Fig. 7: Aspirin synergizes with fludarabine phosphate to inhibit tumor growth.

A Representative immunofluorescence staining of pan-acetyllysine (green) and CS (mitochondrial marker, red) in aspirin treated (5 mM, 24 hours) H1299 cells. Scale bars (bottom right), 10 μm. B Western blot analysis of mitochondrial proteins levels of wild type and SLC25A51 knockdown HCT116 cells treated with aspirin (1 mM) or DMSO for 24 hours. The relative acetylation ratio is normalized to CS. C Western blot analysis of LoVo cells treated with fludarabine phosphate (40 μM) or aspirin (1 mM) or the combination of these two for 24 hours. D LoVo cells were seeded in the 6-well plates at 10³ per well, after cells grew with aspirin or fludarabine phosphate or the combination of these two at indicated concentrations for 7 days, the colonies were fixed and stained with crystal violet. E Wild type and SLC25A51 knockdown LoVo cells were seeded into 6-well plates at 10⁵ per well and grew with DMSO or aspirin (5 mM). The cell numbers were counted every day. F SW480 cells were seeded into 6-well plates at 10⁵ per well and grew with fludarabine phosphate alone or the combination of aspirin and fludarabine phosphate at indicated concentrations. The cell numbers were counted every day. G The representative image of EdU incorporation assay in LoVo cells treated with fludarabine phosphate (10 μM) alone or the combination of fludarabine phosphate (10 μM) and aspirin (2 mM) for 24 hours. Scale bars (bottom right), 50 μm. H The percentage of cells positively stained with EdU was quantified. I BALB/c Nude mice were inoculated with HCT116 cells and inoculated intraperitoneally with vehicle, fludarabine phosphate alone, aspirin alone or the combination of fludarabine phosphate and aspirin as indicated. Tumor volume was measured every other day. J The representative image of HCT116 tumors that dissected at the end of experiments. K The tumor weight was determined at the final day. L Western blot analysis of the phosphorylation levels of AKT, mTOR and S6K in HCT116 tumors treated as indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are representative of three independent experiments with similar results. Bars, mean ± SD.