210 Histochem Cell Biol (2012) 137:205–216 Fig. 1 Perilipin 5 protein expression in whole muscle homogenates, isolated mitochondria and muscle sections. a Western blotting of per- ilipin 5 in whole skeletal muscle lysates from heart muscle, extensor digitorum longus (EDL) and tibialis anterior muscles and mitochondria isolated from tibialis anterior muscle. b Western blots incubated with the PLIN2 antibody did not show any bands in isolated mitochondria, indicating that the mitochondrial fraction was devoid of lipid droplets measured PLIN5 protein content in total muscle protein lysates as well as in mitochondrial fractions from rat skele- tal muscle (Fig. 1a). A double band representing PLIN5 was observed in whole muscle lysates and more strikingly also in isolated mitochondria. To exclude the ability that the PLIN5 signal detected in the mitochondria originated from contamination of the mitochondrial fraction with ER or LD derived membrane fractions we examined protein expression of ADRP/PLIN2 in our mitochondrial fractions (Fig. 1b). The absence of an ADRP/PLIN2 band indicates that the mitochondrial fractions used were devoid of ER or LD membrane structures, substantiating our notion that PLIN5 indeed localizes to mitochondria. We further substantiated mitochondrial localisation of PLIN5 using immunogold electron microscopy, the gold standard methodology for subcellular localisation studies of single proteins. In all muscles examined (soleus, EDL and cardiac muscle) PLIN5 was present on the LDs, at the inter- face of LDs with mitochondria, as well as in the mitochon- dria (Fig. 1c, d), whereas other structures were almost completely devoid of gold particles. Negative controls by omission of the primary antibody or by using non-immune serum did not show any immunogold staining. Immuno- gold staining using the ADRP/PLIN2 antibody only revealed staining associated with LD (data not shown). 123 and was not contaminated with fragments of the ER. HEK cells trans- fected with PLIN2 plasmid were used as positive control. c PLIN5 im- munogold labelling in rat soleus muscle. PLIN5 coats the lipid droplets and is present at mitochondria but not at myoWbrils. d PLIN5 immuno- gold labelling in rat left ventricle showing mitochondrial localization of PLIN5. LD lipid droplet, M mitochondrion PLIN5 overexpression in rat tibialis anterior muscle To examine the eVect of overexpression of PLIN5 on its sub- cellular localisation, a CMV-driven full-length PLIN5 con- struct was used for unilateral overexpression of PLIN5 in rat tibialis anterior muscle by gene electroporation using the contralateral empty vector-electroporated leg as an internal control. Upon transfection in HEK cells the construct used resulted in the anticipated double band on the Western, repre- senting two PLIN5 isoforms (Fig. 2a). In rat tibialis anterior muscle, PLIN5 overexpression doubled PLIN5 protein con- tent in whole muscle homogenates (P = 0.0006, n = 14, paired t test) (Fig. 2b). Consistent with results obtained in vitro (Dalen et al. 2007; Wolins et al. 2006), PLIN5 overexpression profoundly augmented intramyocellular lipid storage in the transfected muscle (Fig. 2c). Immunohisto- chemistry revealed that PLIN5 was present on LDs and showed punctuate mitochondrial localisation. Double immu- noXuorescence using an antibody cocktail against structural components of all 5 OXPHOS complexes conWrmed co- localisation of PLIN5 with mitochondria (Fig. 2d). Ultra- structural analysis by electron microscopy conWrmed the light microscopy based observation of augmented lipid stor- age upon PLIN5 overexpression and clearly showed increased LD size (Fig. 3). In addition, almost all LD appear