Additional file 1 of Aspirin reprogrammes colorectal cancer cell metabolism and sensitises to glutaminase inhibition
Creators: name: Holt, Amy K.
name: Najumudeen, Arafath K.
name: Collard, Tracey J.
name: Li, Hao
name: Millett, Laura M.
name: Hoskin, Ashley J.
name: Legge, Danny N.
name: Mortensson, Eleanor M. H.
name: Flanagan, Dustin J.
name: Jones, Nicholas
name: Kollareddy, Madhu
name: Timms, Penny
name: Hitchings, Matthew D.
name: Cronin, James
name: Sansom, Owen J.
name: Williams, Ann C.
name: Vincent, Emma E.
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Virology

description: Additional file 1: Figure S1. Aspirin treatment reprogrammes nutrient utilisation in CRC cells. Proportion of 13C labelling in downstream metabolites after 8 h incubation with either U-[13C]-Glc or U-[13C]-Q in LS174T and HCA7 cells with long-term 4mM aspirin treatment compared to control cells. Error bars represent SD (n = 3 technical replicates). Asterisks refer to adjusted p-values obtained from multiple t tests (* = p &lt; 0.05, ** = p &lt; 0.01, *** = p &lt; 0.001, **** = p &lt; 0.0001). Figure S2. Long-term aspirin treatment regulates metabolic enzyme expression in SW620 cells. a-e) qPCR analysis and quantification of immunoblot of metabolic gene expression in long-term (52 week) aspirin exposed SW620 cells. Error bars represent SEM (n≥3 independent experiments). Asterisks refer to p-values obtained from one-sample t tests, comparing to a hypothetical mean of 1 (* = p &lt; 0.05, ** = p &lt; 0.01, *** = p &lt; 0.001). a) qPCR analysis of ATF4 target gene expression. b) qPCR and quantified immunoblot analysis of ATF4 expression. Immunoblot image shows a representative blot of 3 independent experiments, α-tubulin is used as a loading control. c) Quantification of immunoblots for proteins involved in glutamine metabolism. d) Quantification of immunoblot for proteins involved in central carbon metabolism. GLS1GAC = GAC splice isoform of glutaminase 1. e) Representation immunoblot image of 3 independent experiments and quantified immunoblot analysis of HK1 and GLUT1 expression, α-tubulin is used as a loading control. f) Relative mRNA levels, determined by qPCR of genes involved in central carbon metabolism. Figure S3. Long-term and short-term aspirin treatment regulates metabolic enzyme expression in three CRC cell lines. a-b) Graphs show quantification of immunoblotting, error bars represent SEM (n = 3 independent experiments). Asterisks refer to p-values obtained from one-sample t tests, comparing to a hypothetical mean of 1 (* = p &lt; 0.05, *** = p &lt; 0.001) Immunoblot images are representative of three independent experiments, α-tubulin is used as a loading control. GLS1GAC = GAC splice isoform of glutaminase 1. a) Short-term (72 h) and long-term aspirin (52 week) treated LS174T and HCA7 cells. b) Short-term (72 h) aspirin treated SW620 cells. c) qPCR analysis of ATF4 target genes in short-term (72 h) aspirin treated SW620 cells. Error bars represent SEM (n = 3 independent experiments), asterisks refer to p-values obtained from one-sample t tests, comparing to a hypothetical mean of 1 (* = p &lt; 0.05, ** = p &lt; 0.01). Figure S4. Aspirin sensitises CRC cells to metabolic inhibitors. a-d) Cell proliferation assays of long-term (52 week) aspirin treated SW620 (a) LS174T (b) and HCA7 cells (c) and short-term (72 h) aspirin treated SW620 cells (d) with increasing concentrations of either inhibitor 968, CB-839 or UK-5099. Graphs show relative cell number in each aspirin condition as measured by crystal violet staining at 72 h of drug treatment compared to vehicle control. Error bars show SEM (n = 5 independent experiments for HCA7 with CB-839, n = 3 independent experiments for all other experiments). Asterisks refer to p-values obtained using one-way ANOVAs with Dunnett’s multiple comparisons tests at each drug concentration (* = p &lt; 0.05, ** = p &lt; 0.01, *** = p &lt; 0.001). Images show representative wells in each condition after 72 h of treatment. e) Quantification of green fluorescent nuclei indicating apoptotic SW620 cells with activated caspase-3/7, with 2µM ABT-737 treatment compared to control, as a positive control for apoptosis. Error bars represent SD (n = 3 technical replicates). f) Representative images of 2µM ABT-737 treated and control SW620 cells at the end of the assay (~ 96 h). Scale bar represents 300µm. Figure S5. Aspirin and CB-389 in combination reduce colon crypt proliferation in vivo. Quantification of the villi length in small intestine of VillinCreER Apcfl/fl mice treated with CB-839 (200mg/kg) and/or aspirin (2.6mg/ml in drinking water). Error bars show SEM (n = 4 mice per experimental arm). Each dot represents the average length of villi for each mouse. Statistical analysis (ns, p &gt; 0.05), one-way ANOVA with Tukey or Bonferroni post hoc test.