Figure 8.
Exogenous modulation of neuronal differentiation using NNAT-targeting antisense oligonucleotide. A, Western blot showing the expression of NNATx and NNAT in cells transfected with control or +43 ASO. Quantification (mean ± SD) of the band intensities from three independent experiments is shown in the graphs. RT-PCR results show the levels of NNAT mRNA in ASO-transfected cells. B, cytoplasmic Ca2+ level in control- or +43 ASO-transfected Neuro-2a cells represented as fluorescence intensity. Cells were treated with differentiation medium containing 2 μM retinoic acid for 24 h and the cytoplasmic Ca2+ level was measured using Fluo-4 AM. The graph (mean ± SD) is a representative of two independent experiments performed with six samples in each set. C and D, qRT-PCR results showing the expression of CHAT and RBFOX3 mRNAs relative to ACTB mRNA in Neuro-2a cells transfected with control or +43 ASO. Transfected cells were treated with differentiation medium containing 2 μM retinoic acid for 24 h before qRT-PCR was performed. E, qRT-PCR results showing the expression of CHAT relative to ACTB mRNA in Neuro-2a cells transfected with combinations of shRNAs and ASOs as indicated. Graphs (mean ± SD) represent results of three independent experiments performed with triplicate samples. Statistical significance was calculated using two-sided paired (A, C, and D) or unpaired (B and E) two-tailed Student’s t test. ∗, Welch’s correction was applied. CHAT, choline acetyltransferase; qRT-PCR, quantitative real-time PCR.