Article Figures & Data
Figures
- Figure 1.
DJ-1 interacts with VHL. A, SH-SY5Y cells were lysed and endogenous VHL was immunoprecipitated with VHL antibody and analyzed by Western blot using DJ-1 antibody. B, SH-SY5Y cells expressing FLAG-VHL-GFP (F-VHL-G) were treated with 25 μm H2O2, then lysed. FLAG-VHL-GFP was immunoprecipitated with FLAG antibody and subjected to SDS-PAGE analysis using DJ-1 antibody. F, FLAG antibody; I, IgG control.
- Figure 2.
Functional link between DJ-1 and VHL. A, B, WT or DJ-1 KO cortical neurons (A) or MEFs (B) were incubated in normal condition or in hypoxia (1% oxygen) for 180 min (neurons) or 40 min (MEFs). C, Cortical neurons were treated with 15 μm H2O2 for 2 h. D, WT and DJ-1 KO MEFs were also exposed to 0 or 25 μm H2O2 for 24 h. Cells were lysed and HIF-1α protein level was assessed with Western blot using HIF-1α antibody. Densitometry analysis of HIF-1α protein for each experiment is demonstrated in the right side of each panel. Data are the mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01, ANOVA with Tukey's post-test. E, Lymphoblasts extracted from two PD cases, one with a deletion in DJ-1 (Del) and one with a point mutation in DJ-1 (L166P), or from control (Ctr) individual were exposed to 50 μm hydrogen peroxide for 24 h and their HIF-1α response was examined with Western blot analysis using HIF-1α antibody.
- Figure 3.
HIF-1α expression is equal in DJ-1 WT and KO neurons and its response is VHL dependent. A, B, WT or DJ-1 KO cortical neurons were exposed to hypoxia for 40 and 180 min (A) or to different concentrations of H2O2 (0, 5, 10, and 15 μm) for 2 h (B). HIF-1α transcripts were amplified by RT-PCR. The densitometry analysis of each experiment is demonstrated in the middle and right panels. C, HIF-1α mRNA levels from WT or DJ-1 KO cortical neurons exposed to oxidative stress (left, 0 and 15 μm for 2 h) and hypoxia (right, 0 and 180 min) was also analyzed using qRT-PCR. D, WT and DJ-1 KO cortical neurons were treated with either 15 μm H2O2 for 2 h or 1 mm DMOG for 3 h. HIF-1α level was assessed by Western blot using HIF-1α antibody. The densitometry analysis is demonstrated in the right panel. Each bar is the mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 ANOVA.
- Figure 4.
HIF-1α ubiquitination is regulated by DJ-1 and HIF-1 overexpression protects neurons against MPP+. A, B, In vitro VHL ubiquitination assay was performed by incubating GST-HIF-1α protein with the components of its ubiquitination pathway, including hUBA1, hUBC5A, and VHL complex, in presence or absence of DJ-1 protein, at 30°C for 2 h. Groups without VHL or ubiquitin were used as controls. After reaction, GST-HIF-1α (G-HIF-1α) was precipitated from the assay mixture with glutathione Sepharose beads, and its ubiquitination level and ability to interact with VHL were assessed by Western blot analysis using ubiquitin and VHL antibodies respectively. Total content of VHL and GST-HIF-1α in each reaction, before starting ubiquitination, is shown as input. Densitometry analysis of ubiquitination and VHL–HIF-1α interaction is shown in the middle and right panels. C, D, In vivo ubiquitination of HIF-1α was assessed by stabilizing ubiquitinated HIF-1α (Ubq-HIF-1α) in WT or DJ-1 KO MEFs treated with MG-132 (20 μm for 1 h) and exposed to hypoxia (1 h). HIF-1α levels were assessed by Western blot analysis using HIF-1α antibody. Quantification of Ubq-HIF-1α was performed by normalizing the signal density of Ubq-HIF-1α from hypoxia plus MG-132 of each genotype to that of MG-132 of the same genotype, and then to the signal density of WT in MG-132-plus-hypoxia group. E, F, Primary cortical neurons derived from DJ-1 KO or WT mouse embryos were pretreated with DMOG for 2 h and then treated with MPP+ (20 μm) for 48 h. Neurons were either (E) lysed and the intact nuclei were counted (n = 4) or (F) assessed by MTT survival assay (n = 3). G, Stable form of HIF-1α (GFP-SHIF-1α) or GFP alone as control was overexpressed in WT and DJ-1 KO cortical neurons by GFP-SHIF-1α and GFP-expressing adenovirus. Cells were then exposed to 20 μm MPP+ for 48 h and their fixed nuclei were stained with Hoechst. H, Their viability was examined by calculating the ratio of neurons with intact nuclei and GFP signal (a and b) to the total number of GFP-expressing cells (n = 3). H, Left, Adenoviral expression of GFP-SHIF-1α in cultured cortical neurons (Ha and Hc) and Hoechst staining to detect intact versus apoptotic nuclei (Hb and Hd). H, Right, Detection of GFP-SHIF-1α by Western blot, using HIF-1α antibody, in RCC cells infected with GFP-SHIF-1α-expressing adenovirus. Each bar is the mean ± SEM of three or four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 ANOVA test and Tukey's post-test.
