Proteomic Analysis of Cellular Response

The cells were harvested for proteomic analysis as per Santos et al. [27 (link)] for liquid chromatography-tandem mass spectrometry (LC-MS/MS), at time points of 0, 3, 6 and 24 h post treatment, were completed in biological and technical triplicates. Culture media was collected from each well in 2 mL Eppendorf tubes and stored at −80 °C for later Bioplex and toxicity assays. Cells were rinsed twice in 5 mL of 1× phosphate buffered saline (PBS, Merck KGaA, Darmstadt, Germany) for 5 min each at 37 °C and aspirated. Cells were then scraped into 1 mL of 1× PBS using a cell scraper (Sarstedt, Numbrecht, Germany) and the liberated cells were collected into an Eppendorf tube and centrifuged at 4000× g for 10 min. The supernatant was then discarded, and the cell pellets were stored at −80 °C until processing.