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  • br Direct infusion mass spectrometry DIMS analysis of


    2.14.2. Direct-infusion mass spectrometry (DIMS) analysis of 15-keto PGE2 15-Keto PGE2 was reconstituted in 0.1% formic L-NAME hydrochloride in 50% acet-onitrile and loaded in a Hamilton syringe, injected by a syringe pump with a flow rate of 1 μl/min into the heated electrospray ionization (HESI) source and measured for 0.5 min on a Q-Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific Inc., Germany). The operating source conditions for MS scan in positive ESI mode were optimized as follows: spray voltage, 3.6 KV; heated capillary temperature, 320 °C Nitrogen was used as damping gas. For higher-energy collision dissociation (HCD) experiments, keeping MS1 static, the precursor ion of interest was selected using the orbitrap analyzer, and the product ions were analyzed. The normalized collision energy (NCE) was set at 27. Resolutions of ions were set at 70,000 for MS1 and 17,500 for HCD experiments. The top 3 precursor ions in the MS scan were selected by orbitrap analyzer for subsequent MS/MS
    For LC-MS analysis, Ultimate 3000 UHPLC system (Thermo Fisher Scientific Inc., Germany) was coupled to Q-Exactive™ Plus Hybrid
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    Fig. 2. Effects of 15-keto PGE2 on phosphorylation, dimerization, nuclear localization, and transcriptional activities of STAT3 in MCF10A-ras cells. A. MCF10A-ras cells were treated with indicated concentrations of 15-keto PGE2 for 24 h. The expression levels of STAT3 and P-STAT3Y705 were determined by Western blot analysis. **p < 0.01. B. MCF10A-ras cells were incubated with 15-keto PGE2 (20 μM) for indicated time periods. The protein level of phosphorylated STAT3 was measured by Western blotting. C. Whole lysates from MCF10A-ras cells treated with or without 15-keto PGE2 (20 μM) for 12 h were prepared for im-munoprecipitation of STAT3 protein using protein A/G agarose conjugated C-terminus anti-STAT3 antibody. The STAT3 homo-dimerization was analyzed by Western blot analysis using N-terminus anti-STAT3 antibody. ***p < 0.001. D. MCF10A-ras cells were treated with 15-keto PGE2 (20 μM) for 24 h, and nuclear extracts were immunoblotted for detection of P-STAT3Y705. Lamin B was used as a nuclear protein marker. **p < 0.01. E. Immunocytochemical analysis was performed using antibodies against P-STAT3Y705. MCF10A-ras cells were treated with or without 15-keto PGE2 (20 μM) for 24 h. Nuclei were stained with DAPI and visualized under a confocal microscope. Two images were merged to verify co-localization. F. The luciferase assay was performed with HeLa/P-STAT3-luc reporter cells. The cells were treated with 15-keto PGE2 (10 or 20 μM) for 24 h and then stimulated with OSM (10 ng/ml) for another 6 h. Cells were then analyzed by a microplate luminometer. **p < 0.01, ***p < 0.001.
    The typical operating source conditions for MS scan in positive ESI mode were optimized as follows: spray voltage, 2.0 KV; heated capillary 
    temperature, 250 °C; and nitrogen was used as damping gas. All the spectra were recorded under identical experimental conditions. The scan range was set from m/z 400–2000, and resolution of precursor ions was set at 70,000. The 10 most abundant ions were fragmented by data-dependent mode at a resolution of 17,500 with the exclusion duration of 30 s and the isolation window was performed with 2.0 m/z. The NCE were set at 27.
    BALB/c (nu/nu) athymic mice were purchased from Charles River Laboratories (Skokie, IL, USA) and kept in a room at constant tem-perature (24 ± 2 °C) and humidity (50 ± 10%). The animal study was approved by Seoul National University Institutional Animal Care and
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    Use Committees (IACUC). Seven-week old female BALB/c nude mice were subcutaneously injected with 2 × 106 MDA-MB-231 cells per mouse at both flanks. After 10 days of treatment, mice were randomly assigned to three groups (seven mice per group) and were treated with vehicle (5% DMSO in PBS), 15-keto PGE2 (70 μg/kg) or 15-keto PGE2 (140 μg/kg) daily by subcutaneous injection for 4 weeks. Tumor vo-lumes were measured every other day with a digital caliper and cal-culated by formula 0.52 x length x width2. Mice were weighed three times a week.