Proteomic Analysis of Cellular Response to Drug Treatment

The cell cultivation was completed in a 6 mm plate; the number of cells accounted for roughly 30–40% of an orifice. We used 2 μΜ of drug and 0.1% DMSO. Each concentration had triplicate experiments over 48 h. Cells were resuspended in lysis buffer combined with a 1 X PMSF proteinase inhibitor cocktail (Thermo Scientific, USA). The total protein concentration was measured using Thermo Scientific NanoDrop One (protein A280). Then, equal amounts of proteins (approximately 1 mg) were diluted with 450 µL of NH₄HCO₃ (50 mM) and added to Ultra filtration tubes (Amicon Ultra-4, PLGC Ultracel-PL ultrafiltration membrane, 10 kDa), and we added a 10 mM DTT reduction (A2948; AppliChem), 20 mM IAA alkylation (I2273; Sigma). Each sample was centrifuged at 14,000× g to pass the solution through a filter. The resuspension was added before the total intracellular protein was hydrolyzed by trypsin (90057; Thermo Scientific™) at 37 °C (12–14 h) and desalted using a Thermo Scientific Pierce C18 tip. We freeze-dried samples and used 1% TFA resuspensions (see Supplementary Materials for details). They were analyzed by mass spectrometry (Orbitrap Fusion Lumos, Shanghai, China). Screening for significantly different proteins was analyzed by DAVID (https://david.ncifcrf.gov (accessed on 12 March 2021)), STRNG (https://string-db.org (accessed on 12 March 2021)), and Cystoscope [50 (link)].