Fig. 5.

Abolished LDMC because of PLIN5(Δ461–463) overexpression similarly interfered with re-esterification and FA release compared with cells overexpressing PLIN5. Transgenic COS-7 cells were cultured in medium containing 0.4 mM OA-BSA for 20 h, using ³H-labeled OA-BSA as tracer (pulse). A: Incorporation of radioactivity into TG during the pulse period was determined by separation of lipid extracts via TLC, followed by excision and liquid scintillation counting of TG-standard corresponding bands. B: Total cellular label incorporation during the FA pulse was determined by scintillation counting of medium supernatants prior to and after the pulse period. C: Following the pulse period, the cells were preincubated for 1 h in complete medium containing DMSO (vehicle) or 5 μM triacsin C. Subsequently, the cells were cultured for 3.5 h in serum-free DMEM (1 g/l glucose) containing 20 μM forskolin and 3% (w/v) FA-free BSA together with either DMSO or 5 μM triacsin C (chase). The FA release into the medium was quantified by liquid scintillation counting. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired Student’s t-test (ns = not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. corresponding β-Gal conditions; ###P < 0.001 vs. corresponding DMSO conditions).