A map of AAV2 construct used to knockdown PTEN.

<p>The shRNA.PTEN was inserted into the gap between BamHI and EcoRI, expressed under the control of an U6 promoter. A robust and non-cell-specific chicken β-actin (CBA) promoter controlled the expression of ZsGreen acting as control reporter.</p

FluoroGold retrograde labeling evaluating the transduction efficiency of AAV2 vectors 4 weeks after injection.

<p>Retinal whole-mounts displayed FluoroGold-labeled (white) RGCs and gene-transduced (green) RGCs from the same retinal regions of rats intravitreally injected Wt AAV2-shRNA.PTEN-GFP or Y444F AAV2-shRNA.PTEN-GFP vector (A-H). Quantifying the percentage of GFP-positive cells in FluoroGold-labeled cells revealed the transduction capacity of Y444F AAV2-shRNA.PTEN-GFP was significantly stronger than that of Wt AAV2-shRNA.PTEN-GFP (I). ** <i>P</i> < 0.01 as tested by Student’s <i>t</i>-test. Scale bar, 100μm.</p

FluoroGold retrograde labeling of RGCs identifying the complete axotomy.

<p>No RGCs was labeled with FluoroGold in retinal longitudinal sections (A) and whole-mounts (C) of axotomized rats while numerous RGCs were labeled with FluoroGold in retinal longitudinal sections (B)and whole-mounts (D) of intact rats. Scale bar, 100μm.</p

Immunofluorescence testing PTEN in retinas 4 weeks after intravitreal injection of AAV2 vectors.

<p>Immunofluorescence of retinal sections showed PTEN expression in retinas following Wt AAV2-GFP (A-C), Y444FA AV2-shRNA.PTEN (D-F), or Wt AAV2-shRNA.PTEN (G-I) injection. Quantification of PTEN expression, measured by ImageJ densitometry method, revealed a significant difference in GCL and INL treated with Wt AAV2-GFP, Wt AAV2-shRNA.PTEN, and Y444F AAV2-shRNA.PTEN respectively (J). **<i>P</i> < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 100μm.</p

PTEN knockdown promoting axons regeneration in optic nerve 6 weeks after axotomy.

<p>Fluorescent images of optic nerve longitudinal sections showed CTB-FITC labeled regenerating axons of rats treated with Y444F AAV2-shRNA.PTEN (A), Wt AAV2-shRNA.PTEN (B), and Wt AAV2-GFP (C) respectively. Quantification of the fluorescence intensity at different distances proximal to and distal to the ONA site showed the significant difference in Y444F AAV2-shRNA.PTEN, Wt AAV2-shRNA.PTEN and Wt AAV2-GFP groups (D). *<i>P</i> < 0.05, **<i>P</i> < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 100μm. Arrow, ONA site.</p

Western-blotting for the expression of GLAST in retina 6 weeks after axotomy.

<p>Compared to intact control, ONA resulted in dramatic down-regulation of GLAST, yet Y444F AAV2-shRNA.PTEN, compared with Wt AAV2-shRNA.PTEN or Wt AAV2-GFP, inhibited the reduction significantly. *<i>P</i> < 0.05, **<i>P</i> < 0.01 in ANOVA followed by Bonferroni’s post-test.</p

Immunofluorescence displaying RGCs and Müller cells transgene expressing GFP 4 weeks after intravitreal injection.

<p>Merged image showed colocalization of GFP fluorescence and TUJ1 staining in retinal flat-mounts from eyes treated with Y444F AAV2-shRNA.PTEN-GFP (A-C) or Wt AAV2-shRNA.PTEN-GFP (D-F), showed colocalization of GFP fluorescence and GS staining in retinal sections from eyes treated with Y444F AAV2-shRNA.PTEN-GFP (G-I) or Wt AAV2-shRNA.PTEN-GFP (J-L). Scale bar, 50μm.</p

TUJ1 immuno-labeling evaluating RGCs survival 6 weeks after axotomy.

<p>The number of survived RGCs decreased significantly 6 weeks after axotomy. Compared with Wt AAV2-GFP, both Y444F AAV2-shRNA.PTEN and Wt AAV2-shRNA.PTEN significantly prompted RGCs survival, and the pro-survival effect of Y444F AAV2-shRNA.PTEN was stronger than that of Wt AAV2-shRNA.PTEN. **<i>P</i> < 0.01 in Student’s <i>t</i>-test or ANOVA followed by Bonferroni’s post-test. Scale bar, 50μm.</p