Fig. 4.

miR-7-mediated reduction of α-Syn level protects against cell death. (A) Schematic diagram of A53T mutant α-Syn construct without its 3′-UTR, with wild-type 3′-UTR, or miR-7 target site mutant 3′-UTR. (B) Mouse neuroblastoma NS20Y cells were transfected with constructs shown in A along with 50 nM miR-7 or control (scrambled miR sequence). pEGFP-C1 was used as internal control for normalization of transfection efficiency. A representative Western blot using LB509 antibody is shown. The relative levels of α-Syn normalized to EGFP expression were calculated based on band density using NIH Image J software with each scrambled miR transfected sample set to 1.0. (C) NS20Y cells were transfected with α-Syn (A53T) constructs in the presence of scrambled miR (control) or presence of miR-7. pSV-β-Gal plasmid was used as internal control for normalization of transfection efficiency. After transfection, cells were treated with 200 μM H₂O₂ for 16 h. Experiments were performed in triplicates and repeated independently 3 times. *, P < 0.05 for difference between control and miR-7-treated samples.