entire 14 days of adipocyte differentiation (Fig. 1A), consistent with a prior report of a microarray analysis showing that the expression levels of PHBs are increased during 3T3-L1 cell adipogenesis [25]. The sequentially induction of the adipogenic markers, CCAAT/ enhancer-binding protein beta (C/EBPb), peroxisome prolifera- tor-activated receptor gamma (PPARc) and adipocyte Protein 2 (aP2), were observed in these conditions. Additionally, we determined that the alterations of protein levels of PHB1 and PHB2 followed a similar pattern during adipogenesis in human ASC compared to mouse 3T3-L1 cells (Fig. 1B). 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 (Fig. 1C and 1D) 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 (Fig. 1E). In addition, the levels of PHBs in white adipose tissue (WAT) 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 (Fig. S1). Actually, even decrease of the expression of adipogenic genes in obesity has been observed [30], which is Prohibitins Are Required for Adipogenesis 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 were used to target PHB2. Commercially available universal siControl was used as a control. Three days after the transfection of siRNA in 3T3-L1 cells, the mRNA levels of PHB1 were markedly reduced by siPHB1-1 (68%), siPHB1-2 (56%) or siPHB1-3 (25%), whereas the mRNA levels of PHB2 were not significantly changed (Fig. 2A). Similarly, the mRNA levels of PHB2 were decreased by siPHB2-1 (64%), siPHB2-2 (38%) or siPHB2-3 (68%), whereas mRNA levels of PHB1 were virtually identical (Fig. 2B). Interestingly, the protein levels of both PHB1 and PHB2 could be down-regulated by either siPHB1 or siPHB2 transfection (Fig. 2C). These data suggest that, in mouse 3T3-L1 preadipocytes, PHB1 and PHB2 are present in an interdependent complex and are consistent with previous findings in yeast, C. elegans, MEFs, human HeLa cells and MCF-7 cells [12,14,16,17,18]. Our earlier studies showed that Figure 1. Expression of PHBs during adipogenesis. Over-confluent 3T3-L1 preadipocytes (A, C, D) or human ASC (B) were treated with the standard adipocyte-differentiation medium for indicated times. A and B. The protein levels of PHBs and adipogenic markers, C/EBPb, PPARc and aP2, were analyzed by immunoblotting. HSP90 was used as loading control. h, hours; d, days. C and D. The relative mRNA expression levels of PHB1 (C) and PHB2 (D) were analyzed by real-time PCR. 18S rRNA was used as an internal control. * p,0.05, ** p,0.01 compared to 0 h. E. 3T3-L1 cells were treated with DMEM supplemented with 10% FBS and a different combination of dexamethasone (Dex), insulin and IBMX as indicated for 24 hours. The expression levels of PHB1 and PHB2 proteins were analyzed by using Immunoblotting. HSP90 was used as the loading control. doi:10.1371/journal.pone.0034315.g001 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e34315