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. 2020 Jun 1;21:251–263. doi: 10.1016/j.omtn.2020.05.031

Figure 2.

Figure 2

Developmental Decline of miR-19a Is Associated with Increased PTEN Expression in RGCs

(A) Dual luciferase assays of PTEN, mPTEN, and SOCS3 3′ UTR after transfection with miR-19a mimic or scramble oligonucleotides in P6 RGCs. Only PTEN 3′ UTR, but not mPTEN or SOCS3 3′ UTR, was significantly suppressed by miR-19a (PTEN, p = 0.0001; mPTEN, p = 0.987; SOCS3, p = 0.976; n = 3–4 experimental replicates). Firefly luciferase was normalized to Renilla activity. See also Figure S2. (B) Western blots of PTEN and SOCS3 in P6 RGCs after transduction with AAV-miR-19a-EGFP or AAV-EGFP (left). miR-19a significantly reduced PTEN (PTEN, p = 0.001; n = 6 experimental replicates), but not SOCS3 (SOCS3, p = 0.869; n = 7 experimental replicates) (right). Values were normalized to GAPDH or β-actin. (C and D) Immunofluorescence images of PTEN (red) in P6 RGCs with AAV-miR-19a-EGFP (green) (C) or AAV-miR-19aTuD-EGFP (green) (D). Scale bars, 10 μm. Single-cell PTEN intensity was significantly lower in AAV-miR-19a-EGFP-transduced RGCs (p = 0.002; n = 100 RGCs) and higher in AAV-miR-19aTuD-EGFP-transduced RGCs (p < 0.0001; n = 183 RGCs), compared with control (n = 327 RGCs). See also Figure S3. (E) In situ hybridization (ISH) and immunohistofluorescence images of endogenous miR-19a (upper panel) and PTEN (lower panel), respectively, in E21, P6, and P30 retinas. Boxed areas are magnified on the right. Scale bars, 25 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (F) ISH images of endogenous miR-19a (green) in E21, P6, and P30 RGCs. Boxed areas are magnified above. Scale bars, 10 μm. Axon growth cones are indicated by arrowheads. (G) Immunofluorescence images of endogenous PTEN (red) in E21, P6, and P30 RGCs. Boxed areas are magnified above. Scale bars, 10 μm. Axon growth cones are indicated by arrowheads. (H) Single-cell PTEN intensity significantly increased from E21 (n = 78 RGCs) to P6 (E21 vs. P6, p = 0.047; n = 89 RGCs) and to P30 (E21 vs. P30, p < 0.0001; P6 vs. P30, p < 0.0001; n = 88 RGCs). All values are shown as mean ± SEM. Unpaired two-tailed Student’s t test was used for all comparisons: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. N.S., not significant.