Article Figures & Data
Figures
- Figure 1.
The in vivo proteasomal activity reporters UbG76V-GFP and ODDLuc are degraded through P97-dependent and P97-independent mechanisms. Top, UbG76V-GFP is a fusion protein consisting of GFP fused to a non-cleavable molecule of ubiquitin, which acts as a constitutively active degradation signal. UbG76V-GFP is degraded by proteasomes following its polyubiquitination and partial unfolding by the P97-Ufd1-Npl4 complex. Bottom, ODDLuc is a fusion protein consisting of firefly luciferase fused to an alternative degradation signal, the ODD of HIF1α. This reporter is polyubiquitinated and degraded by proteasomes without P97 processing. Conditions of inhibited, impaired, or insufficient UPS function result in the intracellular accumulation of either reporter.
- Figure 2.
Comparative analysis of proteasomal activity reporter accumulation in Gγ1 -/- and P23H retinas. A, The UbG76V-GFP reporter was detected in retinal lysates (25 μg total protein/lane) of three- to four-week-old Gγ1 -/- or P23H mice by Western blotting with an anti-GFP antibody; the densities of the UbG76V-GFP bands were normalized to the Hsc70 loading control. The number of mice analyzed was the following: WT, 8; Gγ1 -/-, 6; P23H, 6. Each dot represents a single data point. B, The ODDLuc reporter was detected in retinal lysates (100 μg total protein/reaction) from three- to four-week-old Gγ1 -/- or P23H mice using the Bright-Glo Luciferase Assay system. Retinal lysates from ODDLuc mice treated with the HIF prolyl hydroxylase inhibitor Roxadustat (Rox) were used as a positive control for reporter accumulation. The number of mice analyzed was the following: WT, 12; Gγ1 -/-, 8; P23H, 5; Rox, 6. Each dot represents a single data point. Data are shown as the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001.
- Figure 3.
Overexpression of P97 affects photoreceptor survival. A, P97 protein level was detected in retinal lysates (2 μg total protein/lane) of one-month-old Gγ1 -/- or P23H mice by Western blotting with an anti-P97 antibody; the densities of the P97 bands were normalized to the Hsc70 loading control. The number of mice analyzed was the following: WT, 6; Gγ1 -/-, 6; P23H, 6. Each dot represents a single data point. B, P97 protein level was detected in retinal lysates (2 μg total protein/lane) from one-month-old WT and P97-overexpressing (P97oe) mice as in A; Hsc70 was used as a loading control. The number of mice analyzed was the following: WT, 5; P97oe, 5. Each dot represents a single data point. C, Spider diagrams representing the number of photoreceptor nuclei in 100-μm segments of the inferior and superior retina at various distances from the optic nerve head. The number of mice analyzed was the following: WT, 5; P97oe, 3. Mice were three months old. D, The total number of nuclei in all eight 100-μm retinal segments represented in spider diagrams of panel C. Each dot represents a single data point. E, Representative images of inferior and superior retina cross-sections from WT and P97oe at the 1-mm distance from the optic nerve head. Mice were three months old. Scale bar = 25 μm. Data are shown as mean ± SD. n.s. indicates p > 0.05. *p < 0.05, **p < 0.01.
- Figure 4.
Overexpression of P97 affects photoreceptor survival in Gγ1 -/- mice. A–F, Morphology of Gγ1 -/- retinas with or without P97 overexpression at three (A–C) or six (D–F) months of age. A, D, Spider diagrams representing the number of photoreceptor nuclei in 100-μm segments of the inferior and superior retina at various distances from the optic nerve head. The number of mice analyzed at three months of age was the following: WT, 3; Gγ1 -/-, 7; Gγ1 -/-/P97oe, 3. The number of mice analyzed at six months of age was the following: WT, 4; Gγ1 -/-, 6; Gγ1 -/-/P97oe, 4. B, E, The total number of nuclei in all eight 100-μm retinal segments represented in panels A, D. C, F, Representative images of inferior and superior retina cross-sections at the 1-mm distance from the optic nerve head. Scale bar = 25 μm. Data are shown as mean ± SD. n.s. indicates p > 0.05. **p < 0.01.
- Figure 5.
Overexpression of P97 causes increased accumulation of the UbG76V-GFP reporter. The UbG76V-GFP reporter was detected in retinal lysates (25 μg total protein/lane) of one-month-old Gγ1 -/- and Gγ1 -/-/P97oe mice by Western blotting with an anti-GFP antibody; the densities of the UbG76V-GFP bands were normalized to the Hsc70 loading control. The number of mice analyzed was the following: Gγ1 -/-, 5; Gγ1 -/-/P97oe, 5. Each dot represents a single data point. Data are shown as the mean ± SD. **p < 0.01.
Tables
Line Figure Comparison Type of test p value a Fig. 2A GFP level in Gγ1 -/- vs WT Mann–Whitney 0.0007 b Fig. 2A GFP level in P23H vs WT Mann–Whitney 0.0007 c Fig. 2A GFP level in Gγ1 -/- vs P23H Mann–Whitney 0.0022 d Fig. 2B Luc activity in Rox WT vs untreated Mann–Whitney 0.0001 e Fig. 2B Luc activity in P23H vs WT Mann–Whitney 0.0002 f Fig. 2B Luc activity in Gγ1 -/- vs WT Mann–Whitney <0.0001 g Fig. 2B Luc activity in Gγ1 -/- vs P23H Mann–Whitney 0.0016 h Fig. 3A P97 level in Gγ1 -/- vs WT Mann–Whitney 0.8182 i Fig. 3A P97 level in P23H vs WT Mann–Whitney 0.7056 j Fig. 3B P97 level in P97oe vs WT Mann–Whitney 0.0079 k Fig. 3D Nuclei in P97oe vs WT at three months Mann–Whitney 0.0357 l Fig. 4B Nuclei in Gγ1 -/-/P97oe vs Gγ1 -/- at three months Mann–Whitney 0.2 m Fig. 4E Nuclei in Gγ1 -/-/P97oe vs Gγ1 -/- at six months Mann–Whitney 0.0095 n Fig. 5 GFP level in Gγ1 -/-/P97oe vs Gγ1 -/- Mann–Whitney 0.0079
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