shRNA expression reduced the proliferation of cells and prevented colony formation on soft agar plates, indicative of a defect in anchorage independent growth. These cells lines enabled us to fine-tune PHB1 and PHB2 silencing under standardized conditions and to investigate the mechanism underlying the strong phenotype associated with prohibitin depletion. We first analyzed the effect of prohibitin depletion on the levels of mitochondrial fusion protein OPA1 as previous publications have shown that prohibitins are necessary for stable expression of OPA1. We could show that OPA1 expression is dependent on the levels of prohibitins. A slight reduction of prohibitins, seen early after induction of shRNA production, led to a partial fragmentation of the fusion competent OPA1 fragments a and b. At later time points of induction and consequently stronger depletion of prohibitins, OPA1 fragmentation increased and mitochondria appeared fragmented. An incomplete reduction of prohibitin proteins, as seen with expression of shRNAs shPHB1-0 and shPHB2-0, only resulted in mild OPA1 fragmentation and no fragmentation of the mitochondrial network. Further analysis revealed that even strong and long-lasting depletion of PHB1 e.g. in cells producing shPHB1-3, did not cause the dissipation of MMP. To date, the effects of prohibitin loss in mammalian cells are still unclear: Schleicher et al. and Ross et al. show a reduction in MMP upon PHB1 and PHB1/2 depletion via RNAi in primary endothelial cells and a T-cell line, respectively; in contrast, Merkwirth and colleagues demonstrated that although PHB2 depletion in MEFs causes OPA1 fragmentation, respiratory activity and MMP are not affected. Treatment of cells with the ionophore CCCP resulted in the rapid loss of MMP accompanied by a gradual and consecutive fragmentation of the OPA1 fusion-competent protein fragment and the mitochondrial network. Different patterns of OPA1 fragmentation were observed in CCCP treated cells and prohibitin-depleted cells: All fusion incompetent fragments were upregulated upon CCCP treatment, but not as a consequence of prohibitin reduction. In addition, fragment d was downregulated when prohibitin levels were reduced, but upregulated upon CCCP treatment. Thus, our data confirm the findings of Merkwirth at al., showing downregulation of fragment d in PHB2-depleted MEFs. Studies in yeast have shown prohibitins interact with mAAA protease and are important for the VE-822 proper integration of respiratory chain complex proteins. Furthermore, rhomboid like protease PARL is thought to be involved in OPA1 processing. Considering the fragmentation pattern of OPA1 in prohibitin knockdown cells, it seems most likely that multiple proteases are involved in OPA1 processing and not all of them are affected by prohibitin loss. This would be in accordance with our observation that both mitochondrial cristae morphology and ATP synthesis were unaffected by a loss of prohibitins. Mitochondrial fragmentation only occurred in cell with a strong depletion of prohibtins as we saw it upon shPHB1-3 expression.