nematode markedly reduces mitochondrial membrane potential and fat content early in adulthood [27]. However, the effects of PHBs during adipogenesis in mammals are still unknown. In the current study, we demonstrate that PHB silencing results in mitochondrial fragmentation and adipogenic reduction in 3T3-L1 cells, uncovering for the first time a role for PHBs in mammalian adipogenesis. Materials and Methods Cell culture and adipocyte differentiation The murine preadipocyte 3T3-L1 cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured according to the manufacturer’s instructions. Briefly, 3T3-L1 cells were cultured in growth medium, consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen; Carlsbad, CA) supplemented with 10% cattle bovine serum (CBS), 100 U/ml penicillin and 100 ug/ml streptomycin. The cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37uC. The medium was replaced every 2–3 days. Adipocyte differentiation of 3T3-L1 cells was induced by using standard hormonal cocktail. In brief, at two days post-confluence (day0), cells were treated with a differentiation medium containing DMEM supplemented with 10% fetal bovine serum (FBS), 0.25 mmol/L dexamethasone (Sigma; St. Louis, MO), 5 mg/ml insulin (Sigma) and 0.5 mmol/L isobutyl-methylxanthine (IBMX) (Sigma). At day 2, the medium was replaced with adipogenic medium containing DMEM supplemented with 10% FBS and 5 mg/ml insulin, which was changed every two days thereafter until analysis. Human adipose tissue derived stem cells (ASC) and their culture medium were purchased from Invitrogen and cultured according to the manufacturer’s manual. Briefly, human ASC were cultured in ASC growth medium containing basal medium, growth supplement and 2 mmol/L L-glutamine. The cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37uC. The medium was replaced every 2–3 days. Passages 5–6 were used for all experiments. To initiate differentiation, two days post-confluent ASC (day0) were treated with a differentiation medium containing ASC basal medium, 10% FBS, 2 mmol/L L- glutamine, 1 mmol/L dexamethasone, 10 mmol/L insulin, 0.5 mmol/L IBMX, 200 mmol/L Indomethacin (Sigma), 100 U/ml penicillin and 100 ug/ml streptomycin. The differen- tiation medium was changed every three days thereafter until the indicated times. Immunoblotting Cells were lysed in mammalian protein extraction reagent (Thermo Scientific) supplemented with protease inhibitor cocktail (Sigma). Additionally, phosphatase inhibitor cocktail I and II were added for phospho-ERK (p-ERK) detection. The cell lysates were resolved by electrophoresis on 10% or 4–12% precast Bis-Tris gel (Invitrogen). Proteins were transferred from the gel to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). Specific proteins were detected by immunoblotting using primary antibodies anti-PHB1 (BioLegend, San Diego, CA ), anti-PHB2 (Bethyl, Montgomery, TX), anti-C/EBPb (Cell Signaling; Danvers, MA), anti-PPARc (Cell Signaling), anti-aP2 (Abcam; Cambridge, MA), anti-HSP90 (Santa Cruz Biotechnol- ogy; Santa Cruz, CA) anti-b-actin (Santa Cruz), anti-ERK (Cell Signaling), anti-p-ERK (Cell Signaling) and anti-porin (Abcam). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG were used as secondary antibodies (Jackson Prohibitins Are Required for Adipogenesis ImmunoResearch; West Grove, PA). Blots were revealed with enhanced chemiluminescent reagents (Thermo Scientific). Real-time PCR analysis Real-time PCR analysis was performed as previously described with slight modifications [28]. Briefly, total RNA was isolated from 3T3-L1 cells at the indicated times using the RNeasy Mini Kit (Qiagen; Valencia, CA). Isolated RNA was reverse-transcribed with oligo-dT primer using the Advantage RT-for-PCR kit (Clontech; Mountain View, CA). Real-time PCR was performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics; Indianapolis, IN) and a LightCycler real-time thermal cycler (Roche Diagnostics). The primer pairs used were: 59-CCGTGGCGTACAGGACATTG-39 and 59- AAGGCAG- GACCCGCTCATCATAGT-39 for mouse PHB1; 59- CGTGGAAGGCGGTCATAGAGC-39 and 59-TCGGGACAG- CACACGCAGGGAGAT-39 for mouse PHB2; 59-GGAAGGG- CACCACCAGGAGT-39 and 59-TGCAGCCCCGGACATC- TAAG-39 for mouse 18S rRNA as an internal control. The amplified products were analyzed by electrophoresis on 2% agarose gels containing ethidium bromide (E-Gels; Invitrogen) to confirm primer specificity and PCR product size. Transfection of siRNA in 3T3-L1 cells One day before transfection, 3T3-L1 cells were seeded in the growth medium without antibiotics so that they would be 50–70% confluent at the time of transfection. Cells were transfected with 10 nmol/L siRNA using Lipofectamine RNAiMAX, according to the manufacturer’s protocol (Invitrogen). The three siRNAs, siPHB1-1, siPHB1-2 and siPHB1-3, target the following distinct sequences in mouse PHB1 mRNA: 59-GCUGUCAUCUUU- GACCGAUTT-39, 59-GCUUCCUCGUAUCUACACCTT-39, and 59-GCCGCUGUCAAUAAAUGACTT-39, respectively. The three siRNAs, siPHB2-1, siPHB2-2 and siPHB2-3, target the following distinct sequences in mouse PHB2 mRNA: 59- GCACUGAGCAAGAAUCCUGTT-39, 59-GCAAGAAUC- CUGGCUAUAUTT-39 and 59-GGCAUCAUGUGAUGGA- CUUTT-39, respectively. A universal siRNA Control (siControl) was used as the negative control. All siRNAs were obtained from Invitrogen. Oil Red O staining Oil red O (Sigma) staining was performed as suggested by the manufacturer with minor modifications. Seven days after the induction of adipocyte differentiation, 3T3-L1 cells in 60 mm dishes were washed with PBS and fixed with 10% formalin. The dishes were washed once with 60% isopropanol and left to dry completely. The cells were then stained in 0.2% Oil Red O for 10 minutes, rinsed with 60% isopropanol once, and thoroughly washed with water four times. The dishes were subsequently scanned to get the pictures. After extracting the Oil Red O with 100% isopropanol, the extracted dye was quantified on a spectrophotometer (Molecular Devices; Sunnyvale, CA) by reading the absorbance at 510 nm wave length. Immunocytochemistry The procedure used for immunofluorescence microscopy of cells to detect PHBs has been previously described in detail [19,20]. In brief, cover glasses were autoclaved and coated with attachment factor solution (Cell Applications; San Diego, CA) in a 6-well plate. 3T3-L1 cells were seeded and treated with differentiation medium in the plate as described above. The cells were then fixed with 4% paraformaldehyde in PBS for 30 min, PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34315