Fig. 5.

Impact of the in vivo roles of the perilipin proteins in basal lipolysis. The 3T3-L1 preadipocytes stably expressed empty vector (EV), WT, or chimeric forms of perilipin (Plin). (A) Perilipin and ABHD5 protein expression were assessed by immunoblotting (Left). Stable cell lines were loaded with [¹⁴C]oleic acid and the release of incorporated radioactivity was measured. Lipolysis is expressed as the quantity of radioactivity released into the medium as a percentage of the total radioactivity incorporated (Right). Results from three independent experiments are expressed as mean ± SD. ***P < 0.001. (B) Differentiated 3T3-L1 adipocytes transfected with control or two independent siRNAs for perilipin 1 (Plin1) were fixed for immunofluorescence, harvested for protein, or assessed for lipolysis. Endogenous localization of perilipins 1 and 2 and ABHD5 was examined in perilipin 1 (siRNA 98176) knockdown adipocytes (Upper). Note that cells in the boxed area display a loss of LD staining for perilipin 1 and ABHD5, whereas perilipin 2 staining is increased. Other cells in the same field were presumably not transfected with the siRNA so retain perilipin 1 expression. Perilipin 1 or perilipin 2 protein expression was detected by immunoblotting (Lower Left). Blots are representative of three separate experiments. Lipolysis was determined by quantifying glycerol release into the medium (Lower Right). The data are mean ± SD of four independent experiments and are presented as fold increase of glycerol release compared with control siRNA treatment (set as 1). ***P < 0.001. (C) Differentiated 3T3-L1 adipocytes were transfected with control, two independent siRNAs for perilipin 2 (Plin2), or in conjunction with perilipin 1 siRNA (siRNA 98176). ABHD5, perilipin 1, or perilipin 2 protein expression was detected by immunoblotting (Left) and lipolysis (Right) assayed as described in B. Blots are representative and lipolysis data are a result of three separate experiments.