Figure 3. High glucose levels induce mitochondrial ROS overproduction in hypoxia, which can be rescued by promoting HIF-1 function.
(A) Mitochondrial ROS levels were measured as mitosox intensity in mIMCD-3 cells cultured in normal (5.5 mM) or high (30 mM) glucose media in normoxia (N) or hypoxia (H) for 24 hr in the presence of DMOG or vehicle (n = 5). (B–D) mIMCD-3 cells were transfected with von Hippel–Lindau tumour suppressor (VHL) or control (Ctrl) siRNA, and exposed to hypoxia (H) and 30 mM glucose for 24 hr. VHL gene expression (B, n = 3), endogenous HIF-1α expression (red) and DAPI staining (blue) (C) and mitochondrial ROS levels (D, n = 5) were assessed using quantitative RT-PCR, fluorescent immunocytochemistry and flow cytometry, respectively. (E and F) mIMCD-3 cells were transfected with plasmids encoding GFP or GFP-HIF-1α,and exposed to hypoxia and 30 mM glucose for 24 hr. (E) Expression of GFP and GFP-HIF-1α (green) were detected using confocal microscopy. The nuclear HIF-1α expression was confirmed by immucytochemistry using anti-HIF-1α antibody (red). Nuclei were stained blue with DAPI. (F) Mitochondrial ROS levels are shown (n = 6). The mitosox intensity of cells cultured under control conditions were considered as 100%. Data are shown as mean ± SEM. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 using one-way ANOVA followed by Bonferroni’s post hoc test (A), and unpaired two-sided Student t-test (B, D and F). This figure has two figure supplements. Source data are shown in Figure 3—source data 1. Scale bar: 50 μm.
Figure 3—figure supplement 1. Flow cytometry gating strategy for the evaluation of mitosox intensity.
Figure 3—figure supplement 2. Flow cytometry gating strategy for the evaluation of mitosox intensity in mIMCD3 cells transfected with plasmids encoding GFP or GFP-fused protein.
