the adipogenic markers, C/EBPb at day 1, PPARc and aP2 at day 7, and the accumulation of lipids were significantly decreased after adipogenic induction in PHB1- or PHB2-silenced 3T3-L1 cells. Additionally, based on the reports that PHB is required for Ras-induced RafMEK-ERK pathway buy 181223-80-3 activation in epithelial cells and that activation of MEK/ERK signaling is necessary to initiate the preadipocyte in the differentiation process during the early phase, we determined the level of phosphorylated ERK1/2 at the early stage of adipogenic induction upon PHB-silencing in 3T3-L1 cells. Our data demonstrated that the phosphorylation of ERK1/2 is remarkably inhibited post-adipogenic induction in either PHB1 or PHB2-silencing 3T3-L1 cells, suggesting that the effect of PHBs in adipogenesis might be via ERK phosphorylation in 3T3-L1 cells. Interestingly, when using the gain-of-function strategy, a slight decrease in adipocyte markers and lipid accumulation were observed upon overexpression of PHB1 in adipocyte-differentiating human ASC. These observations were in agreement with a recent report in 3T3-L1 cells with uncertain mechanisms. Enhanced levels of mitochondrial PHBs and mtDNA in adipogenic 3T3-L1 cells The essential role of mitochondria in adipocyte differentiation is well described, which led us to examine the changes in the levels of mitochondrial PHBs before and after 3T3-L1 cell adipogenesis. Our immunocytochemistry results showed that either PHB1 or PHB2 in mitochondria was increased after seven days of adipocyte differentiation. PHB1 were mainly present in mitochondria while PHB2 in mitochondria and 5 Prohibitins Are Required for Adipogenesis 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 and slightly increased in nuclear fraction, 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 induction. The increments of mtDNA were partially suppressed upon PHB1- or PHB2- silencing in 3T3-L1 cells, whether or not subject to adipogenic induction. These results, in accordance with the observations in HeLa cells, 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 3T3L1 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. To further investigate the effects of mitochondrial PHBs during adipogenesis, the mitochondrial morphology of PHBsilencing 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 a