Fig. 4

Increased cellular lipid levels are fueled by enhanced lipid uptake. a LNCaP cells were treated for the indicated times with Enz (10 μM) including the final 72 h in the presence of ¹³C-acetate. Incorporation of ¹³C into cholesterol via lipogenesis was measured by GC/MS (n = 3, mean ± SD). b Protein (blue) and mRNA (gray) expression of indicated enzymes was measured by quantitative proteomics with isobaric mass tag labels and microarray analysis, respectively (n = 3, mean ± SD). c LNCaP cells were treated as described in a, and cellular uptake of indicated fluorescent lipid probes was measured by qSCI based on the mean fluorescence intensity (MFI) of NBD-cholesterol, LDL(DiL), acetylated-LDL(DiL), or d NBD-PE and PC-A2 in LNCaP and C4-2B cells (n > 9000 cells, mean ± SD, representative of 2 independent experiments). e Fold-change mRNA expression changes of indicated lipid transporters relative to D0 of Enz-treatment were calculated from the microarray data described in Fig. 2a (top panel) or measured by qRT-PCR (bottom panel). f LNCaP cells were treated for the indicated times with Enz (10 μM), and protein expression of lipid transporters SCARB1 (left) and LDLR (right) was measured using immunofluorescence microscopy (IgG = antibody control, n > 9000 cells, mean ± SD, representative result from 2 independent experiments). g LNCaP cells were treated with Enz (10 μM) or grown in androgen-depleted media (CSS, charcoal-stripped serum) for the indicated times, and FA uptake was measured by qSCI based on the MFI of cellular Bodipy-C16:0 (n > 9000 cells, mean ± SD, representative result from 3 independent experiments). h LNCaP cells were treated for the indicated times with Enz (10 μM), and fatty acid content was analyzed by GC/MS FAME (n = 2, mean ± SD). Statistical analysis: a–h one-way ANOVA followed by Dunnett’s multiple comparisons test compared to D0; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001