Extended Data Fig. 8: Acute DFO insult disrupts mitochondrial metabolism without decreased cell growth.
a, Cell growth assay in HCT116 and SW480 cells 24 hours after DFO treatment (10μM) and assessed by MTT (N = 3 biologically independent cell replicates for each treatment and cell line). b and c, Seahorse analysis of mitochondrial metabolism in DLD1 (b) and RKO (c) cells 24 hours after DFO administration (10μM) (N = 4 biologically independent cell replicates for each treatment condition). d and e, Western blot analysis of doxycycline (Dox)-inducible Lactobacillus NADH-oxidase (LbNox) and mitochondrial NADH-oxidase (Mito-LbNox) in stably generated in HCT116 (d) and SW480 (e) cells. f and g, MTT cell growth assays in LbNox and Mito-LbNox expressing cells following DFO (10μM) in (f) HCT116 (p = 0.014) and (g) SW480 cells (p = 0.0076) (N = 6 biologically independent cell replicates for each cell line and treatment). h and i, MTT cell growth assay in cells stably expressing yeast NADH-ubiquinone reductase (NDI1) following DFO (10μM) or phenformin (62.5μM) treatment in (h) HCT116 (p = 0.0006) and (i) SW480 (p = 0.0003) cells (N = 3 biologically independent cell replicates for each cell line and treatment). j and k, Western blot analysis of mitochondrial enzymes following 24-hours of DFO (10 µM) treatment in DLD1 (j) and RKO (k) cells. Data represent the mean ± SEM. Significance was determined by 2-tailed, unpaired t test (a, h, i)) or one-way ANOVA (f–g) followed by Tukey’s post hoc. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared to vehicle.
