Figure 7.
NNATx deficient cells show enhanced differentiation.A, Western blot showing the levels of NNATx and NNAT in NNATx-deficient (ΔNNATx) Neuro-2a cells. The graphs show densitometric analysis (mean ± SD). B, cytoplasmic Ca2+ level in ΔNNATx Neuro-2a cells represented as fluorescence intensity. Cells were treated with differentiation medium for 24 h and the cytoplasmic Ca2+ level was measured using Fluo-4 AM. The graph (mean ± SD) is a representative of three independent experiments performed with triplicate samples. C and D, qRT-PCR results showing the expression of CHAT and RBFOX3 mRNAs relative to ACTB mRNA in ΔNNATx Neuro-2a cells after 4 days of differentiation. Results are representatives of three independent experiments. E, images of WT and ΔNNATx Neuro-2a cells showing neurites after 4 days of differentiation. F, quantification of neurites length (mean ± SD, n = 3 fields). At least 65 cells in each group were analyzed to quantify the length of neurites. WT Neuro-2a cells. Statistical significance was calculated using two-sided paired (A, C, and D) or unpaired (B and F) two-tailed Student’s t test. ∗, Welch’s correction was applied. CHAT, choline acetyltransferase; qRT-PCR, quantitative real-time PCR.