The metabolic alterations in media were measured with proton NMR spectroscopy
After recovery, the culture media of SKOV3 cells were collected. One day before the NMR experiment, media samples were thawed at the room temperature. The sample with 540 μL was collected and mixed with 60 μL potassium phosphate buffer (pH = 7.4) containing 1.5 M KH2PO4, 1mM NaN3, 0.1% TSP and D2O. A total of 580 μL mixture was placed into an NMR tube (Bruker Corporation, Rheinstetten, Germany) with an outer diameter of 5 mm. 1H-NMR spectra of media samples were obtained using a Bruker 600 MHz spectrometer (Bruker Corporation, Rheinstetten, Germany) at the operating 1H frequency of 600.13 MHz at a temperature of 300 K. A standard NMR pulse sequence (recycle delay-90º-t1-90º-tm-90º acquisition) was applied to acquire 1H-NMR spectral data (t1 = 3 μs, tm = 100 ms). The water peak suppression was achieved using selective irradiation during a recycle delay of 4 s and tm. A 90º pulse was adjusted to ~10 μs. A total of 32 scans for cell media were collected into 64 k data points with a spectral width of 20 ppm.1H-NMR spectral data were acquired using TopSpin 4.0.9 (Bruker Corporation, Rheinstetten, Germany). The spectral data were imported into MATLAB R2018a (MathWorks, Natick, Massachusetts, USA) and SIMCA (Sartorius, Gottingen, Germany) for multivariate statistical analysis. The chemical shift ranged from 4.7 to 5.0, and from -1 to 0.3 was cut. The spectral data was aligned with recursive segment-wise peak alignment method and normalised with probabilistic quotient normalisation method (Dieterle, Ross, Schlotterbeck, & Senn, 2006; Veselkov et al., 2009). Metabolites were identified using Chenomx software (CHENOMX, Edmonton, Canada).