Figure 2. In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A: Real time PCR for Egr1 in 661W cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, Table 1). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B: PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C: In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D: Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E: Schematic representations of the Egr1 downregulation (AAV2-shEgr1, top), Egr1 overexpression (AAV2-mEgr1, middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW: human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.