siRNA-mediated knockdown of PHB2 led to fragmentation of the mitochondrial network. However, it is still not clear whether prohibitin expression stabilizes the mitochondrial membrane potential. Here we show prohibitins play a role in maintaining cellular homoeostasis and proliferation in cancer cells and primary cells. Depletion of prohibitins had no major effect on mitochondrial function but did affect adhesion of cells to the extracellular matrix. Since the discovery of their anti-proliferative activity, prohibitins have been implicated in multiple cellular functions. However, several lines of indirect evidence, such as their overexpressionin numerous cancer cells, indicated that prohibitins werenot functioningasa classical tumorsuppressor.Here,weshowprohibitins are a crucial prerequisite for cancer cell proliferation and survival. We used lentivirus-mediated shRNA expression to specifically reduce PHB1 and PHB2 mRNA and protein levels, respectively. Using the constitutive expression system pLL3.7, we demonstrated that, in all tested cancer cell lines, numbers of cells, expressing a PHB1 mRNA targeting shRNA were decreased from within a pool of transduced and non transduced cells. Furthermore, PHB1-depleted cells were unable to grow under anchorage-independent conditions. Our results are in accordance with two recent studies demonstrating that RNAimediated silencing of PHB1 or PHB2, respectively, leads to reduced proliferation in primary endothelial cells and mouse embryonic fibroblasts. These data are in stark contrast to a previous study showing increased proliferation in the breast cancer melanoma cell line MCF-7 upon siRNA-mediated silencing of PHB1. It is possible prohibitins play a different role in breast cancer cells in comparison to other cancer cell types. In agreement with the initial finding that the RNA of 39UTR of PHB1 exerts cytotoxic effects on cancer cells, the expression of a T-allelic PHB1 39UTR has been linked with an increased susceptibility to breast cancer : however, several clinical studies have contradicted these findings. In addition, sequencing of the PHB1 39UTR of different cancer cell lines, including MCF-7, revealed a higher frequency of overall sequence variation in comparison to primary non-cancer cells rather than a predominant expression of a specific T-allele. These findings suggest that prohibitins do play a role in carcinogenesis, but the level at which the effect is VE-821 exerted, i.e. 39UTR RNA or protein, remains unclear: Silencing of either PHB1 or PHB2 mRNA by RNAi resulted in reduction of the respective 39UTR but also in a knockdown of both prohibitin proteins. Since expression of all tested shRNAs led to a slowed proliferation rate, this phenotype is most likely a consequence of the loss of prohibitin proteins. To assess the long-term effect of prohibitin depletion, we generated HeLa cell lines with inducible shRNA expression. In these cells PHB1 and PHB2 mRNAs were efficiently and selectively down-regulated upon induction of the gene-specific shRNA and prohibitin proteins were reversibly suppressed.