PHB silencing could increase cellular sensitivity to apoptosis [20]. In the current study, we did not see the induction of cleaved caspase-3 in 3T3-L1 cells by simply silencing PHB1 or PHB2 without an apoptotic inducer (data not shown), thus consistent with the observation in PHB2-deficient MEFs [18]. For all of the following experiments, siPHB1-1 and siPHB2-3 were used to knockdown PHB1 and PHB2, respectively. Using the loss-of-function strategy, our results demonstrated that expression levels of the adipogenic markers, C/EBPb at day 1, PPARc and aP2 at day 7 (Fig. 2D), and the accumulation of lipids (Fig. 2E) 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 Raf- MEK-ERK pathway activation in epithelial cells [31] and that activation of MEK/ERK signaling is necessary to initiate the preadipocyte in the differentiation process during the early phase [32,33], 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 Prohibitins Are Required for Adipogenesis either PHB1 or PHB2-silencing 3T3-L1 cells (Fig. 2F), 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 (80% of control) were observed upon overexpression of PHB1 in adipocyte-differentiating human ASC (Fig.S2). These observations were in agreement with a recent report in 3T3-L1 cells with uncertain mechanisms [34]. Enhanced levels of mitochondrial PHBs and mtDNA in adipogenic 3T3-L1 cells The essential role of mitochondria in adipocyte differentiation is well described [4,5,6,7,35], 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 (Fig. 3A and 3B). PHB1 were mainly present in mitochondria while PHB2 in mitochondria and Figure 2. Silencing of PHBs reduced the adipogenesis in 3T3-L1 cells. A, B and C. 3T3-L1 preadipocytes were transfected with the siControl or siRNAs targeting PHB1 (siPHB1-1, siPHB1-2 and siPHB1-3) or PHB2 (siPHB2-1, siPHB2-2 and siPHB2-3), respectively. A and B. Cells were harvested for total RNA isolation and quantitative real-time PCR to determine the mRNA expression levels of PHB1 (A) and PHB2 (B) three days post transfection. 18S rRNA was used as an internal control. ** p,0.01 compared to siControl. C. Cells were harvested to determine the protein expression levels of PHB1 and PHB2 with Immunoblotting three days post transfection. b-actin was used as a loading control. D, E and F. 3T3-L1 cells were treated with adipogenic inducers (day 0) three days post transfection of siRNAs targeting PHB1or PHB2. siControl was used as the control for siPHBs. D. The levels of C/EBPb at day1, and the levels of PHB1, PHB2, PPARc and aP2 at day 7 were determined using immunoblotting. HSP90 was used as a loading control. E. The cells were stained with Oil Red O dye at day 7. The quantification of accumulated lipid was performed by readings on a spectrophotometer at 510 nm for cell-released dye. **p,0.01 compared to siControl. F. Phospho-ERK (p-ERK) in siRNA transfected 3T3-L1 cells was detected using immunoblotting at the indicated times of treatment with adipogenic inducer cocktail. ERK was used as a loading control. doi:10.1371/journal.pone.0034315.g002 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34315