| Autor: |
Sprowl-Tanio, Stephanie, Habowski, Amber, Pate, Kira, McQuade, Miriam, Kehui Wang, Edwards, Robert, Grun, Felix, Lyou, Yung, Waterman, Marian |
| Rok vydání: |
2016 |
| DOI: |
10.6084/m9.figshare.c.3631172_d3.v1 |
| Popis: |
Disruption of oncogenic signaling leads to decreased lactate production and increased GSSG levels. SW480 cell cultures treated with vehicle (DMSO) or XAV inhibitor, or established cell lines transfected with either empty vector (mock) or a dnLEF-1 construct, were grown to 80 % confluency in 10-cm tissue culture plates in 10 % FBS supplemented DMEM medium. Cells and conditioned media were collected for metabolite extraction as follows. Briefly, cells were harvested from plates by trypsinization, counted and 4–5 replicate aliquots of 10e6 cells/sample prepared. Cells or media were collected by centrifugation at 1200×g for 5 min, rinsed with phosphate buffered saline, and extracted with 75 % ethanol/10 mM HEPES pH 7.4 buffer (final) at 80 °C for 5 min. Lysates were cleared by centrifugation at 12,000×g for 10 min at 4 °C. Supernatants were lyophilized, resuspended in 10 mM ammonium formate buffer, and cleared supernatant transferred to low volume mass spectrometry sample vials. Panel (A) lactic acid and panel (B) oxidized glutathione (GSSG) were quantitated by UPLC ESI MSMS on a Waters Quattro Premier XE instrument using ESI− and ESI+ ion modes, respectively. Analyte concentrations of lactic acid and GSSG were calculated from 8-point calibration standard curves in the range of 0.1–300 μM, quadratic fit, r 2 > 0.98. Data are shown for intracellular and media concentrations as scatter plots with means ± SEM, n = 4–5. **p |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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