Figure 2. The PHD-HIF axis up-regulates glycolysis under ammonia stress.

(A) HIF-1α and HIF-2α levels were increased by NH₄Cl treatment in a dose- and time-dependent manner. SKOV3 cells treated with NH₄Cl at the indicated concentrations and durations. β-actin expression was shown as the loading control. (B) Kinetic PFKFB3 mRNA expressions in PEO1 CD90⁺ cells upon NH₄Cl treatment. The PFKFB3 up-regulation by NH₄Cl observed in control cells (shNT) was almost completely abolished by two independent shRNA knockdown of HIF1A (HIF-1α). Each duplicated experiment was repeated twice. A two-way ANOVA and post-hoc tukey comparisons revealed a group effect between NT and KDs (P < 0.0001). (C) Protein expressions of HIFs, a HIF target GLUT1 and α-tubulin as a loading control were determined in SKOV3 cells by immunoblot upon NH₄Cl treatment (10 mM) for 16 hrs. (D) Glucose uptake assay with NH₄Cl treatment at 0, 2 or 5 mM for 16 hours in CD90⁻ and CD90⁺ PEO1 cells. Intracellular glucose levels were determined using 2-NBDG fluorescence signals. (E) Immunoblot of A498 cells and their derivative cells expressing WT-VHL upon treatment with 10 mM NH₄Cl. Arrow head indicates the ectopic expression of WT-VHL. (F) Comparison of prolyl-hydroxylation levels of HIF-1α between the indicated treatments. SKOV3 cells were treated with NH₄Cl (10 mM for 16 hrs), CoCl₂ (150 μM for 1 hr), MG132 (10 μM for 2 hrs) or hypoxia (1% O₂ for 2 hrs). The amount of protein loaded was adjusted to show approximately the same level of whole HIF-1α protein (refer to β-actin levels). (G) Cellular NO levels were determined by the fluorescent signal from DAF2-DA dye. (H) Effect of L-NAME or SOD on HIF-1α expression levels by NH₄Cl. The data in D and G show the mean of at least n = 3 independent experiments. Error bars indicate s.e.m. ^*P < 0.05; (Student’s t-test).