Table 1.
List of primers used for qRT-PCR.
Fig 1.
Cellular growth of undifferentiated and differentiated SH-SY5Y and BE(2)-M17 cells.
All of the examined drugs inhibited cell proliferation. In SH-SY5Y cells the effect of staurosporine was the most pronounced, while in BE(2)-M17 cells the strongest inhibition was achieved with RA treatment. The number of cells is shown as the mean ± S.E.M. of three experiments. Differences between differentiated and undifferentiated cells were tested for significance using Student’s t-test. (*P<0.05, **P<0.01, ***P<0.001).
Fig 2.
Cellular morphology of undifferentiated and differentiated SH-SY5Y and BE(2)-M17 cells after 7 days of differentiation.
Representative phase contrast images showed that after 7 days of treatment, staurosporine and RA promoted the most remarkable neurite extensions in SH-SY5Y cells and BE(2)-M17 cells, respectively. Scale bar = 100 μM.
Fig 3.
Neurite outgrowth in undifferentiated and differentiated SH-SY5Y and BE(2)-M17 cells after 7 days of differentiation.
Staurosporine and RA promoted the most remarkable increase in neurite length in the SH-SY5Y cells and BE(2)-M17 cells, respectively. RA treatment also increased neurite branching in BE(2)-M17 cells. Data are expressed as the mean ± SEM of three experiments. At least 90 cells were analyzed for each conditions. Differences between differentiated and undifferentiated cells were tested for significance using Student’s t-test. (*P<0.05, **P<0.01, ***P<0.001).
Fig 4.
Immunofluorescence staining for the neuronal markers neurofilament and β-III tubulin.
The comparison was made between undifferentiated and differentiated SH-SY5Y and BE(2)-M17 cells after 7 days of treatment. RA and staurosporine differentiation induced the formation of long processes positive for neurofilament and β-III tubulin in both cell lines. Blue: Hoechst; Green: neurofilaments; Red: β-III tubulin; Gray: phase contrast. Scale bar = 50 μM.
Fig 5.
Gene expression profile of DAergic, cholinergic and glutamatergic markers in SH-SY5Y and BE(2)-M17 cells.
TH, AADC, VMAT2, and DβH mRNAs were analyzed in undifferentiated cells and after 7 days of treatment with TPA, RA and staurosporine using semi-quantitative RT-PCR.
Table 2.
Gene expression profile of the primary catecholaminergic markers after differentiation.
Fig 6.
Gene expression profile of CAergic markers in differentiated SH-SY5Y and BE(2)-M17 cells.
After 4 and 7 days of differentiation with TPA, RA and staurosporine, TH, AADC, VMAT2 and DβH mRNA levels were compared with their corresponding levels in undifferentiated cells using qRT-PCR. Expression was displayed on a Log2 scale. Positive and negative values indicated up and downregulation of the genes with respect to control cells (undifferentiated cells), respectively. For each gene, differences between differentiated and undifferentiated cells were tested for significance using Student’s t-test. (*P<0.05, **P<0.01, ***P<0.001).
Table 3.
Cathecolamine contents detected in SH-SY5Y and BE(2)-M17 cell lines before and after differentiation for 7 days.