Fig. 7
ROS/PI3K/AKT pathway activation boosts HIF-1α levels and induces 5-FU resistance. a ROS levels in 5-FU-R and WT cells. Results shown as mean MFI (FITC channel) ± SEM. b Western blots of ROS scavenging enzymes. β-Actin was used as the internal reference. c Western blots of PI3K/AKT pathway members from WT and 5-FU-R CRC cells. β-Actin was used as the internal reference. d Correlations between levels of p-Akt/Akt and HIF-1α proteins by Pearson correlation analysis. e 5-FU-R cells were treated with 25 μM LY294002 for 48 h. Western blots of HIF-1α, AKT, and p-AKT. β-Actin was used as the internal reference. f HCT8 (WT) cells were treated with titrated concentrations of t-BHP (0 μM to 200 μM) for 4 h. Western blots of HIF-1α and PI3K/AKT pathway members. β-Actin was used as the internal reference. g 5-FU-R cells were treated with 750 μM NAC for 48 h. Western blots of HIF-1α and PI3K/AKT pathway members. β-Actin was used as the internal reference. h Effect on 5-FU sensitivity as determined by CCK8 assays. WT cells were treated with t-BHP (100 μM, 4 h) and 5-FU-R cells were treated with NAC (750 μM, 48 h). Cells were cultured with increasing concentrations of 5-FU for 72 h after t-BHP or NCA treatment. i Effect of modulating ROS levels on 2-NBDG uptake. j Effect of modulating ROS levels on lactate release. Normalized lactate release counts using the total protein concentrations. k Effect of modulating ROS levels on expression of the key glycolytic enzymes GLUT1 and MCT4. β-Actin was used as the internal reference. For all studies n ≥ 3. Data are presented as means ± SEM. Data were analyzed by Student’s t-test or ANOVA (ns = not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001). R2 denotes the Pearson correlation coefficient and the P value indicates the significance of the correlation