Vnmr 500 spectrometer

High-resolution ³¹P NMR spectra were recorded on a Varian VNMR-500 spectrometer, operating at 202.45 MHz. Broadband proton irradiation (waltz 16) was applied to eliminate ¹H–³¹P NMR coupling.
Routinely 256 transients, with 1 s delay time, 90° pulse width, were collected for a total acquisition time of about 11 min for each sample. The probe temperature was 298 ± 1 K. All measurements were performed with D₂O as the internal field/frequency lock. Spectra were processed, using the MestReNova 12.0.2 software, with 2 Hz apodization, manually phased and base line corrected. Chemical shift assignments were referenced relative to hexamethylphosphoramide (HMPA) at δ 29.90 ppm and were attributed with the help of standards or literature data. Concentrations were calculated from the spectra by comparing the peak integrals of phosphorylated metabolites resonances with HMPA (2 mM) peak area.

Stock cultures of human MCF-7 breast cancer cells were purchased from The American Type Culture Collection. DMEM and FBS used in cell culture were products of Gibco; Thermo Fisher Scientific, Inc. Glutamine, penicillin and streptomycin were obtained from Quality Biological, Inc. The JC-1 MitoScreen kit was supplied by BD Pharmigen; BD Biosciences. ELISA kits used to detect the concentrations of matrix metalloproteinase (MMP)-2, MMP-9, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, mTOR and soluble intercellular adhesion molecule (sICAM)1 were obtained from Wuhan EIAab Science Co., Ltd. (cat. nos. E0100 h, E0553 h, E0133 h, E0699 h, E14969 h and EH0161). Etoposide was obtained from Sigma-Aldrich; Merck KGaA and the purity of the compound was >98%. Monoclonal anti-MUC1 antibody (cat. no. MA1-06503) was a product of Thermo Fisher Scientific, Inc.
Nuclear magnetic resonance (NMR) spectra were recorded using a Varian VNMR500 spectrometer (Varian, Inc.). Chemical shifts are quoted in parts per million relative to TMS for ¹H and toluene-d₈ for ¹³C NMR. Coupling constants J are reported in Hz. Mass spectra were recorded using an AMD-604 Intectra GmbH mass spectrometer (Waters Corporation).