Fig. 3

Enzalutamide increased cellular lipid content. LNCaP cells were treated for the indicated times with Enz (10 μM) and analyzed by qSCI based on the mean fluorescence intensity (MFI) of Nile Red to measure a cellular neutral lipid and phospholipid content and b number of lipid droplets (LDs)/cell and total area of LDs/cell (n > 9000 cells/treatment, mean ± SD, representative result of 3 independent experiments). Perilipin mRNA expression was measured by qRT-PCR (n = 3, mean ± SD). c Representative images of D0 and D21 showing phospholipid and DNA staining as described in a. d LNCaP cells were treated as in a or grown in androgen-depleted media (CSS, charcoal-stripped serum) for the indicated times, stained with Filipin, and free cholesterol was measured by qSCI (n > 9000 cells/treatment, mean ± SD, representative result from 2 independent experiments). e LC/MS shotgun lipidomics of intact lipid species of LNCaP cells treated as described in a. Total combined sum compositions of indicated lipid classes (left panel, SM, sphingomyelin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; CE, cholesterol ester; TAG, triacylglycerol), and individual CE and TAG species (right panel) are reported as fold change relative to D0 (n = 2, mean ± SD). Statistical analysis: a–d one-way ANOVA followed by Dunnett’s multiple comparisons test compared to D0; e two-way ANOVA followed by Tukey’s multiple comparisons test relative to D0; ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001