re shown. Immunoreactivity of this protein in wild type cells is assumed as 100%, n = 4, p<0.001. Comparison of three loading markers level in MEFwt and MEFMfn2-/- cells. Mitochondrial mass, transcription factors and respiratory chain complexes Aforementioned suggestion that MEFMfn2-/- fibroblasts may have more mitochondria than MEFwt cells was supported by Western blotting indicating that Mfn2-depleted cells exhibit significantly higher level of TOM20 which is an accepted mitochondrial marker protein . Moreover, more intense staining of MEFMfn2-/- cells with NAO probe which binds to cardiolipin, a specific mitochondrial inner membrane phospholipid enforces this hypothesis. Further confirmation of this suggestion comes from Western blot analysis of TFAM which is a mitochondrial transcription factor and a key activator of mitochondrial DNA replication. As shown in Fig 6, TFAM level in MEFMfn2-/- cells is much higher than in MEFwt fibroblasts. Furthermore, the same was observed when PGC-1 protein level was analyzed. Both proteins are involved in mitochondrial biogenesis and increased level of them is considered as a marker of increased rate of this process. Thus, these data convincingly indicate more intense "mitochondriogenesis" which may compensate some mitochondrial dysfunction shown hitherto and described as a consequences of mtDNA copy number reduction by others. In line with this assumption, Western blot analysis of selected subunits of the mitochondrial respiratory chain complexes has revealed reproducibly higher level of complexes III, IV and V in mitofusin 2-depleted fibroblasts than in the MEFwt cells. Interestingly, respiratory complex I level seems to be the same in both cell lines 9 / 18 Mitofusin, Mitochondria and Energy Metabolism in MEF Cells Fig 6. Effect of mitofusin deficiency on the amount of TFAM and PGC-1 protein in MEF cells. The cells were grown in the medium supplemented with FBS. Relative amounts of both proteins in MEFwt and MEFMfn2-/- cells were tested immunochemically by Western blotting and normalized to immunoreactivity of PCNA. Representative blots and mean values of immunoreactivity S.D. are shown. Immunoreactivity of these proteins in wild type cells is assumed as 100%, n = 4, p<0.01. p<0.05. doi:10.1371/journal.pone.0134162.g006 tested. This may suggest its relatively low impact on the metabolic control of the respiratory chain in mitofusin 2-positive fibroblasts. It is also possible that slightly but reproducibly increased ROS formation in mitofusin 2-deficient cells could reflect relatively higher rate-controlling participation of the complex I to the electron flow than in mitofusin 2-positive cells. Increased amount of the complex V in MEFMfn2-/- cells probably counterbalances, improper composition of this protein complex. Altogether, data shown here indicate that mitochondrial metabolism of MEF cells with depleted mitofusin 2-encoding gene differs from that found in the wild type MEF and these change may be attributed to adaptations reflected PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755667 by enhanced mitochondrial biogenesis. ATP content and lactate formation Difference in mitochondrial metabolism could also influence an efficiency of the oxidative phosphorylation. As shown in Fig 8 ATP content 
in MEFMfn2-/- cells seems to be PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19754774 less MedChemExpress GLYX13 sensitive to oligomycin and more sensitive to iodoacetate than in the MEFwt fibroblasts. These differences are rather minor and indicate only slight switch from aerobic to anaerobic ADP 10 / 18 Mitofusin, Mitochondria a