nuclei. The contents of PHBs were further determined in isolated mitochondria and nuclei. Our data demonstrated that the protein levels of PHB1 and PHB2 were remarkably increased in mitochondrial fraction (Fig. 3C) and slightly increased in nuclear fraction (Fig. S3), seven days post adipogenic induction. These findings indicate that the recruitment of PHBs to the mitochondria is enhanced during adipogenesis and that this is not simply because of increased mitochondrial biogenesis. To investigate the effects of PHBs on mitochondrial biogenesis during adipogenesis, the relative contents of mitochondrial mtDNA were examined in 3T3-L1 cells upon PHB knockdown and adipocyte-differentiation induction. Our results showed that the contents of relative mtDNA were significantly increased in 3T3-L1 cells subject to adipogenic Figure 3. Content of mitochondrial PHBs and mtDNA in 3T3-L1 cells pre- and post-adipogenesis. A, B and C. Over-confluent 3T3- L1 cells (Day 0) were induced for adipocyte differentiation for 7 days. A, PHB1 (green) or B, PHB2 (green) was detected by using immunocyto- chemistry. CytC (red) was used as the mitochondrial marker. Bar = 10 mm. C. The levels of PHB1 and PHB2 in isolated mitochondria were detected using the immunoblotting analysis. The mitochondrial marker porin, also called the voltage dependent anion channel (VDAC), was used as a loading control. D. 3T3-L1 cells were transfected with indicated siRNAs and cultured for three days. The relative mtDNA content was evaluated by a ratio of the DNA level of mitochondrial Complex I to the DNA level of nuclear 18SrRNA. The relative mtDNA content in 3T3-L1 cells transfected with siControl was set to 1. *p,0.05, **p,0.01 compared to siControl (Day 0). #p,0.05, ##p,0.01 compared to siControl (Day 7). doi:10.1371/journal.pone.0034315.g003 Prohibitins Are Required for Adipogenesis induction. The increments of mtDNA were partially suppressed upon PHB1- or PHB2- silencing in 3T3-L1 cells, whether or not subject to adipogenic induction (Fig. 3D). These results, in accordance with the observations in HeLa cells [21], suggest that PHBs are required in maintaining mitochondrial contents. Effects of PHB silencing on mitochondria in 3T3-L1 cells The ultrastructure of mitochondria in siRNA transfected 3T3- L1 cells was examined using electron microscopy. We observed that the regular lamellar cristae in mitochondria were lost in siPHB1- or siPHB2-transfected 3T3-L1 cells, whereas siControl transfection did not affect the ultrastructure of mitochondria (Fig. 4A). To further investigate the effects of mitochondrial PHBs during adipogenesis, the mitochondrial morphology of PHB- silencing 3T3-L1 cells was compared before and after adipogenic induction. MitoTracker analysis revealed that, instead of normally tubular mitochondria, about 40% of the PHB1 or PHB2 knockdown cells consisted of fragmented mitochondria either before or after the cellular adipogenesis (Fig. 4B). Actually, punctiform mitochondria have been observed in our previous study upon PHB1-knockdown in ovarian cancer cells [20]. Because a lack of PHB1 or PHB2 might reduce mitochondrial membrane integrity, disrupt OXPHOS and therefore result in increased levels of ROS [21,23,36,37], we examined the ATP accumulation and ROS formation upon PHB1- or PHB2-silencing in 3T3-L1 preadipocytes. Our results showed that ROS levels were significantly increased in either PHB1- or PHB2-knockdown 3T3-L1 cells, whereas the contents of ATP were unaffected. The increase of ROS could be ablated when the cells were preincubated with PEG-catalase, a hydrogen peroxide scavenger, indicating the specificity of the DCF-DA signal for hydrogen peroxide (Fig. 5A and 5B). These results, in agreement with the effects of PHB deficiency on wild type C. elegans [27], suggest the damage of mitochondrial OXPHOS system upon PHB-silencing in 3T3-L1 cells. To further investigate the underlying mechanisms of the extra ROS generation, mitochondrial complex I activity was examined. Our data demonstrated a reduction of the complex I activity in PHB1- or PHB2-knockdown 3T3-L1 cells (Fig. 5C). This result is in accordance with the observation in the PHB1- depleted endothelial cells [23], indicating the affection of mitochondrial electron transport in the OXPHOS system. Discussion It is well established and reviewed that mitochondrial biogenesis is essential during adipocyte differentiation; and that PHBs complexes, located in the inner membrane of mitochondria, play a crucial role in mitochondrial morphology and dynamics [22,35,38,39]. A recent study provides the evidence that depletion of PHB1 or PHB2 in C. elegans significantly decreases mitochon- drial membrane potential and adipose accumulation in young adults [27]. However, studies on the effects of PHBs in mammalian adipogenesis are currently lacking. Data presented here indicate that the levels of PHBs are remarkably increased during adipogenesis in 3T3-L1 cells, and silencing of PHBs causes mitochondrial fragmentation and adipogenic reduction. Either PHB1 or PHB2-knockout mice exhibit early embryonic lethality, indicating that these proteins have fundamentally important functions [14,40]. In primary mouse adipocytes, PHB1 decreases insulin-stimulated oxidation of glucose and fatty acid, implying that PHB1 may play a role in promoting fat accumulation [41]. Indeed, our results showed the incremental mRNA and protein expression of PHBs in a time course manner using real-time PCR and immunoblotting, which is consistent with PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34315