Figure 6

Top IIβ deficiency inhibits neurite outgrowth and growth cone formation of primary cultured MDNs. A. ICRF-193 inhibits neurite outgrowth of MDNs. VM neurons isolated and cultured on PDL-coated cover slips. After 2 hours, ICRF-193 was added to a final concentration of 20 μM or 40 μM and the incubation was continued for another 5 days. Neurons were fixed and stained with anti-TH or anti-MAP2 antibodies. Scale bar = 50 μm. B. Statistic analysis of neurite lengths. Neurite outgrowth of TH⁺ neurons in the presence and absence of ICRF-193 showed significant difference, while neurite lengths of the total neurons (MAP2⁺) showed no significant difference. C. and D. No significant cell number change was found of the total neuron numbers (MAP2⁺) or dopaminergic neuron numbers (TH⁺) after the ICRF-193 treatment by cell counting. E. Growth cone formation was abnormal in the presence of ICRF-193. VM neurons isolated and treated with 20 μM, 40 μM ICRF-193 or vehicle for 24 hours. Cells were fixed and stained with Alexa Fluor 488-conjugated phalloidin (for F-ACTIN) and anti-TH antibody. Red: TH, green: F-ACTIN and blue: DAPi. Scale bar = 10 μm. F. Statistic analysis of the growth cone size. Area measurement of growth cones was performed by tracing the perimeter of the growth cones using Image J software (NIH, USA). G. Neurite outgrowth of MDNs was blocked by Top IIβ siRNA plasmids. Primary cultured MDNs were transfected with pSUPER-random or pSUPER-Top IIβ siRNA plasmids before seeding using AMAXA nucleofector instrument and cultured for 5 days. Neurons were fixed and stained with anti-TH and anti-GFP antibodies. Red: TH and green: GFP. Scale bar = 50 μm. H. Measurement of the neurite lengths of the transfected dopaminergic neurons showed pSUPER-Top IIβ siRNA plasmids significantly inhibited the neurite outgrowth. The average neurite length and growth cone size were determined from more than 100 cells. Bars represent the mean ± SEM resulted from 3 independent experiments. **p < 0.01 vs. control.