To unravel pathways crucial for the onset of toxicity, we systematically investigated the changes in the proteome composition upon FQ addition via LC-MS/MS proteomics using labelfree quantification (LFQ). In pilot studies performed with Ciprofloxacin in HEK-293 cells we first tested high (75 µM, resembling peak plasma levels) and low (7.5 µM, resembling regular plasma levels) concentrations of the drug co-incubated with cells for 3 hours, 3 days, 7 days and 2 weeks representing a typical therapeutic regime (Figure , S1, S2) . Interestingly, both high and low concentrations evoked the significant up-regulation of proteins involved in iron transport and homeostasis (HMOX1, HBA1, HPX, LTF) , cellular stress (HMOX1, AATF, GPX3, MST1, PEX10, VNN1) and collagen fibril organization (COL18A1, COL1A1, COL1A2, COL1A5, FMOD, FN1, ITGA-1) already after 3 h (Figure ). The functions of these proteins correlate well with the known adverse effects of Ciprofloxacin to engage in metal-ion complexation, enhancing cellular stress, e.g. via reactive oxygen species (ROS) and leading to tendon and ligament damage. Interestingly, multiple target gene products and proteins involved in NF-κB signaling were detected to be upregulated within the thresholds (foldchange ≥ 1, -log(P-value) ≥ 1.3; see Figure ). Following the time course further, we observed the downregulation of proteins relevant for the electron transport chain (ETC), crucial for oxidative phosphorylation, especially of complex I and of complex IV (Figure , S2). At high concentrations, the downregulation already started at 3 h and was strongly pronounced after 3 days, peaking after 7 days (Figure ). Incubation of cells with 7.5 µM Ciprofloxacin showed a weaker shift of complex I and IV subunits after 3 and 7 days, but importantly after 14 days the downregulation became more prominent (Figure ). The extent and identity of the top regulated protein pathways after 3 days at 75 µM and 14 days at 7.5 µM was comparable, with strongly impaired oxidative phosphorylation, enhanced mitochondrial dysfunction and pronounced granzyme A and sirtuin signaling, e.g. involved in inflammation and redox homeostasis (Figure ). As tendon/ligament-derived cells more closely represent the physiological environment of these tissues and ideally provide additional insights into the origin of FQ-mediated tendon rupture, we performed similar whole proteome studies with human periodontal ligament (PDL) cells. We used as a model system an established cell line, namely PDL-hTERT, which was immortalized via stable expression of human telomerase reverse transcriptase. We here focused on the 3-day time point at high and low Ciprofloxacin concentrations and additionally performed offline cation-exchange chromatography fractionation to achieve deeper proteome coverage (Figure ). Interestingly, similar effects were recorded as in our previous analysis in HEK-293 cells. In order to investigate if these regulated proteins are a general hallmark of FQs, we performed the same study with Levofloxacin which revealed a large fraction of comparable changes (Figure ). Despite the deviating structure of Levofloxacin, the downregulation of complex I and IV was also observed using the same concentrations, although to a lesser extent. Of note, plasma concentrations of Levofloxacin are reported to be higher compared to Ciprofloxacin. For both Ciprofloxacin and Levofloxacin the top regulated protein pathways in PDL-hTERT are consistent with the results in HEK-293 cells (Figure , S3, S6). 3 (two-sided two-sample t-test, n = 3 replicates per group). Subunits of the electron transport chain (ETC) complexes I and IV are depicted in blue and green, respectively. A pronounced and time-dependent down-regulation of both complexes was detected. Similar effects were observed for the reported plasma concentration (7.5 µM), however, shifted to later onset (see Figure ). (C) Volcano plot depicting the proteome regulation of periodontal ligament cell line (PDL-hTERT) treated with Ciprofloxacin (75 µM) for 3 days versus vehicle control-treated cells featuring significant down-regulation of complex I (blue) and complex IV (green) subunits of the ETC. Threshold lines represent a log2 regulation of ±1 and -log10 (P-value) of 1.3 (two-sided two-sample t-test, n = 4 replicates per group). (D) Alphabetically sorted table of top up-regulated proteins (threshold: log2 regulation ≥ 1.5). (E) Alphabetically sorted table of top downregulated proteins (threshold: log2 regulation ≤ -1.5). (F) Top 5 most strongly regulated pathways (detailed pathway analysis in Figure ).