Fig. 4

Aspirin treatment sensitises CRC cells to metabolic inhibitors. a Schematic showing the mechanism of action of metabolic inhibitors; CB-839 and UK-5099. Created with BioRender.com. b Cell proliferation assay of long-term (52 weeks) aspirin-treated SW620 cells with increasing concentration of CB-839. The graph shows the relative cell number in each aspirin condition, measured by crystal violet staining at 72 h compared to vehicle control. Error bars show SEM (n = 3 independent experiments). Asterisks refer to p values obtained using one-way ANOVAs with Dunnett’s multiple comparisons tests at each CB-839 concentration (*p < 0.05, **p < 0.01, ***p < 0.001). Images show representative wells in each condition at 72 h. c, d Confluency and relative apoptotic cells in long-term 4-mM aspirin-treated SW620 cells in combination with 5µM CB-839, in comparison to controls and to each drug alone. Error bars show SEM (n = 3 distinct passages of cells analysed on the same experimental plate). Relative apoptotic cells were measured by green fluorescent nuclei (indicating cells with activated caspase-3/7) relative to cell confluency. Line graphs show values over time, and the bar graph shows the values at the experiment endpoint (75 h after treatment). Error bars show SEM (n = 3 distinct passages of cells analysed on the same experimental plate). Asterisks refer to p values obtained using a one-way ANOVA with Tukey’s multiple comparisons test (****p < 0.0001). e, f Proliferation assay of SW620 cells treated with aspirin and/or CB-839 compared to vehicle control for 72 h, performed in Human Plasma-Like Medium (HPLM). e Cell number over time relative to the 0 h time point. f Relative cell number at 72 h, 5µM CB-839 treatment condition is shown relative to vehicle control in the same aspirin treatment condition. Error bars show SEM (n = 3 independent experiments). Asterisks refer to p values obtained using one sample t tests, comparing to a hypothetical mean of 1 (**p < 0.01). g Cell proliferation assay of long-term aspirin-treated SW620 cells with increasing concentration of UK-5099. The graph shows a relative cell number in each aspirin condition measured by crystal violet staining at 72 h. Error bars show SEM (n = 3 independent experiments). Asterisks refer to p values obtained using one-way ANOVAs with Dunnett’s multiple comparisons tests at each UK-5099 concentration (*p < 0.05, **p < 0.01, ***p < 0.001). Images show representative wells in each condition at 72 h