may influence and limit the development of adipose tissue. Our data provide the evidence that the contents of ROS are enhanced in either PHB1 or PHB2-knockdown 3T3-L1 preadipocytes, which is consistent with the observation in PHB1-deficient endothelial cells and in PHB1- or PHB2- deficient nematodes [23,27]. It is reported that the reason for the extra ROS generation may be the inhibition of mitochondrial complex I activity in PHB-depleted cells, and therefore affects mitochondrial electron transport in the OXPHOS system [22,23]. Indeed, our results demonstrate a reduction of mitochondrial complex I activity in 3T3-L1 cells upon knockdown of PHB1 or PHB2. To maintain cytochrome oxidase activity and overall ATP produc- tion, there are compensatory mechanisms at play in mitochondria, involving an increase in electron flow through complex II and/or complex III, which may explain the unaffected ATP levels in this situation [23]. In summary, enhanced expression and mitochondrial recruit- ment of PHBs are required for maintaining mitochondrial morphology and inducing adipocyte differentiation in 3T3-L1 cells. These findings underscore the emerging concept of mitochondrial PHBs as important molecules in modulating fat metabolism [27]. Since both mitochondrial biogenesis and adipocyte differentiation have been linked to obesity, PHBs may become interesting candidates for further studies in this field. Supporting Information Figure S1 Expression of PHBs in mouse WAT. The relative mRNA expression levels of PHB1 and PHB2 in WAT from male or female of wild type (ob2/2), heterozygous (ob+/2) and homozygous (ob2/2) obese mice were analyzed with real-time PCR. The materials and methods were described in Text S1. The 36B4 was used for normalization (n = 5). (TIF) References 1. Johnson PR, Hirsch J (1972) Cellularity of adipose depots in six strains of genetically obese mice. J Lipid Res 13: 2–11. 2. 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