Fig. 5
HIF-1α is a master regulator of 5-FU resistance and glucose metabolic reprogramming in 5-FU-R CRC cells. a Western blots of HIF-1α in WT and 5-FU-R cells. β-Actin was used as the internal reference. b RT-qPCR analysis of HIF1A gene expression in 5-FU-R cells relative to WT cells. ACTB was used as the internal reference. c Immunocytofluorescence staining of HIF-1α (red). Merged images represent overlays of HIF-1α and nuclear staining in blue by DAPI. Scale bar = 20 μm. d WT and 5-FU-R cells cultured with 100 μM CHX for 1 h, 2 h, and 6 h to compare the stability of HIF-1α. β-Actin was used as the internal reference. e Effect of HIF1A knock-down on 5-FU sensitivity of 5-FU-R CRC cells. Viability was measured by CCK8 assay after treating cells with increasing concentrations of 5-FU for 72 h. f Correlations between HIF-1α expression and IC50 of 5-FU in 11 CRC cell lines by Pearson correlation analysis. g Effect of HIF1A knock-down on 2-NBDG uptake by 5-FU-R CRC cells. h Effect of HIF1A knock-down on lactate release from 5-FU-R CRC cells. Normalized lactate release counts using the total protein concentrations. i Measurement of OCR and ECAR of HIF1A knock-down 5-FU-R cells and control cells. j Comparison of basal respiration, maximal respiration, glycolysis and glycolytic capacity in HIF1A knock-down and control 5-FU-R CRC cells. k Effect of HIF1A knock-down 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 means ± SEM. Bar chart data were compared 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