Supplementary MaterialsAdditional document 1: The graphs display the proliferation price (BrdU; in % of control cells) for the seven different breasts cancers cell lines cultured in moderate containing 1. evaluating the IC50 acquired for cells cultured with 3?mM 3-OHB (grey blots) with the control cells grown in medium free of 3-OHB (black boxes). Each blot represents 3C4 independent dose-response experiments with 6 replicate wells per experiment. None of the differences are statistically significant; however a strong tendency to a reduction in IC50 of paclitaxel is seen for T47D grown in 3-OHB medium compared to the control. (PPTX 104 kb) 40170_2018_180_MOESM2_ESM.pptx (105K) GUID:?3413BD6F-FB5B-4210-9F99-DF890ED37C31 Additional file 3: Columns represent mean??SEM of cell proliferation after irradiation shown for the seven cell lines at 21% and 5% oxygen concentration (gray column?=?with 3-OHB; black column without 3-OHB) (summarized in Fig.?6). The BT20, BT474 and T47D cell lines cultured in the presence of 3-OHB showed a trend towards increased radio-resistance at 21% oxygen (with some significant results at single doses). In contrast, in MCF-7 and MDA-MB 468, 3-OHB cultured cells showed a trend towards impaired cell proliferation following radiation at the same oxygen concentration. At 5% oxygen concentration, 3-OHB seemed to have a sensitizing effect to radiation in some cell lines. Columns represent mean??SEM of 3 independent tests with 6 replicate wells per test. * ?0.05, **not released *Mutations shown here are described by [63C65] Turnover of metabolites For quantification of glucose, lactate, and 3-OHB metabolism, cells were seeded and cultured at conditions described in the cell proliferation assay. After 5?days, supernatants were collected and the levels of 3-OHB were analyzed by the PrecisionXceed? instrument with the corresponding test strips FreeStyle Precision? -Ketone (Abbott, Wiesbaden, Germany). The concentrations of glucose and lactate were measured with the Cobas 8000 modular analyzer series (Roche Diagnostics; Mannheim, Germany) at the central laboratory of the University Hospital of Wrzburg. Concentrations of metabolites were expressed in millimolar per optical density (OD) of crystal violet dye extracts in each well at day 3 or 5 of culture. The amount of solubilized dye in OD is usually directly proportional to the cell number. Therefore, after removing the supernatant carefully for metabolite quantification, adherent cells were fixed with 100?l methanol (Sigma-Aldrich) for 10?min at room temperature (RT) and then dried. Cells were stained by incubation in 100 l crystal violet solution per well (0.4% crystal violet [Merck, Darmstadt, Germany] in 1:3 methanol: phosphate-buffered saline) for 10?min at RT and then washed several FLT3-IN-2 times with distilled water. Crystal violet was extracted from cells with 100?l of 10% acetic acid per well on a plate shaker for 30?min, and OD was determined at 570?nm by using a standard ELISA-Plate reader. Energetic profiling by Seahorse technique The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed using the Seahorse XF Cell Mito Tension Test (Component #103015-100; Agilent Technology, Santa Clara, CA, USA) within a Seahorse XFe96 Analyzer (Agilent Technology). The entire time prior to the test, 40,000 cells per well had been plated within a 96-well FLT3-IN-2 Seahorse dish in 100?l DMEM/10% FCS/Gentamycin/5?mM blood sugar moderate with or without 3?mM 3-OHB (sodium-hydroxybutyrate, Sigma-Aldrich). The Agilent Seahorse XFe96 Sensor Cartridge was hydrated with 200?l/well of XF calibrant option within a non-CO2 incubator in 37 over night?C. On the entire time from the test, 100?ml of FLT3-IN-2 Seahorse assay moderate containing 1?mM pyruvate, 2?mM glutamine, and 5?mM blood sugar was ready. The pH from the pre-warmed (37?C) moderate was adjusted to 7.4 with 0.1?N NaOH. Twenty milliliters from the assay moderate was used to get ready 3?mM 3-OHB, as well as the pH was readjusted to 7.4 with 0.1?N HCl. Cells were washed with 200 twice?l MPL from the corresponding Seahorse moderate and incubated in 175?l from the respective Seahorse moderate per well within a non-CO2 incubator in 37?C for 1?h. In the meantime, the Seahorse sensor cartridge slots had been packed with 25?l of inhibitors to truly have a final focus of 2?M oligomycin (interface A, Calbiochem), 1?M FCCP (interface B, Sigma-Aldrich), and 0.5?M rotenone/antimycin A (interface C, Sigma-Aldrich). The experimental style was set up using the WAVE computer software, and measurements had been performed in the Seahorse XFe96 Analyzer. Following the dimension, supernatant through the cells was taken out as well as FLT3-IN-2 the cells had been set by addition of 100?l methanol (Sigma-Aldrich) for 10?min in atmosphere and RT dried. Subsequently, the cells had been stained using crystal violet option as referred to for the colony development assay (discover below). For quantification, stained plates had been incubated with 200?l of 10% acetic acidity per good with shaking for 15?min as well as the resulting option was analyzed in a plate reader (Tecan GENios plus, Tecan Deutschland GmbH, Crailsheim, Germany) at 630?nm. Cell proliferation assay Adherent growing cells were seeded in 96-well flat bottom plates (TPP) at cell numbers determined for each cell line to reach semiconfluency after 3?days under the respective oxygen and low glucose conditions and to reach confluency after 5?days via preliminary testing. Thus, 250C350 cells per well, depending on the cell line, were seeded in 200?l DMEM/10%FCS/Gentamycin/5?mM.