Fig. 8

The HIF-1α/β-catenin transcriptional complex contributes to 5-FU resistance and glycolytic activity. a Western blots of Wnt/β-catenin signaling pathway members from WT and 5-FU-R cells. β-Actin was used as the internal reference. b Immunocytofluorescence staining of β-catenin and HIF-1α. Merged images show the overlap of β-catenin (red), HIF-1α (green) and nuclear staining by DAPI (blue). c Effect of siRNA CTNNB1 knock-down on distribution of HIF-1α analyzed by Western blotting. Lamin B1 is a nuclear protein reference, and β-Actin is a cytoplasmic marker. d Effect of siRNA CTNNB1 knock-down in 5-FU-R cells on the stability of HIF-1α as assessed by Western blotting. β-Actin was used as the internal reference. e Anti-β-catenin co-immunoprecipitation experiment to identify interactions between β-catenin, HIF-1α, TCF1, and TCF4 in WT and 5-FU-R HCT8 cells. f Anti-HIF-1α co-immunoprecipitation experiment to identify interactions between HIF-1α, β-catenin, TCF1, and TCF4 in WT and 5-FU-R HCT8 cells. g Correlations between levels of β-catenin and HIF-1α proteins by Spearman rank analysis. h Correlations between levels of TCF1 and HIF-1α proteins by Spearman rank analysis. i Effect of 10 μM MSAB treatment for 72 h on 5-FU sensitivity of 5-FU-R cells. Cell viability was measured by the CCK8 assay after treating with increasing concentrations of 5-FU for 72 h. j Effect of 10 μM MSAB treatment on 2-NBDG uptake. k Effect of 10 μM MSAB treatment on lactate release. Normalized lactate release counts using the total protein concentrations. l Effect of 10 μM MSAB treatment 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 ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.001). r denotes Spearman rank correlation value and the p value denotes the significance level