Histochem Cell Biol (2012) 137:205–216 211 Fig. 2 PLIN5 overexpression (gene electroporation) in rat muscle using an empty vector as internal control. a SpeciWcity of the PLIN5 antibody (Progen). A full-length PLIN5 vector was transfected in cells without endogenous PLIN5 expression (HEK cells), this resulted in ectopic expression of PLIN5. b PLIN5 protein content in protein lysates from electroporated tibialis anterior muscle (n = 16). Error bars represent SEM, *P < 0.01. The samples were derived from the same experiment and were simultaneously processed in random order on two gels/blots. EV: empty vector. c Shows the eVect of PLIN5 overexpression on lipid storage (upper right hand panel) and PLIN5 to interact tightly with mitochondria in the PLIN5-over- expressing muscle and sometimes even appear to envelop the lipid droplets (Fig. 3). Immunogold labelling of PLIN5 in electroporated muscle conWrmed the localization of PLIN5 in mitochondria, on LDs and on the LD-mitochondrial interface (Fig. 4). PLIN5 overexpression does not aVect mitochondrial density To examine the putative eVect of PLIN5 overexpression on mitochondrial density, we measured in whole muscle homogenates the protein content of PGC1 (Fig. 5a), VDAC (Fig. 5b) and 5 distinct structural components of subunits of the individual OXPHOS complexes (complex I content (lower right hand panel) compared to the empty vector control (upper and lower left hand panels). LDs are stained in red), PLIN5 in green and cell membranes in blue. Bars represent 100 m. d Overlay of PLIN5 and mitochondrial proteins upon PLIN5 overexpression. PLIN5 overexpression increases LD (in red) and PLIN5 (in green) content (upper right hand and middle left hand panel, respectively). Mitochondria are shown in magenta (middle left hand panel). The low- er left hand panel shows LD decorated with PLIN5 while the lower right hand panel shows PLIN5 interaction with mitochondria. The bar represents 20 m. Representative image are shown subunit NDUFB8, complex II subunit 30 kDa, complex III subunit Core 2, complex IV subunit I and complex V ATP synthase subunit alpha (Fig. 5c). Although we observed a non-signiWcant (P = 0.14) increase in PGC1 , a key co- activator for mitochondrial biogenesis, protein content of VDAC and OXPHOS complexes remained unaltered, indi- cating that 8 days after overexpressing PLIN5 mitochon- drial density was not aVected. PLIN5 overexpression does not improve respiration of isolated mitochondria Mitochondrial respirometry assays in the mitochondria iso- lated from PLIN5 overexpressing muscles relative to mus- cles isolated from empty vector-electroporated muscles did 123