Article Figures & Data
Figures
- Figure 1.
Point mutation of Sox11 lysine 91 to alanine (Sox11K91A) attenuates expression of SUMOylated isoforms. A, B, HEK cells (2 × 105 cells/well) and (C) hippocampal neurons (106 cells/well) were infected with GFP control, Sox11, and Sox11K91A AAV2 viruses. Fluorescence imaging suggested similar expression levels between Sox11 viral vectors (A) and Sox11 and Sox11K91A protein expression were also similar when assayed by Western blotting (B, C). D, For SUMOylation detection, P2 RGCs (5 × 105 cells/well) were infected with GFP-conjugated control, Sox11, and Sox11K91A viruses. Quantification of higher molecular weight bands corresponding to total Sox11 protein was detected by Western blotting against Sox11, which detected both endogenous and mutant protein. Scale bar: 100 μm.
- Figure 2.
Sox11 and Sox11K91A decrease RGC viability and axon outgrowth in vitro. Primary RGCs were transduced with Sox11 or control AAVs as marked, cultured for 3 d, and immunostained for β-III tubulin (E7) and GFP to identified transduced neurons, and counterstained with the nuclear dye DAPI (A). Exogenous expression of either Sox11 or Sox11K91A reduced cell survival (B) and axon outgrowth (C) in primary RGCs (N ≥ 3 experimental replicates, *p < 0.05, by one-way ANOVA and post hoc t test with Tukey correction; mean ± SEM shown; scale bar: 200 μm).
- Figure 3.
Exogenous Sox11K91A leads to more RGC death than Sox11 in vivo. Control AAV2-GFP-Ctrl, AAV2-Sox11, or AAV2-Sox11K91A were injected intravitreally in control eyes and in eyes two weeks before optic nerve crush. Two weeks after injection in controls, or two weeks after optic nerve crush (four weeks after injection), eyes were harvested and retinas flat-mounted and immunostained against RGC-specific marker RBPMS (A). Both Sox11 and Sox11K91A significantly increased RGC death in sham (B) and optic nerve crush (C) eyes, with Sox11K91A showing significantly greater effect than wild-type Sox11 (N ≥ 4 experimental replicates, *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA with post hoc t test with Tukey correction; mean ± SEM shown; scale bar: 100 μm).
- Figure 4.
Sox11K91A promotes more axon regeneration than Sox11 in vivo. Intravitreal AAV2 injection was performed as in Figure 3. Regenerating axons were visualized by CTB-555 injection anterograde labeling 2 d before euthanasia (A). Both Sox11 and Sox11K91A significantly promoted more short distance (200 and 600 μm) axon regeneration than controls, with Sox11K91A showing significantly greater effect than wild-type Sox11 (B). The arrows indicate crush sites. N ≥ 5 animals per group, * compares with AAV2-GFP-Ctrl group; # compares with AAV2-Sox11 group; *p < 0.05 versus control, **p < 0.01 versus control, #p < 0.05 versus AAV2-Sox11, by one-way ANOVA with post hoc t test with Tukey correction. Mean ± SEM shown. Scale bar: 200 μm.
- Figure 5.
Gene regulation by Sox11 and Sox11K91A in primary RGCs. A, Sox11 and Sox11K91A upregulate several signaling pathways related to neuronal and axon growth. B, A volcano plot highlighting selected genes that are more downregulated by Sox11K91A than by Sox11 (in blue, 173 genes) and selected genes that are more upregulated by Sox11K91A than by Sox11 (in red, 181 genes). C, Spp1 and Opn4 were significantly downregulated in Sox11-treated and Sox11K91A-treated groups, confirmed by qRT-PCR (N = 3, *p < 0.05, **p < 0.01; N.S., no significant difference, by one-way ANOVA with post hoc t test with Tukey correction; mean ± SEM shown).
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