buffer. The enzyme activities were then calculated by measuring the optical density of precipitation, colorimetric enzyme reaction products, using the NIH ImageJ software. Standard curves were created from multiple determinations of complex activities in cultured 3T3-L1 cell extracts. Transmission electron microscopy Three days post transfection of siRNA, 3T3-L1 cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate LY341495 site buffer for two hours at 4uC, and post-fixed with 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer for one hour at 4uC. The specimens were then incubated in 0.5% aqueous uranyl acetate for 2 hours at room temperature for en bloc staining, and in a graded series of ethanol for dehydration. Thereafter, the specimens were embedded in Embed 812 resin. Ultrathin sections were cut and post-stained with uranyl acetate and lead citrate. These sections were examined using a JEOL 1200EX transmission electron microscope. Statistics All samples were at least prepared in triplicate. Results from the quantitative studies are expressed in terms of mean and standard deviation of three independent experiments. Statistical analyses were performed by one-way ANOVA and comparisons between groups were performed using the Student’s t test. Differences were considered significant at p,0.05. Mitotracker staining and confocal microscopy MitoTracker Red CMXRos, a mitochondriaspecific cationic fluorescent dye, was used to label mitochondria. 3T3-L1 cells in Lab-Tek chamber slides were stained with 250 nmol/L MitoTracker in serum-free DMEM for 15 minutes at 37uC according to the manufacturer’s instructions. A Leica TCS SP5 Confocal Microscopy System, consistent with a prior report of a microarray analysis showing that the expression levels of PHBs are increased during 3T3-L1 cell adipogenesis. The sequentially induction of the adipogenic markers, CCAAT/ enhancer-binding protein beta, peroxisome proliferator-activated receptor gamma and adipocyte Protein 2, were observed in these conditions. Additionally, we determined ” that the alterations of protein levels of PHB1 and PHB2 followed 11118042” a similar pattern during adipogenesis in human ASC compared to mouse 3T3-L1 cells. When we examined the mRNA levels of PHBs in differentiating 3T3-L1 cells, we found that both PHB1 and PHB2 were significantly increased as early as six hours post adipogenic induction and peaked at day two and fell to basal levels by one to two weeks, suggesting post-translational protein stabilization. Among the three hormone ingredients in the adipogenic cocktail, the PHBs expression was mainly induced by IBMX and insulin rather than dexamethasone in 3T3-L1 cells. In addition, the levels of PHBs in white adipose tissue from wild-type, heterozygous and homozygous obese mice, both female and male, were compared. Interestingly, the expression levels of PHBs in WAT from obese mice were not higher than that from wild type ones. Actually, even decrease of the expression of adipogenic genes in obesity has been observed, which is considered as the consequence of the dedifferentiation in WAT from obese mice. PHBs are required for PPARc expression and adipocyte differentiation in 3T3-L1 cells To investigate the possible roles of increasing PHBs during adipogenesis, we screened siRNA for effective knockdown of PHBs expression in this model. Three different siRNA oligonucleotides, siPHB1-1, siPHB1-2 and siPHB1-3, were used to target PHB1; while siPHB2-1, siPHB2-2 and siPHB2-3 w