Figure 1.

hGX sPLA₂promotes enrichment of LDs with long-chain PUFA-containing triglycerides. (A, B) LD levels in control cells and cells treated with 10 nM recombinant hGX sPLA₂ for 48 h in serum-rich medium. (A) Neutral lipid content was quantified by Nile Red staining and flow cytometry (n = 3 independent experiments; 20000 cells per treatment). (B) Representative live-cell confocal microscopy images of LDs stained with BODIPY 493/503 (green) and nuclei with Hoechst 33342 (blue). (C) PLA2G10 mRNA expression in samples from cancer patients (TCGA PanCancer Atlas Studies, cBioportal). mRNA levels were batch normalized from Illumina HiSeq_RNASeqV2. Dashed line indicates median. (D) Diagram illustrating the hypothesis that unsaturated fatty acids (FAs) released through hGX sPLA₂ membrane hydrolysis are incorporated into triacylglycerols (TAGs) and lead to lasting changes in LD TAG acyl-chain composition. (E) hGX-sPLA₂-induced changes in the incorporation of radiolabelled oleate into cellular TAGs in MDA-MB-231 cells grown in complete medium for 24 h (Fed), in complete medium for 24 h followed by 96 h of serum starvation (Fed + Starved), and in serum-free medium for 96 h (Starved) (n = 4 independent experiments). (F) Diagram illustrating the experimental treatments used for lipidomic analysis in (G)–(L). (G–L) TAG lipidomic analyses of MDA-MB-231 cells treated with recombinant hGX sPLA₂ in complete medium for 48 h (Fed; G–I, K, L) or in the absence of serum for 96 h (Starved; G, J). Cell lysates were collected and analysed by UPLC/qTOF-MS (n = 4 independent experiments). (H–J) hGX sPLA₂-induced changes in the levels of individual TAG species presented as a representative z-score heat-map (H) and volcano plots (I, J) prepared by log₂ data transformation and multiple t-test analyses (n = 4 independent experiments). Statistically significant changes (–log₁₀(P value) > 1.30) in TAGs containing mostly saturated and mono-unsaturated FAs (SFA/MUFA-TAGs with 0–2 double bonds; blue) and those containing polyunsaturated FAs (PUFA-TAGs with 3–12 double bonds; red) are shown. (K, L) hGX sPLA₂-induced changes in relative levels of TAG species grouped by number of acyl-chain C-atoms (chain length) and double bonds (chain unsaturation). (A–L) Data are means ± SEM of at least three independent experiments. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (unpaired t-tests).