Additional file 4 of ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer

Additional file 4: Figure S4. HIF-1α is a prognostic biomarker and regulator of 5-FU resistance in CRC, related to Figs. 4 and 5. a. HIF1A mRNA expressions were obtained and compared from 2 GEO datasets. b. Nucleus and cytoplasm distribution of HIF-1α was analyzed by Western blots. Lamin B1 is a nuclear protein reference, and β-Actin is a cytoplasmic marker. c. Expression of HIF-1a in WT CRC cells versus 5-FU-R CRC cells. Cells were cultured under normoxia (20% oxygen) or hypoxia (1% oxygen). β-Actin was used as an internal reference. d. WT and 5-FU-R CRC cells were stably knocked down for HIF1A using shRNA. HIF-1a was confirmed by Western blotting, using β-Actin as a loading control. e. Western blots of HIF-1α in 11 CRC cell lines (HCT8, DLD-1, HCT116, HT29, SW480, SW1116, DiFi, Caco-2, HCT15, T84, and LoVo). β-Actin was used as the internal reference. f. CCK8 assays to assess 5-FU sensitivity of 11 CRC cell lines, and cells were treated with an increasing concentrations of 5-FU for 72 h. All experiments were performed with 6 replicates. Data are presented as means ± SEM. Bar chart data were compared by Student’s t-test (ns = not significant).