Prohibitins Are Required for Adipogenesis Figure 4. Effect of PHB knockdown on the morphology of mitochondria in 3T3-L1 cells. 3T3-L1 cells were transfected with siRNA targeting PHB1 or PHB2, and cultured for three days. siControl was used as the control. A. The cristae morphology of mitochondria (arrows) in 3T3-L1 cells, transfected with the indicated siRNAs, was assessed by transmission electron microscopy (TEM) at 80 kV acceleration voltage and 50 k magnification. Scale bar, 100 nm. B. The siRNA-transfected 3T3-L1 cells were subject to adipogenic induction for 7 days. The cells at day 0 and day 7 were stained with MitoTracker Red. The mitochondrial morphology was analyzed with a Confocal Microscopy System. Scale bar, 10 mm. High-power magnification insets are included to emphasize the comparison of tubular mitochondria to fragmented ones. The bar graph represents the percentage of cell populations with fragmented mitochondria. **p,0.01 compared to siControl at day 0; ##p,0.01 compared to siControl at day 7. doi:10.1371/journal.pone.0034315.g004 the prior observations that intracellular expression of PHBs is increased and extracellular secretion of PHBs is decreased during 3T3-L1 adipocyte differentiation upon genetic and proteomic approaches [25,26]. In addition, we observed that the PHBs expression was mainly induced by insulin and IBMX rather than glucocorticoid among the three components in adipogenic induction cocktail. Insulin is known to act through the insulin- like growth factor 1 (IGF-1) receptor [42]. Stimulation of the IGF- Figure 5. PHB deficiency induces the accumulation of ROS without affecting the formation of ATP. 3T3-L1 cells were transfected with indicated siRNAs and cultured for three days. A. ATP concentration was detected using a luminescence assay, and was normalized to protein concentration. B. Cells were preincubated with/without 250 U/ml PEG-catalase for two hours. ROS formation was detected by using DCFA-DA staining and flow cytometry. *p,0.05, **p,0.01 compared to siControl without PEG-catalase preincubation; #p,0.05 compared to siPHB2-3 without catalase preincubation; ##p,0.01 compared to siPHB1-1 without catalase preincubation. C. Activities of complexes I were determined in whole cell lysates of 3T3-L1 with the Mitochondrial Dipstick Assay kit. The Complex I activity in 3T3-L1 cells transfected with siControl was set to 100%. *p,0.05, compared to siControl. doi:10.1371/journal.pone.0034315.g005 PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e34315