followed by PBS wash and subsequent treatment with cold absolute methanol for 5 min at 220uC. The cells were rinsed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After blocking with 5% BSA in PBS for 1 hour, the cover glasses were incubated with anti-PHB1 (Abcam, 1:100), anti- PHB2 (Abcam, 1:50) or anti-Cytochrome C (CytC) (Invitrogen, 1:500) antibody in 0.1% BSA in PBS at room temperature for two hours. The cells were then washed with PBS and incubated with Rhodamine (Invitrogen, 1:250) or Alexa Fluor 488 (Invitrogen, 1:500) conjugated secondary antibody in 0.1% BSA in PBS at room temperature for 1 hour. Thereafter, the cover glasses were mounted upside down on microscope slides containing mounting medium (Vector Laboratories; Burlingame, CA). The mounted slides were examined under an Olympus BX41 microscope equipped with an Optronics Magnafire digital camera and Prior Proscan motorized driven stage (Olympus, Melville, NY). For each image, specific antibody staining was merged with CytC (red) using Soft Imaging System software that results in virtually no pixel shifting during the image merge. Representative photomi- crographs were arranged using Adobe Photoshop without any further adjustment to maintain the true nature of the findings. Isolation of mitochondria Isolation of mitochondria was performed using a mitochondria isolation kit (Thermo Scientific) according to the manufacturer’s protocol and previous descriptions [19,20]. Briefly, pre- and seven days post-adipocyte differentiation 3T3-L1 cells in 100 mm dishes were harvested using trypsinization. A reagent-based method was used for cell homogenization allowing multiple samples to be processed concurrently. To obtain a more purified fraction of mitochondria with more than 50% reduction of lysosomal and peroxisomal contaminants, the last step for pelleting mitochondria was performed using centrifugation for 15 minutes at 3,0006 g rather than at standard 12,0006 g. Detection of mitochondrial DNA content Total DNA in 3T3-L1 cells was isolated with the DNeasy DNA isolation kit (Qiagen). The DNA levels of mitochondrial Complex I and nuclear 18S rRNA were determined by real-time PCR quantification. The relative mtDNA content was evaluated by the ratio of DNA levels between mitochondrial Complex I and nuclear 18S rRNA as previously described [29]. Transmission electron microscopy (TEM) Three days post transfection of siRNA, 3T3-L1 cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for two hours at 4uC, and post-fixed with 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer for one hour at 4uC. The specimens were then incubated in 0.5% aqueous uranyl acetate for 2 hours at room temperature for en bloc staining, and in a graded series of ethanol for dehydration. Thereafter, the specimens were embedded in Embed 812 resin. Ultrathin sections were cut and post-stained with uranyl acetate and lead citrate. These sections were examined using a JEOL 1200EX transmission electron microscope (Tokyo, Japan). Mitotracker staining and confocal microscopy MitoTracker Red CMXRos (Invitrogen), a mitochondria- specific cationic fluorescent dye, was used to label mitochondria. 3T3-L1 cells in Lab-Tek chamber slides (Thermo Scientific) were stained with 250 nmol/L MitoTracker in serum-free DMEM for 15 minutes at 37uC according to the manufacturer’s instructions. A Leica TCS SP5 Confocal Microscopy System (Leica Micro- Prohibitins Are Required for Adipogenesis systems; Bannockburn, IL) equipped with a 636/1.40 oil- immersion objective lens was used to characterize the optical properties of these samples. Images were captured with a scanning speed of 400 Hz and image resolution of 5126512 pixels, and then analyzed using Leica Application Suite, Advanced Fluores- cence (LAS AF) software. Measurement of ATP concentration The adenosine triphosphate (ATP) concentration was measured with an ENLITEN ATP assay system bioluminescence detection kit (Promega; Madison, WI). Briefly, three days after transfection of 3T3-L1 cells in a 96-well plate with siRNA oligonucleotides, 0.5% trichloroacetic acid (TCA) was added for ATP efficient release. Then, 25 mmol/L Tris-acetate (pH 7.75) was used for neutralization. After addition of recombinant Luciferase/Luciferin reagent (rL/L), luminescence was measured using a 10-second integration time with a microplate luminometer (Lmax) and SOFTmax PRO software (Molecular Devices), and was normal- ized to protein concentration. The ATP standard curve was generated by using the ATP standard (1027 mol/L) included in the kit. Reactive oxygen species (ROS) detection ROS were detected with the cell-permeable, peroxide-sensitive fluorophore, 29,79-dichlorofluorescein diacetate (DCF-DA) (Sig- ma). In brief, 3T3-L1 cells in a 6-well plate were incubated in 0.2 mmol/L DCF-DA at 37uC for 30 minutes. Cells were then washed with prewarmed PBS twice and allowed to recover in growth medium for 20 minutes at 37uC in an atmosphere of 5% CO2. Afterwards, the cells were trypsinized, washed, resuspended in PBS and analyzed by running ExpressPlus assay on a flow cytometer, Guava PCA-96 AFP (Millipore; Billerica, MA). To ensure that DCF-DA was detecting hydrogen peroxide, cells were preincubated with 250 U/ml cell-permeable polyethylene glycol (PEG)-catalase (Sigma) at 37uC for two hours. Detection of mitochondrial complex I activity The activity of complex I was determined in whole cell lysates of 3T3-L1 with the Mitochondrial Dipstick Assay kit according to the manufacturer’s instructions (Abcam). Twenty-five micrograms of proteins were allowed to wick up through the dipstick membrane. The dipsticks, with complex I immunocaptured, were transferred into the complex I enzyme substrate buffer. The enzyme activities were then calculated by measuring the optical density of precipitation, colorimetric enzyme reaction products, using the NIH ImageJ software. Standard curves were created from multiple determinations of complex activities in cultured 3T3-L1 cell extracts. Statistics All samples were at least prepared in triplicate. Results from the quantitative studies are expressed in terms of mean and standard deviation (mean 6 SD) of three independent experiments. Statistical analyses were performed by one-way ANOVA and comparisons between groups were performed using the Student’s t test. Differences were considered significant at p,0.05. Results PHBs are up-regulated during adipogenesis of mouse and human preadipocytes In the 3T3-L1 adipogenic model, we observed that the protein levels of both PHB1 and PHB2 continuously increased during the PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34315