Figure 2
HIFs are activated in RCC4-EV cells in normoxic conditions. (a) RCC4-EV and RCC4-VHL cells were exposed to normoxic (20% O2) or hypoxic (1% O2) conditions for 4 h. Whole-cell lysates were immunoblotted (IB) using anti-HIF-1α, HIF-2α, HIF-1β, and β-actin antibodies. Experiments were repeated twice and representative blots are shown. (b) RCC4-EV and RCC4-EV cells were cultured for 4 h under 20% O2 or 1% O2 conditions prior to analysis of hypoxia-inducible factor 1α (hif1α), hypoxia-inducible factor 2α (hif2α), hypoxia-inducible factor 1β (hif1β), glucose transporter 1 (glut1), lactate dehydrogenase A (lhda), and pyruvate dehydrogenase kinase 1 (pdk1) mRNA levels using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Fold expression was calculated relative to the values measured for RCC4-VHL cells incubated in 20% O2. Data presented are expressed as mean ± standard deviation (SD). *p < 0.05 compared to the control cell population of RCC4-VHL cells under 20% O2 conditons. # p < 0.05 compared between indicated groups. (c) Graphic depiction of the cell viability levels for treated and untreated cells at each time point (n = 4). Differences between results were evaluated by two-way ANOVA followed by Dunnett’s test for multiple comparisons. (d) ATP content of RCC4-EV and RCC4-VHL cells was measured. Differences between results were evaluated by t-test. # p < 0.05 compared between indicated groups. (e) Equal numbers of RCC4 and RCC4-VHL cells were stained with MitoGreenTM and analyzed by flow cytometry to measure mitochondrial mass. Differences between results were evaluated by t-test.
