Fig 1: mRNA and mature Dynorphin B expression in differentiated and undifferentiated SH-SY5Y following scAAV-pDyn transduction. SH-SY5Y cells were transduced with scAAV-pDyn at three different MOIs (5,000, 1,500, and 500) and harvested 96 h post-transduction. pDyn mRNA expression (A) and Dyn B levels in the medium (B) and in the cell extract (C) were analyzed in parallel from each infection well (n = 5) of differentiated and undifferentiated SH-SY5Y cells. (A) mRNA expression of pDyn was normalized to GAPDH in differentiated cells () and undifferentiated cells (). (B) Amount of DynB was quantified per well in the medium of differentiated cells () and undifferentiated cells (). (C) Amount of DynB was quantified per well in the cell extract of differentiated cells () and undifferentiated cells (). Data show five biological replicates with mean ± SD (****P-value ≤ 0.0001; ***P-value ≤ 0.001; **P-value ≤ 0.01; *P-value ≤ 0.05; ns—non-significant following a two-way ANOVA).
Fig 2: mRNA expression kinetics of preprodynorphin following ssAAV-pDyn/scAAV-pDyn transduction in differentiated SH-SY5Y cells. (A) The time course of pDyn mRNA expression from ssAAV-pDyn is depicted. Cells were transduced at decreasing MOIs: 1,000 (), 100 (), 10 (), 1 (), or mock infected (). Cells were harvested at different time points (days 1, 3, 5, and 8 post-AAV transduction). mRNA expression levels were analyzed for pDyn by RT-ddPCR and normalized to the housekeeping gene GAPDH. Data show three biological replicates with mean ± SD. Analysis of variance with Tukey's multiple comparison test was performed for MOI 1,000 comparing the different time points (***P-value = 0.0003; **P-value = 0.0031; *P-value = 0.0110; ns, non-significant). (B) pDyn mRNA expression profiles following transduction of scAAV-pDyn () at an MOI of 500 and ssAAV-pDyn () at an MOI of 5,000 are compared. Cells were harvested 96 h (±2 h) after infection and analyzed for pDyn mRNA expression as in (A). Data are shown as nine technical replicates each from two biological replicates.
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