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. 2021 Jun 15;131(12):e143691. doi: 10.1172/JCI143691

Figure 5. ROS generation and iron accumulation are involved in DMF and FG4592–mediated cell death in CRC cells.

Figure 5

Cell death assay in HCT116 and SW480 cells treated with DMF (25 and 75 μM) (A) cotreated with DMF and FG4592 (100 μM) (B) cultured under hypoxia with or without NAC (5 mM). Data are represented as mean ± SD from 3 independent experiments. Statistical significance was calculated using unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ROS measurements in HCT116 and SW480 cells (C) treated with FG4592 (100 μM), DMF (50 μM), or DMF and FG4592 with or without NAC. (D) Cells treated with DMF and cultured in normoxia and hypoxia with or without NAC. Data are plotted as the mean ± SEM from 3 independent experiments. Statistical significance was calculated using 1-way ANOVA with Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (E) ROS measurements in shRNA-mediated HIF-1α, HIF-2α knockdown, and non–target scrambled HCT116 and SW480 cells treated with DMF (50 μM) either alone or in combination with FG4592 (100 μM). Statistical significance was calculated using 2-way ANOVA with Tukey’s multiple comparison. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (F) Heatmap showing the relative abundance of mitochondrial metabolites in FG4592-treated (100 μM) and DMF-treated (50 μM) HCT116 and SW480 cells. (G) Cell death and (H) ROS measurements using FG4592 (100 μM) and DMF (75 μM) either alone or under cotreated conditions in the presence of normal iron (control) and low iron. Statistical significance was calculated using unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.