Next, in-depth validation studies for apoptosis-inducing factor 1 (AIFM1) were performed. AIFM1 is a mitochondrial intermembrane space (IMS) protein anchored to the inner mitochondrial membrane. Among its function in non-caspase-mediated cell death, AIFM1 is crucially involved in the IMS import machinery. AIFM1 facilitates the binding and translocation of MIA40 (also called CHCHD4), the key component of the mitochondrial disulfide relay system, into the IMS. Substrates of this redox-regulated system are small nuclear encoded cysteine-containing proteins important for mitochondrial processes, such as ETC complex biogenesis, mitochondrial translation and lipid homeostasis, that are imported into the IMS and oxidatively folded by MIA40. Partial loss of AIFM1, such as in hypomorphic harlequin (Hq) mice, therefore results in severe mitochondrial dysfunction including complex I and IV dysfunction. In accordance, whole proteome analyses of AIFM1 knockout (KO) HEK-293 cells revealed a strikingly similar proteotype to FQ-treated cells with a strong complex I and IV down-regulation. In order to study the effects of FQ on AIFM1 in cells directly, we compared proteome changes of Ciprofloxacin-treated and untreated HEK-293 wt with treated and untreated KO cells (Figure ). In accordance with the literature, we see overall similar pattern of protein regulation of treated wt cells, in particular confirming downregulation of complex I and IV. Strikingly, proteomic comparison between Ciprofloxacin treated and untreated KO cells show only little additional alterations highlighting that Ciprofloxacin needs the presence of AIFM1 to affect respiratory chain complex levels (Figure ).