), ML210 (for 2h, 3 μM), WMK-1 (5 μM, for 3h or 6h), ZL-1-180 (20 μM, for 7h), in the presence or absence of Lip-1 (1 μM). For lipidomic analysis, 1 µL of the prepared tissue lipidomics samples were loaded into an ACQUITY UPLC BEH C8 column (100 × 2.1 mm,1.7 μm, 130 Å, Waters, Cat# 186002878), and separated chromatographically with a 25-min gradient elution at a flow rate of 0.3 mL/min. The mobile phase for the positive ion acquisition mode contains H2O with 0.1% (v/v) FA and 10 mM ammonium acetate for Phase A, and ACN/IPA (80:20) with 0.1% (v/v) FA for Phase B. The mobile phase for the negative ion mode is similar to the ones for the positive ion mode except excluding formic acid in the buffer. The LipidSearch software (v4.2.21, Thermo Fisher Scientific) was used for lipidomics peak detection, peak alignment, and compound annotation. Specifically, we used a 0.5-min retention time interval for peak detection with the isotope compositions recalculated. 5 ppm and 8 ppm deviations were employed as tolerance ranges for precursor and product ions, respectively, during Database searches. The minimum relative fragment ion intensity threshold was set at 1%. Isomer peaks were filtered out. In parallel, the precursor ion quantitation was performed with a 5 ppm tolerance and a 0.5-min retention time range. Lipidomics data normalization:Before downstream analysis of lipidomics data, the following criteria was employed to exclude unreliable ion peaks: 1) exclusion of peaks with a peak-quality (PQ) score < 0.9 or peak area score < 0.7; 2) manual inspection of peak shapes, excluding those with abnormal shapes or insufficient data points; 3) filtering out ions with a relative standard deviation (RSD) > 0.3 across all QC samples in each experimental set; 4) exclusion of lipid species with intensity lower than five times of that of blank samples; For downstream analysis, the raw intensity data were normalized by dividing by the median intensity of the corresponding sample and scaled by a scaling factor, 10^6. Then, the fold change of each lipid ion between compound treatment (ML210, WMK-1, ZL-1-180 or Lip-1 rescue) and Vehicle treatment was calculated. P value was calculated using two-tailed, non-parametric, unpaired Student’s t-test. Lipid species with fold change >= 1.5 and P value <= 0.05 were considered as significantly changed lipid species after WMK-1 treatment and were then used for plotting