Figure 7.

Effects of SPL overexpression on free fatty acid-mediated lipid droplet formation and Plin2 gene expression. INS1E cells were stably transfected with the pcDNA3-huSPL-GFP vector, followed by clone selection by G418. Cells were incubated in the absence or presence of 500 µM PA, OA or a combination of PA and OA (500 µM of each) for 24 h. Thereafter, (a) cells were stained with Oil Red O and DAPI (nuclear staining), fixed and analyzed using a CellR/Olympus IX81 inverted microscope system (objective 40×, Olympus, Hamburg, Germany). To quantify lipid droplet formation in single cells, images were analyzed with xCellence Rt software. The fluorescence of lipid droplets is shown in relation to the total cell area and measured at 560/630 nm, and representative pictures are shown in (b) (red: Oil Red O, blue: DAPI, bar: 20 µm). Shown in (c) is the mRNA expression of Plin2. RNA was isolated, cDNA was synthetized and real-time PCR was performed. Shown are MEANS ± SEM from n = 4–6 independent experiments; each condition was measured in triplicate. ANOVA followed by Bonferroni, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated, # p < 0.05 vs. INS1E-ctr cells treated in the same way.